HPV-16感染对喉咽鳞癌和FaDu细胞的生物学行为影响及miRNAs表达的调控
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摘要
背景与目的
人乳头状瘤病毒(Human papillomavirus, HPV)可引起一系列上皮病变,有些型别HPV与癌前病变相关,称为高危型HPV,部分人感染高危型HPV后可发生癌变。国际癌症研究机构在1995年认定HPV-16, HPV-18两种型别在人类有致癌作用。宫颈癌与高危型HPV感染的相关性已得到普遍认可。流行病学、分子和临床证据表明,高危型HPV感染除了与宫颈癌有强烈相关性之外,还与部分头颈部鳞状细胞癌(Head and neck Squamous cell carcinoma, HNSCC)的发生密切相关。
在HPV相关的HNSCC中,目前研究最多的是口咽部鳞状细胞癌(Oropharyngeal squamous cell carcinoma, OSCC)。临床证据表明,HPV相关OSCC通常不具备此类疾病中典型的大量抽烟和／或饮酒病史,在生物学行为上不同于经典的OSCC, HPV感染与否对OSCC病人的预后有显着影响,无论是治疗效果还是生存率,HPV阳性OSCC病人都优于HPV阴性OSCC病人。
喉鳞癌和喉咽鳞癌是HNSCC中两种常见恶性肿瘤,多发于中老年男性,大量抽烟和／或饮酒是其公认的致癌因素,然而有一部分患者并无大量抽烟和／或饮酒的病史,针对这部分患者的发病原因,研究者将目光转向了HPV感染。许多研究机构对喉鳞癌和喉咽鳞癌组织中HPV感染情况进行了检测,以往的研究多为回顾性研究,检测的癌组织来自病理存档标本,病人临床资料的获得受到诸多限制。此外,喉部与喉咽部解剖位置毗邻,部分晚期癌肿仅凭病理标本不易鉴别原发位置,鉴于喉鳞癌发病率显着高于喉咽鳞癌,大多数研究者便将喉咽鳞癌归入喉鳞癌一并研究。因此,报道喉鳞癌HPV感染率的文献很多,但鲜有喉咽鳞癌HPV感染情况的报道,HPV在喉咽鳞癌中的感染率及常见感染型别均不明确。
喉咽鳞癌和喉鳞癌的临床表现有一定的相似性,但却是原发于不同解剖部位的两种HNSCC,二者的分化程度、局部淋巴结转移率及5年生存率有明显不同,喉咽鳞癌的预后更差。而且目前研究认为HPV感染主要与HNSCC的预后相关,因此在开展HPV感染相关性研究时,有必要将喉咽鳞癌与喉鳞癌进行区分,将喉咽鳞癌作为独立的疾病进行相关研究。本课题拟对收集的28例喉咽鳞癌新鲜冰冻组织标本进行HPV DNA检测并分型,了解人喉咽鳞癌组织中HPV感染情况及常见HPV型别。
E6、E7基因为HPV基因组早期区基因,编码E6、E7蛋白,HPV-16编码的E6、E7蛋白是其发挥致癌作用的主要癌蛋白。慢病毒载体(Lentivirus vector)可以高效率且稳定的感染各期细胞,是将基因整合入细胞的良好运载工具,且具有操作简便、感染效率高、作用稳定、安全性好等优点。本课题拟利用重组HPV-16E6-E7基因的慢病毒感染人喉咽鳞癌FaDu细胞,观察HPV-16E6-E7基因整合对FaDu细胞生物学行为的影响。
微小RNA (miRNA或microRNA)参与了人体细胞的分化、增殖、凋亡、侵袭、及迁移等几乎所有的细胞生物学过程。miRNAs的异常表达与许多肿瘤的发生、发展相关,可发挥癌基因或抑癌基因的作用。然而目前对HPV相关HNSCC的]miRNAs表达情况了解有限。本课题拟对仅感染HPV-16和HPV阴性的喉口咽鳞癌组织miRNAs表达水平进行检测,同时检测人喉咽鳞癌FaDu细胞整合HPV-16E6-E7前后miRNAs表达水平的变化,分析HPV-16感染对人喉咽鳞癌组织和细胞株miRNAs表达的调控。
第一部分人喉咽鳞癌新鲜冰冻组织中HPV检测及分型
方法
1.收集28例人喉咽鳞癌新鲜组织标本,液氮速冻后移入-80℃低温冰箱保存。
2.应用基因组DNA小量试剂盒提取新鲜冰冻喉咽鳞癌组织标本基因组DNA。
3.PCR-反向点杂交法检测标本中HPV DNA并分型
4.将HPV感染情况与病人临床资料相结合,进行相关性分析。
结果
1.28例喉咽鳞癌标本HPV感染情况如下：HPV阳性者7例,总阳性率为25.0%(7/28)。5例为单一型别HPV感染,HPV-16型3例,HPV-18型1例,HPV-52型1例；2例为两种型别HPV同时感染,HPV-16,33型1例,HPV-16,52型1例。
2.HPV阳性与HPV阴性病人相比较,大量抽烟和／或饮酒病史有显着性差异(P<0.05)；年龄、性别、病理分型和肿瘤T分期之间的差异无统计学意义(P>0.05)
第二部分HPV-16E6-E7基因整合对FaDu细胞生物学行为的影响
方法
1.重组HPV-16E6-E7慢病毒的制备：全基因合成HPV-16E6-E7基因,并在两端分别加上HindⅢ和KpnⅠ限制性核酸内切酶位点,与慢病毒载体pLV-EGFP-C在T4连接酶的作用下连接,将连接体系转化入DH5a感受态细菌,挑选长出的菌落进行测序鉴定,即得到重组HPV-16E6-E7慢病毒。
2.重组HPV-16E6-E7慢病毒的包装：
>制备用于包装的质粒DNA溶液：将慢病毒包装载体pH1、pH2与重组HPV-16E6-E7'慢病毒质粒按照一定比率混合于50mL无血清DMEM培养基中。
》制备转染试剂稀释液：取一定体积的转染液Polyfect-V与500μL无血清DMEM培养基混合。
>将转染试剂稀释液加入用于包装的质粒DNA溶液中,立刻充分混匀,即制备成为转染混合液。室温孵育转染混合液15min,消化HEK293T细胞,每lml转染混合液加入10ml细胞悬液,轻轻吹吸细胞混匀。将细胞悬液加入10cm培养皿,放入37℃培养24h。去除含有转染试剂的培养基,用10ml病毒培养基换液。转染后48h培养上清即含病毒颗粒,收集并测定病毒滴度,.80-C保存。
3.重组HPV-16E6-E7漫病毒感染人喉咽鳞癌FaDu细胞：复苏FaDu细胞,用含有重组HPV-16E6-E7慢病毒颗粒的慢病毒液感染FaDu细胞。48h后观察荧光表达情况,用含有0.5μg/mL puromycin的DMEM高糖培养基进行筛选培养,3周后得到稳定转染细胞。PCR检测FaDu细胞中HPV-16E6、E7DNA, RT-PCR检测FaDu细胞中HPV-16E6、E7mRNA, Western-blot检测细胞中E6、E7蛋白。
4.将细胞分为3组：实验组(重组HPV-16E6-E7慢病毒稳定感染的FaDu细胞)、载体对照组(慢病毒空载体稳定感染的FaDu细胞)和空白对照组(未感染慢病毒的FaDu细胞)。
5. CCK-8试剂盒检测各组细胞增殖情况,分别测定实验组、载体对照组和空白对照组细胞孔内的吸光度值(A),绘制各组细胞的生长曲线。
6.收集各组细胞,通过PI染色用流式细胞仪检测细胞周期的变化；通过PI和Annexin-V联合染色检测细胞的凋亡。
7.分别收集实验组、载体对照组和空白对照组细胞,酶联免疫吸附法检测细胞Caspase-3、Caspase-9活性。
8. Transwell侵袭实验,检测各组细胞的体外侵袭能力变化。将Matrigel胶均匀铺于聚碳酸酯膜内表面,在上室分别加入三组细胞悬液各l00μL,在下室中加.A.500-L含趋化因子的DMEM完全培养基,37℃培养箱孵育48h,计算侵袭细胞数。
结果
1.重组HPV-16E6-E7慢病毒测序结果与设计序列进行同源性比较分析,结果显示目的DNA E6-E7序列与设计序列完全一致；
2.慢病毒滴度测定结果：重组HPV-16E6-E7漫病毒滴度为1.15×108MOI,空载体慢病毒滴度为2.09×108MOI。
3.重组HPV-16E6-E7慢病毒感染人喉咽鳞癌FaDu细胞及筛选结果显示：重组HPV-16E6-E7'慢病毒和空载体慢病毒感染FaDu细胞48h后用荧光显微镜观查,在感染上述慢病毒的细胞中可见明亮的绿色荧光蛋白EGFP表达。说明得到稳定感染重组HPV-16E6-E7'慢病毒和空载体慢病毒的FaDu细胞。
4.收集重组HPV-16E6-E7慢病毒感染后的FaDu细胞,提取细胞基因组DNA,用E6和E7基因上下游引物分别进行PCR扩增鉴定。1.5%琼脂糖凝胶电泳可见,在接近500bp处有一明亮条带,与HPV-16E6基因序列的理论值474bp一致；在接近250bp处也有一明亮条带,与HPV-16E7序列的理论值297bp一致。
5. RT-PCR结果显示：重组HPV-16E6-E7'慢病毒稳定感染的FaDu细胞中HPV-16E6、E7mRNA相对表达量分别为(2.6±0.22和1.8±0.12),明显高于空载体组(0.003±0.0001和0.003±0.0002)和未转染组(0.002±0.0002和0.005±0.0001),有显着性差异(P<0.05)。
6.免疫印迹结果显示重组HPV-16E6-E7曼病毒稳定感染的FaDu细胞有明显的E6. E7蛋白印迹条带。与RT-PCR检测mRNA结果一致,说明制备的重组HPV-16E6-E7'慢病毒感染FaDu细胞后能够高效表达E6、E7蛋白。
7. CCK-8细胞增殖检测试剂盒分别测定各组细胞孔内的吸光度值(A),实验组在第2d、3d、4d、5d的吸光度值分别为0.6418±0.0391、1.0808±0.0878、1.3598±0.0464、1.5936±0.1107与载体对照组(0.5442±0.01490、0.8952±0.0358、1.2002±0.0549、1.3848±0.0388)和空白对照组(0.5268±0.0320、0.888±0.03302、1.2162±0.0320、1.4228±0.0444)比较,吸光度值(A)明显升高,有显着性差异(p<0.05)。
8.流式细胞术(FCM)检测各组细胞凋亡结果显示：实验组(7.246±0.815)与载体对照组(13.464±0.609)和空白对照组(13.298±1.324)比较,细胞凋亡率显着降低,均有显着性差异(p＜0.01)；空白对照组与载体对照组比较,平均细胞凋亡率无显着性差异(p＞0.05)。说明HPV-16E6-E7整合可抑制FaDu细胞的凋亡。
9.酶联免疫吸附法检测细胞Caspase-3、Caspase-9活性结果显示：实验组、载体对照组和空白对照组Caspase-3相对活性分别为1.398±0.106、2.538±0.24和2.587±0.19; Caspase-9相对活性分别为1.268±0.083、1.992±0.13和1.984±0.11。实验组与2个对照组(空白对照组和载体对照组)细胞相比较,Caspase-3、Caspase-9相对活性均显着降低,均有显着性差异(p<0.01)。
10.流式细胞术分析细胞周期结果显示：实验组GO-G1期、S期和G2-M期各期所占比率分别为53.816±1.665、28.836±1.013和17.348±0.878；载体对照组分别为62.284±1.609、22.292±0.970和15.424±1.236；空白对照组分别为62.262±2.139、22.664±1.551和15.074±1.451。空白对照组与载体对照组细胞S期、G2/M期和G0/G1细胞比例比较,差异无显着性(p>0.05)实验组与2个对照组(空白对照组和载体对照组)比较,S期和G2/M期细胞比例增加,G0/G1期细胞比例减少,有显着性差异(p＜0.05)。说明FaDu细胞基因组整合HPV-16E6-E7后,S期和G2/M期细胞比例增加,G0/G1期细胞比例减少,促进细胞分裂增殖。
11.细胞Transwell实验结果显示：实验组体外侵袭实验视野内平均细胞数为117.4±14.8,载体对照组和空白对照组分别为68.4±8.2和72±8.1,实验组与2个对照组(载体对照组和空白对照组)比较,穿透Matrigel的平均细胞数显着增加,有显着性差异(p＜0.01)。说明FaDu细胞基因组整合HPV-16E6-E7后,FaDu细胞穿透Matrigel的能力显着增强,细胞侵袭转移能力增强。
第三部分HPV-16感染对喉咽鳞癌组织及FaDu细胞miRNA表达的调控
方法
1.稳定培养实验组、载体对照组和空白对照组FaDu细胞,确认3例仅HPV-16感染及21例HPV阴性的新鲜冰冻喉咽鳞癌组织。
2.提取人喉咽鳞癌组织及各组细胞总RNA。
3. qRT-PCR检测各组标本及细胞总RNA中miR-363、miR-15a和miR-155的表达水平。
结果
1.仅感染HPV-16的喉咽鳞癌组织标本中IniR-363、miR-15a和miR-155的表达水平分别为5.62±3.48、1.97±0.48和0.34±0.08；HPV阴性的喉咽鳞癌组织标本中各miRNA表达水平分别为0.90±1.53、1.09±1.31和1.92±1.47；实验组细胞中各,miRNA表达水平分别为6.75±0.17、4.15±0.13和0.16±0.01；载体对照组细胞中各miRNA表达水平分别为0.09±0.01、0.20±0.01和7.01±1.04；空白对照组细胞中各miRNA表达水平分别为0.09±±.0.003、0.21±0.003和4.14±0.02。
2.与HPV-16阴性的人喉咽鳞癌组织相比,HPV-16阳性人喉咽鳞癌组织中miR-363、miR-15a表达显着上调,miR-155表达无显着变化；与未转染HPV-16E6-E7基因的FaDu细胞相比,转染HPV-16E6-E7基因的FaDu细胞中miR-363、miR-15a表达显着上调,miR-155表达无显着变化；HPV-16阳性的人喉咽鳞癌组织与整合HPV-16E6-E7的FaDu细胞中miR-363、miR-15a和miR-155表达水平无显着性差异；HPV-16阴性的人喉咽鳞癌组织与未整合HPV-16E6-E7的FaDu细胞中niR-363、miR-15a和miR-155表达水平亦无显着性差异。
结论
1.喉咽鳞癌组织中存在HPV感染,本组标本中HPV阳性率为25.0%(7/28),HPV-16是最常见的感染型别；
2.在本课题包含的28例喉咽鳞癌病人中,与HPV阴性病人相比,HPV阳性病人中具有大量抽烟和/或饮酒病史者较少,差异有显着性；
3.人喉咽鳞癌FaDu细胞基因组整合HPV-16E6-E7后,细胞的增殖能力和侵袭力增强,细胞凋亡受到抑制,S期和G2/M期细胞比例增加,G0/G1期细胞比例减少。
4.在HPV-16阳性喉咽鳞癌组织和转染HPV-16E6-E7基因的FaDu细胞中,miR-363、miR-15a表达显着上调,miR-155表达无明显变化。
Background and objectives
Human papilloma virus (Human papilloma virus, HPV) can cause a series of epithelial lesions. Some types of HPV associated with precancerous lesions, referred to as a high-risk type HPV. And some people infected with these high-risk HPV types can occur canceration. In1995, the International Agency for Research on Cancer (IARC) recognized that HPV-16and HPV-18were carcinogenic in human. The correlations between cervical cancer and high risk HPV types have been widely recognized. Epidemiology, molecular and clinical evidence suggested that the high-risk HPV infection was strongly associated with cervical cancer. Also, parts of the head and neck squamous cell carcinoma (Head and neck Squamous cell carcinoma, HNSCC) were closely related with high-risk HPV.
At present, most of the HNSCC of HPV related research is oropharyngeal squamous cell carcinoma (Oropharyngeal squamous cell carcinoma, OSCC). Clinical evidences suggested that HPV-related OSCC usually did not have such typical history as heavy consumption of alcohol and/or tobacco use, which different from the classic OSCC in biological behavior. HPV infection or not has a significant influence on the prognosis of OSCC patients, therapeutic effect and survival rate. HPV positive patients with OSCC were superior to HPV negative OSCC patients.
Laryngeal squamous cell carcinoma and hypopharyngeal squamous cell carcinomas are two common malignancies of HNSCC, which occurs mainly in middle-aged men. Heavy consumption of alcohol and/or tobacco use was recognized as carcinogenic factors. However, some patients do not have the history of heavy consumption of alcohol and/or tobacco use. For this part of the patients, the researchers turned to the HPV infection. Many research institutions detected the overall rate of HPV infection in laryngeal squamous cell carcinoma and hypopharynx squamous cell carcinoma. While these previous studies were always retrospective study, the detection of cancerous tissue from the pathology archive specimens and the patients clinical data obtained subject to many limitations. In addition, anatomical position of the larynx and hypopharynx are adjacent. Some advanced cancers alone with pathological specimens were not easy to identify the primary position. Given the incidence of laryngeal squamous cell carcinoma of were significantly higher than the hypopharyngeal squamous cell carcinoma, most researchers put hypopharyngeal squamous cell carcinoma classified as laryngeal squamous cell carcinoma. Therefore, lots of the literatures reported laryngeal squamous cell carcinoma of HPV infection, but rarely hypopharyngeal squamous cell carcinoma HPV infection. HPV infection rates and common infection in hypopharyngeal squamous cell type is not clear.
The clinical manifestations of hypopharyngeal squamous cell carcinoma and laryngeal squamous cell carcinoma are similar, but primary in two different anatomical sites. The degree of differentiation, regional lymph node metastasis rate and5-year survival rate were significantly different. Hypopharyngeal squamous cell carcinoma prognosis is worse. Moreover, studies suggest that HPV infection is mainly associated with HNSCC prognosis. Thus, when carried out the related research of HPV infection, it is necessary to distinguish with hypopharyngeal squamous cell carcinoma and laryngeal squamous cell carcinoma. Hypopharyngeal squamous cell carcinoma will be as an independent disease-related research. In this study, we will test and genotype fresh frozen tissue samples which collected from28cases of hypopharyngeal squamous cell carcinoma HPV DNA, understand human HPV infection cases in laryngeal pharyngeal squamous carcinoma tissues and the common type of HPV.
E6and E7genes were the early region of the HPV genome. Encoding E6and E7protein. The E6and E7protein of HPV-16play a carcinogenic role in encoded oncoprotein. Lentiviral vector (Lentivirus vector) can be high efficiency and stable transfected phases of the cell. It is a good means of delivery in integration of the gene into cells, and has the advantages of easy operation, high efficiency of infection, security. This project intends to use the lentiviral vector genome synthesis HPV-16E6-E7gene to transfect hypopharyngeal squamous cell carcinoma FaDu cells, and observe HPV-16E6-E7genes impact the FaDu cell biology behavior.
MicroRNA are involved in almost all the body biological processes, including cell proliferation, differentiation, apoptosis, invasion, and migration. Abnormal expression of miRNAs associated with the occurrence and development of many tumors. However, the current understanding of HPV related miRNAs expressions in HNSCC are limited. This subject intends to detect the miRNAs expression levels in hypopharyngeal squamous cell carcinoma tissue which only infected with HPV-16and HPV-negative.
Part one HPV detection and genotyping of fresh frozen tissue in the human hypopharyngeal squamous cell carcinoma
Methods
1. Collected fresh tissues of hypopharyngeal squamous cell carcinoma from28cases. Saved in-80℃low temperature refrigerator after frozen in liquid nitrogen.
2. Applied a small amount of genomic DNA kit to extracted frozen hypopharyngeal squamous cell carcinoma tissue genomic DNA.
3. PCR-reverse dot blot hybridization specimens and genotyping of HPV DNA.
4. Combinded the state of HPV infection with the patient's clinical data and made correlation Analysis.
Results
1.28cases of hypopharyngeal squamous cell carcinoma of HPV infection were as follows:HPV positive specimens were7cases, total positive rate was25.0% (7/28). Five cases for a single type. HPV-16in three cases, HPV-18type in1case, HPV-52type in1case.2cases were two types of HPV infection at the same time, HPV-16, HPV-33in type1case, HPV-16,52in type1case.
2. Compared with HPV-positive and HPV-negative patients, less HPV-positive patients had heavy consumption of alcohol and/or tobacco use history (P<0.05), and there were no significant differences between age, gender, pathological type and tumor T stage (P>0.05).
Part two HPV-16E6-E7gene integration affect FaDu cells biological behavior
Methods
1. Preparation of recombinant HPV-16E6-E7lentiviral:Gene synthesis HPV-16E6-E7genes and addition HindⅢ and KpnI restriction endonuclease enzyme sites at both ends respectively. Then, connected with lentiviral vectors pLV-EGFP-C, packaging the reorganization of HPV-16E6-E7lentiviral, determining the lentiviral titer.
2. Packaging of Recombinant HPV-16E6-E7lentiviral
Prepared the packaging plasmid DNA:The lentivirus packaging vectors PHI, PH2, and recombinant lentivirus vectors HPV-16E6-E7in accordance with a certain ratio, mixed in500μL serum-free DMEM medium.
Prepared transfection reagent dilutions:A certain volume of transfection solution Polyfect-V was mixed with500μL serum-free DMEM medium.
Transfection reagent dilution was added into the packaging plasmid DNA solution, and immediately mixes thoroughly, this was transfection mixture. Incubated transfection mixture for15minutes, digestion HEK293T cells, per1ml transfection mixture was added10ml of cell suspension, and gently blowing and suction cell.The cell suspension was added to a10cm petri dish, and cultured for24hours in37℃. Removed the culture medium containing the transfection reagent, the medium was changed with10ml of virus medium. After48hours transfection, the virus particles were collected from the culture supernatan and virus titer was measured, stored at-80℃.
3. Recombinant HPV-16E6-E7lentiviral infection laryngeal pharyngeal squamous carcinoma FaDu cells:recoved FaDu cells, and infected with recombinant virus HPV-16E6-E7lentiviral particles liquid. Fluorescence expression was observed after48hours. DMEM high glucose medium containing0.5μg/mL puromycin was used for screening culture, and stably transfected cells were obtained after3weeks. PCR detected the HPV-16E6-E7DNA in FaDu cells, RT-PCR detected E6, E7mRNA, Western-blot detected E6-E7protein.
4. Cells were divided into three groups:the experimental group (the stable infection FaDu cells with recombinant HPV-16E6-E7lentiviral), lentiviral empty vector (stable FaDu cells infected by the vector control group) and blank control group (not infected with lentivirus FaDu cells).
5. CCK-8kit was used to detect the cell proliferation. It measured the absorbance values hole in the experimental group, vector control group and blank control group cells (A), draw the growth curve of the cells in each group.
6. Collected each group cells. Flow cytometer were used to determine the variation of cell cycles and apoptosis through PI and FITC-Annexin-V dying. The datas were analyzed by CellQuest acquistion and analysis program.
7. Experimental group, vector control group and blank control group were cllected, then, the enzyme-linked immunosorbent assay was used to detect cell Caspase-3, Caspase-9activity.
8. Transwell invasion assay was used to detect changes in vitro invasion capacity of the cells in each group. Matrigel glue was spread evenly on the surface of the polycarbonate membrane.100μL cell suspensions with three groups were added into the upper chamber.500μL DMEM complete medium was added into the lower chamber containing a chemokine. Incubated at37℃incubator for48h, and calculated invasion inhibition rate.
Results
1. The recombinant HPV-16E6-E7lentiviral sequencs were conformed to the design.
2. Lentiviral titer measurement results:reorganization of the HPV-16E6-E7 lentiviral titer was1.15x108MOI, and empty vector control lentiviral titer was2.09x108MOI.
3. The hypopharyngeal squamous cell carcinoma FaDu cells which recombinant HPV-16E6-E7lentiviral infection and screening results show that recombinant HPV-16E6-E7lentiviral liquid and empty vector control lentivirus infection after48hours with a fluorescent microscope view of the investigation, bright green fluorescent protein seen in cells infected with the virus. It showed that stably transfected with the recombinant HPV-16E6-E7Lentiviral FaDu cells stably transfected FaDu cells transfected with empty vector control lentivirus.
4. FaDu cells were collected after infection of recombinant lentivirus. PCR was used to identify the E6and E7genes.1.5%agarose gel electrophoresis showed a bright strip near at500bp and474bp consistent with the theoretical value of the HPV-16E6gene sequence; also have a bright strip near the250bp consistent with the theoretical value of297bp of the sequence of HPV-16E7.
5. RT-PCR confirmed that the lentiviral stability infected FaDu cell E6, E7mRNA were expressed at high levels. The experimental group E6, E7mRNA relative expression level (2.6±0.22and1.8±0.12) was significantly higher than the vector control group (0.003±0.0001and0.003±0.0002) and untransfected group (0.002±0.0002and0.005±0.0001). This lentiviral stable infection FaDu cell E6, E7showed a high level of expression (P<0.05).
6. Western blot analysis showed significantly E6, E7protein blot bands of the lentiviral stability infected FaDu cells. Consistent with the results of the RT-PCR. Preparation of recombinant HPV-16E6-E7infection of human pharyngeal squamous cell carcinoma cells FaDu cells capable of efficient express E6, E7protein.
7. CCK-8cell proliferation assay kit determined the absorbance value of the cells in each group hole (A). Experimental group in2d,3d,4d,5d absorbance values were0.6418±0.0391,1.0808±0.0878,1.3598±0.0464,1.5936±0.1107. Comparing with vector control group (0.5442±0.01490,0.8952±0.0358,1.2002±0.0549,1.3848±0.0388) and blank control group (0.5268±0.0320,0.888±0.03302,1.2162±0.0320,1.4228±0.0444), there is a significant difference (p <0.05).
8. Cell apoptosis was detected by flow cytometry (FCM). It showed that the experimental group (7.246±0.815) comparing with the vector control group (13.464±0.609) and blank control group (13.298±1.324) the apoptosis rate the were significantly reduced, which have a significant difference (p<0.01). Comparing with the blank control group and vector control group, the average rate of apoptosis was no significant difference (p>0.05). Pharyngeal squamous cell carcinoma FaDu cell genomic integrated with HPV E6-E7can inhibit pharyngeal squamous cell carcinoma FaDu cell apoptosis.
9. ELISA was used to detect the Caspase-3, Caspase-9activity:Caspase-3relative activity of the experimental group, vector control group and blank control group were1.398±0.106,2.538±0.24and2.587±0.19. Caspase-9relative activity were1.268±0.083,1.992±0.13and1.984±0.11. Compared with the experimental group and the control group (blank control group and vector control group), the Caspase-3, caspase-9relative activity was significantly reduced, there were significant differences (p<0.01).
10. Flow cytometric analysis of cell cycle:percentage of experimental group cell cycle:G0～G1phase, S phase and G2～M phase were53.816±1.665,28.836±1.013and17.348±0.878. Vector control group were62.284±1.609,22.292±0.970and15.424±1.236; blank control group were62.262±2.139,22.664±1.551and15.074±1.451. Blank control group and vector control group in S phase and G2/M phase and G0/G1, the difference was not significant (p>0.05). The experimental group compared with the control group (blank control group and vector control group), was increasing in S phase and G2/M phase, reducing the proportion of G0/G1phase, there was a significant difference (p<0.05). It suggested that Pharyngeal squamous cell carcinoma FaDu cells genomic integration of HPV E6-E7can increase the proportion of S phase and G2/M phase cells, reduce the proportion of cells in G0/G1phase, promote cell division and proliferation.
11. The Transwell cell experimental results showed:the average number of cells in vitro invasion assay within the field were:the experimental group was117.4± 14.8, and the vector control group and blank control group were68.4±8.2and72±8.1. Comparing with the experimental group and two control groups (vector control groupblank control group), the mean cell number of penetrate the Matrigel was increased significantly, and there were significant differences (p <0.01). It suggested that Pharyngeal squamous cell carcinoma FaDu cell genomic integration of HPV-16E6-E7can significantly increase pharyngeal squamous cell carcinoma FaDu cell.
Part three Regulation of HPV16infection squamous cell carcinoma of the hypopharynx and FaDu cell miRNA expression
Methods
1. Stable cultured the integration of HPV-16E6-E7FaDu cells and uninfected lentiviral FaDu cells. To confirm three only HPV-16infection and twenty-one HPV-negative fresh frozen hypopharynx squamous cell carcinoma specimen.
2. Extracted human hypopharyngeal squamous cell carcinoma tissue and cellular total RNA.
3. qRT-PCR was used to detect the total cellular RNA of miR-363, miR-15a and miR-155expression levels in each group of specimens and cell.
Results
1. Hypopharyngeal squamous cell carcinoma tissue Only infected with HPV-16express miR-363, miR-15a and miR-155. The expression levels were5.62±3.48,1.97±0.48and0.34±0.08. Hypopharyngeal squamous cell carcinoma of HPV-negative specimensin miRNA expression levels were0.90±1.53,1.09±1.31and1.92±1.47. Each miRNA expression levels in the experimental group were6.75±0.17,4.15±0.13and0.16±0.01. miRNA expression levels in the vector control group cells were0.09±0.01,0.20±0.01and7.01±1.04. And the blank control group cells miRNA expression levels were0.09±0.003,0.21±0.003and4.14±0.02.
2. In HPV-16positive human hypopharyngeal squamous cell carcinoma, the miR-363, miR-15a expression were significantly raised, while miR-155 expression was no significant change. Hypopharyngeal squamous cell carcinoma FaDu cells transfected with the HPV-16E6-E7genes, the miR-363, miR-15a expression were significantly raised. While the miR-155expression was no significant change. There was no significant difference between HPV positive and HPV-16E6-E7infected human hypopharyngeal squamous cell carcinoma FaDu cells in miR-363and miR-15a and miR-155expression levels. And slo, There was no significant difference between HPV negative human hypopharyngeal squamous cell carcinoma and FaDu cells in miR-363and miR-15a and miR-155expression levels.
Conclusions
1. Hypopharyngeal squamous cell carcinoma in the presence of HPV infection, HPV-positive specimens is25.0%(7/28), HPV-16is the most common infection type.
2. The subject includ28cases of hypopharyngeal squamous cell carcinoma patients. Compare with HPV-negative patients, less HPV-positive patients have heavy consumption of alcohol and/or tobacco use history. There are significant differences.
3. Human hypopharyngeal squamous cell carcinoma FaDu cell genome integrated with HPV-16E6-E7, the cell proliferation and invasiveness are enhanced, apoptosis is suppressed, the proportion of S phase and G2/M phase is increased, and G0/G1phase is reducded.
4. Comparing with HPV-16positive hypopharyngeal squamous cell carcinoma and FaDu cells transfected with the HPV-16E6-E7genes, there have significantly raised in miR-363, miR-15a expression, while there has no significant change in miR-155.
人乳头状瘤病毒(Human papillomavirus, HPV)可引起一系列上皮病变,有些型别HPV与癌前病变相关,称为高危型HPV,部分人感染高危型HPV后可发生癌变。国际癌症研究机构在1995年认定HPV-16, HPV-18两种型别在人类有致癌作用。宫颈癌与高危型HPV感染的相关性已得到普遍认可。流行病学、分子和临床证据表明,高危型HPV感染除了与宫颈癌有强烈相关性之外,还与部分头颈部鳞状细胞癌(Head and neck Squamous cell carcinoma, HNSCC)的发生密切相关。
在HPV相关的HNSCC中,目前研究最多的是口咽部鳞状细胞癌(Oropharyngeal squamous cell carcinoma, OSCC)。临床证据表明,HPV相关OSCC通常不具备此类疾病中典型的大量抽烟和／或饮酒病史,在生物学行为上不同于经典的OSCC, HPV感染与否对OSCC病人的预后有显着影响,无论是治疗效果还是生存率,HPV阳性OSCC病人都优于HPV阴性OSCC病人。
喉鳞癌和喉咽鳞癌是HNSCC中两种常见恶性肿瘤,多发于中老年男性,大量抽烟和／或饮酒是其公认的致癌因素,然而有一部分患者并无大量抽烟和／或饮酒的病史,针对这部分患者的发病原因,研究者将目光转向了HPV感染。许多研究机构对喉鳞癌和喉咽鳞癌组织中HPV感染情况进行了检测,以往的研究多为回顾性研究,检测的癌组织来自病理存档标本,病人临床资料的获得受到诸多限制。此外,喉部与喉咽部解剖位置毗邻,部分晚期癌肿仅凭病理标本不易鉴别原发位置,鉴于喉鳞癌发病率显着高于喉咽鳞癌,大多数研究者便将喉咽鳞癌归入喉鳞癌一并研究。因此,报道喉鳞癌HPV感染率的文献很多,但鲜有喉咽鳞癌HPV感染情况的报道,HPV在喉咽鳞癌中的感染率及常见感染型别均不明确。
喉咽鳞癌和喉鳞癌的临床表现有一定的相似性,但却是原发于不同解剖部位的两种HNSCC,二者的分化程度、局部淋巴结转移率及5年生存率有明显不同,喉咽鳞癌的预后更差。而且目前研究认为HPV感染主要与HNSCC的预后相关,因此在开展HPV感染相关性研究时,有必要将喉咽鳞癌与喉鳞癌进行区分,将喉咽鳞癌作为独立的疾病进行相关研究。本课题拟对收集的28例喉咽鳞癌新鲜冰冻组织标本进行HPV DNA检测并分型,了解人喉咽鳞癌组织中HPV感染情况及常见HPV型别。
E6、E7基因为HPV基因组早期区基因,编码E6、E7蛋白,HPV-16编码的E6、E7蛋白是其发挥致癌作用的主要癌蛋白。慢病毒载体(Lentivirus vector)可以高效率且稳定的感染各期细胞,是将基因整合入细胞的良好运载工具,且具有操作简便、感染效率高、作用稳定、安全性好等优点。本课题拟利用重组HPV-16E6-E7基因的慢病毒感染人喉咽鳞癌FaDu细胞,观察HPV-16E6-E7基因整合对FaDu细胞生物学行为的影响。
微小RNA (miRNA或microRNA)参与了人体细胞的分化、增殖、凋亡、侵袭、及迁移等几乎所有的细胞生物学过程。miRNAs的异常表达与许多肿瘤的发生、发展相关,可发挥癌基因或抑癌基因的作用。然而目前对HPV相关HNSCC的]miRNAs表达情况了解有限。本课题拟对仅感染HPV-16和HPV阴性的喉口咽鳞癌组织miRNAs表达水平进行检测,同时检测人喉咽鳞癌FaDu细胞整合HPV-16E6-E7前后miRNAs表达水平的变化,分析HPV-16感染对人喉咽鳞癌组织和细胞株miRNAs表达的调控。
第一部分人喉咽鳞癌新鲜冰冻组织中HPV检测及分型
方法
1.收集28例人喉咽鳞癌新鲜组织标本,液氮速冻后移入-80℃低温冰箱保存。
2.应用基因组DNA小量试剂盒提取新鲜冰冻喉咽鳞癌组织标本基因组DNA。
3.PCR-反向点杂交法检测标本中HPV DNA并分型
4.将HPV感染情况与病人临床资料相结合,进行相关性分析。
结果
1.28例喉咽鳞癌标本HPV感染情况如下：HPV阳性者7例,总阳性率为25.0%(7/28)。5例为单一型别HPV感染,HPV-16型3例,HPV-18型1例,HPV-52型1例；2例为两种型别HPV同时感染,HPV-16,33型1例,HPV-16,52型1例。
2.HPV阳性与HPV阴性病人相比较,大量抽烟和／或饮酒病史有显着性差异(P<0.05)；年龄、性别、病理分型和肿瘤T分期之间的差异无统计学意义(P>0.05)
第二部分HPV-16E6-E7基因整合对FaDu细胞生物学行为的影响
方法
1.重组HPV-16E6-E7慢病毒的制备：全基因合成HPV-16E6-E7基因,并在两端分别加上HindⅢ和KpnⅠ限制性核酸内切酶位点,与慢病毒载体pLV-EGFP-C在T4连接酶的作用下连接,将连接体系转化入DH5a感受态细菌,挑选长出的菌落进行测序鉴定,即得到重组HPV-16E6-E7慢病毒。
2.重组HPV-16E6-E7慢病毒的包装：
>制备用于包装的质粒DNA溶液：将慢病毒包装载体pH1、pH2与重组HPV-16E6-E7'慢病毒质粒按照一定比率混合于50mL无血清DMEM培养基中。
》制备转染试剂稀释液：取一定体积的转染液Polyfect-V与500μL无血清DMEM培养基混合。
>将转染试剂稀释液加入用于包装的质粒DNA溶液中,立刻充分混匀,即制备成为转染混合液。室温孵育转染混合液15min,消化HEK293T细胞,每lml转染混合液加入10ml细胞悬液,轻轻吹吸细胞混匀。将细胞悬液加入10cm培养皿,放入37℃培养24h。去除含有转染试剂的培养基,用10ml病毒培养基换液。转染后48h培养上清即含病毒颗粒,收集并测定病毒滴度,.80-C保存。
3.重组HPV-16E6-E7漫病毒感染人喉咽鳞癌FaDu细胞：复苏FaDu细胞,用含有重组HPV-16E6-E7慢病毒颗粒的慢病毒液感染FaDu细胞。48h后观察荧光表达情况,用含有0.5μg/mL puromycin的DMEM高糖培养基进行筛选培养,3周后得到稳定转染细胞。PCR检测FaDu细胞中HPV-16E6、E7DNA, RT-PCR检测FaDu细胞中HPV-16E6、E7mRNA, Western-blot检测细胞中E6、E7蛋白。
4.将细胞分为3组：实验组(重组HPV-16E6-E7慢病毒稳定感染的FaDu细胞)、载体对照组(慢病毒空载体稳定感染的FaDu细胞)和空白对照组(未感染慢病毒的FaDu细胞)。
5. CCK-8试剂盒检测各组细胞增殖情况,分别测定实验组、载体对照组和空白对照组细胞孔内的吸光度值(A),绘制各组细胞的生长曲线。
6.收集各组细胞,通过PI染色用流式细胞仪检测细胞周期的变化；通过PI和Annexin-V联合染色检测细胞的凋亡。
7.分别收集实验组、载体对照组和空白对照组细胞,酶联免疫吸附法检测细胞Caspase-3、Caspase-9活性。
8. Transwell侵袭实验,检测各组细胞的体外侵袭能力变化。将Matrigel胶均匀铺于聚碳酸酯膜内表面,在上室分别加入三组细胞悬液各l00μL,在下室中加.A.500-L含趋化因子的DMEM完全培养基,37℃培养箱孵育48h,计算侵袭细胞数。
结果
1.重组HPV-16E6-E7慢病毒测序结果与设计序列进行同源性比较分析,结果显示目的DNA E6-E7序列与设计序列完全一致；
2.慢病毒滴度测定结果：重组HPV-16E6-E7漫病毒滴度为1.15×108MOI,空载体慢病毒滴度为2.09×108MOI。
3.重组HPV-16E6-E7慢病毒感染人喉咽鳞癌FaDu细胞及筛选结果显示：重组HPV-16E6-E7'慢病毒和空载体慢病毒感染FaDu细胞48h后用荧光显微镜观查,在感染上述慢病毒的细胞中可见明亮的绿色荧光蛋白EGFP表达。说明得到稳定感染重组HPV-16E6-E7'慢病毒和空载体慢病毒的FaDu细胞。
4.收集重组HPV-16E6-E7慢病毒感染后的FaDu细胞,提取细胞基因组DNA,用E6和E7基因上下游引物分别进行PCR扩增鉴定。1.5%琼脂糖凝胶电泳可见,在接近500bp处有一明亮条带,与HPV-16E6基因序列的理论值474bp一致；在接近250bp处也有一明亮条带,与HPV-16E7序列的理论值297bp一致。
5. RT-PCR结果显示：重组HPV-16E6-E7'慢病毒稳定感染的FaDu细胞中HPV-16E6、E7mRNA相对表达量分别为(2.6±0.22和1.8±0.12),明显高于空载体组(0.003±0.0001和0.003±0.0002)和未转染组(0.002±0.0002和0.005±0.0001),有显着性差异(P<0.05)。
6.免疫印迹结果显示重组HPV-16E6-E7曼病毒稳定感染的FaDu细胞有明显的E6. E7蛋白印迹条带。与RT-PCR检测mRNA结果一致,说明制备的重组HPV-16E6-E7'慢病毒感染FaDu细胞后能够高效表达E6、E7蛋白。
7. CCK-8细胞增殖检测试剂盒分别测定各组细胞孔内的吸光度值(A),实验组在第2d、3d、4d、5d的吸光度值分别为0.6418±0.0391、1.0808±0.0878、1.3598±0.0464、1.5936±0.1107与载体对照组(0.5442±0.01490、0.8952±0.0358、1.2002±0.0549、1.3848±0.0388)和空白对照组(0.5268±0.0320、0.888±0.03302、1.2162±0.0320、1.4228±0.0444)比较,吸光度值(A)明显升高,有显着性差异(p<0.05)。
8.流式细胞术(FCM)检测各组细胞凋亡结果显示：实验组(7.246±0.815)与载体对照组(13.464±0.609)和空白对照组(13.298±1.324)比较,细胞凋亡率显着降低,均有显着性差异(p＜0.01)；空白对照组与载体对照组比较,平均细胞凋亡率无显着性差异(p＞0.05)。说明HPV-16E6-E7整合可抑制FaDu细胞的凋亡。
9.酶联免疫吸附法检测细胞Caspase-3、Caspase-9活性结果显示：实验组、载体对照组和空白对照组Caspase-3相对活性分别为1.398±0.106、2.538±0.24和2.587±0.19; Caspase-9相对活性分别为1.268±0.083、1.992±0.13和1.984±0.11。实验组与2个对照组(空白对照组和载体对照组)细胞相比较,Caspase-3、Caspase-9相对活性均显着降低,均有显着性差异(p<0.01)。
10.流式细胞术分析细胞周期结果显示：实验组GO-G1期、S期和G2-M期各期所占比率分别为53.816±1.665、28.836±1.013和17.348±0.878；载体对照组分别为62.284±1.609、22.292±0.970和15.424±1.236；空白对照组分别为62.262±2.139、22.664±1.551和15.074±1.451。空白对照组与载体对照组细胞S期、G2/M期和G0/G1细胞比例比较,差异无显着性(p>0.05)实验组与2个对照组(空白对照组和载体对照组)比较,S期和G2/M期细胞比例增加,G0/G1期细胞比例减少,有显着性差异(p＜0.05)。说明FaDu细胞基因组整合HPV-16E6-E7后,S期和G2/M期细胞比例增加,G0/G1期细胞比例减少,促进细胞分裂增殖。
11.细胞Transwell实验结果显示：实验组体外侵袭实验视野内平均细胞数为117.4±14.8,载体对照组和空白对照组分别为68.4±8.2和72±8.1,实验组与2个对照组(载体对照组和空白对照组)比较,穿透Matrigel的平均细胞数显着增加,有显着性差异(p＜0.01)。说明FaDu细胞基因组整合HPV-16E6-E7后,FaDu细胞穿透Matrigel的能力显着增强,细胞侵袭转移能力增强。
第三部分HPV-16感染对喉咽鳞癌组织及FaDu细胞miRNA表达的调控
方法
1.稳定培养实验组、载体对照组和空白对照组FaDu细胞,确认3例仅HPV-16感染及21例HPV阴性的新鲜冰冻喉咽鳞癌组织。
2.提取人喉咽鳞癌组织及各组细胞总RNA。
3. qRT-PCR检测各组标本及细胞总RNA中miR-363、miR-15a和miR-155的表达水平。
结果
1.仅感染HPV-16的喉咽鳞癌组织标本中IniR-363、miR-15a和miR-155的表达水平分别为5.62±3.48、1.97±0.48和0.34±0.08；HPV阴性的喉咽鳞癌组织标本中各miRNA表达水平分别为0.90±1.53、1.09±1.31和1.92±1.47；实验组细胞中各,miRNA表达水平分别为6.75±0.17、4.15±0.13和0.16±0.01；载体对照组细胞中各miRNA表达水平分别为0.09±0.01、0.20±0.01和7.01±1.04；空白对照组细胞中各miRNA表达水平分别为0.09±±.0.003、0.21±0.003和4.14±0.02。
2.与HPV-16阴性的人喉咽鳞癌组织相比,HPV-16阳性人喉咽鳞癌组织中miR-363、miR-15a表达显着上调,miR-155表达无显着变化；与未转染HPV-16E6-E7基因的FaDu细胞相比,转染HPV-16E6-E7基因的FaDu细胞中miR-363、miR-15a表达显着上调,miR-155表达无显着变化；HPV-16阳性的人喉咽鳞癌组织与整合HPV-16E6-E7的FaDu细胞中miR-363、miR-15a和miR-155表达水平无显着性差异；HPV-16阴性的人喉咽鳞癌组织与未整合HPV-16E6-E7的FaDu细胞中niR-363、miR-15a和miR-155表达水平亦无显着性差异。
结论
1.喉咽鳞癌组织中存在HPV感染,本组标本中HPV阳性率为25.0%(7/28),HPV-16是最常见的感染型别；
2.在本课题包含的28例喉咽鳞癌病人中,与HPV阴性病人相比,HPV阳性病人中具有大量抽烟和/或饮酒病史者较少,差异有显着性；
3.人喉咽鳞癌FaDu细胞基因组整合HPV-16E6-E7后,细胞的增殖能力和侵袭力增强,细胞凋亡受到抑制,S期和G2/M期细胞比例增加,G0/G1期细胞比例减少。
4.在HPV-16阳性喉咽鳞癌组织和转染HPV-16E6-E7基因的FaDu细胞中,miR-363、miR-15a表达显着上调,miR-155表达无明显变化。
Background and objectives
Human papilloma virus (Human papilloma virus, HPV) can cause a series of epithelial lesions. Some types of HPV associated with precancerous lesions, referred to as a high-risk type HPV. And some people infected with these high-risk HPV types can occur canceration. In1995, the International Agency for Research on Cancer (IARC) recognized that HPV-16and HPV-18were carcinogenic in human. The correlations between cervical cancer and high risk HPV types have been widely recognized. Epidemiology, molecular and clinical evidence suggested that the high-risk HPV infection was strongly associated with cervical cancer. Also, parts of the head and neck squamous cell carcinoma (Head and neck Squamous cell carcinoma, HNSCC) were closely related with high-risk HPV.
At present, most of the HNSCC of HPV related research is oropharyngeal squamous cell carcinoma (Oropharyngeal squamous cell carcinoma, OSCC). Clinical evidences suggested that HPV-related OSCC usually did not have such typical history as heavy consumption of alcohol and/or tobacco use, which different from the classic OSCC in biological behavior. HPV infection or not has a significant influence on the prognosis of OSCC patients, therapeutic effect and survival rate. HPV positive patients with OSCC were superior to HPV negative OSCC patients.
Laryngeal squamous cell carcinoma and hypopharyngeal squamous cell carcinomas are two common malignancies of HNSCC, which occurs mainly in middle-aged men. Heavy consumption of alcohol and/or tobacco use was recognized as carcinogenic factors. However, some patients do not have the history of heavy consumption of alcohol and/or tobacco use. For this part of the patients, the researchers turned to the HPV infection. Many research institutions detected the overall rate of HPV infection in laryngeal squamous cell carcinoma and hypopharynx squamous cell carcinoma. While these previous studies were always retrospective study, the detection of cancerous tissue from the pathology archive specimens and the patients clinical data obtained subject to many limitations. In addition, anatomical position of the larynx and hypopharynx are adjacent. Some advanced cancers alone with pathological specimens were not easy to identify the primary position. Given the incidence of laryngeal squamous cell carcinoma of were significantly higher than the hypopharyngeal squamous cell carcinoma, most researchers put hypopharyngeal squamous cell carcinoma classified as laryngeal squamous cell carcinoma. Therefore, lots of the literatures reported laryngeal squamous cell carcinoma of HPV infection, but rarely hypopharyngeal squamous cell carcinoma HPV infection. HPV infection rates and common infection in hypopharyngeal squamous cell type is not clear.
The clinical manifestations of hypopharyngeal squamous cell carcinoma and laryngeal squamous cell carcinoma are similar, but primary in two different anatomical sites. The degree of differentiation, regional lymph node metastasis rate and5-year survival rate were significantly different. Hypopharyngeal squamous cell carcinoma prognosis is worse. Moreover, studies suggest that HPV infection is mainly associated with HNSCC prognosis. Thus, when carried out the related research of HPV infection, it is necessary to distinguish with hypopharyngeal squamous cell carcinoma and laryngeal squamous cell carcinoma. Hypopharyngeal squamous cell carcinoma will be as an independent disease-related research. In this study, we will test and genotype fresh frozen tissue samples which collected from28cases of hypopharyngeal squamous cell carcinoma HPV DNA, understand human HPV infection cases in laryngeal pharyngeal squamous carcinoma tissues and the common type of HPV.
E6and E7genes were the early region of the HPV genome. Encoding E6and E7protein. The E6and E7protein of HPV-16play a carcinogenic role in encoded oncoprotein. Lentiviral vector (Lentivirus vector) can be high efficiency and stable transfected phases of the cell. It is a good means of delivery in integration of the gene into cells, and has the advantages of easy operation, high efficiency of infection, security. This project intends to use the lentiviral vector genome synthesis HPV-16E6-E7gene to transfect hypopharyngeal squamous cell carcinoma FaDu cells, and observe HPV-16E6-E7genes impact the FaDu cell biology behavior.
MicroRNA are involved in almost all the body biological processes, including cell proliferation, differentiation, apoptosis, invasion, and migration. Abnormal expression of miRNAs associated with the occurrence and development of many tumors. However, the current understanding of HPV related miRNAs expressions in HNSCC are limited. This subject intends to detect the miRNAs expression levels in hypopharyngeal squamous cell carcinoma tissue which only infected with HPV-16and HPV-negative.
Part one HPV detection and genotyping of fresh frozen tissue in the human hypopharyngeal squamous cell carcinoma
Methods
1. Collected fresh tissues of hypopharyngeal squamous cell carcinoma from28cases. Saved in-80℃low temperature refrigerator after frozen in liquid nitrogen.
2. Applied a small amount of genomic DNA kit to extracted frozen hypopharyngeal squamous cell carcinoma tissue genomic DNA.
3. PCR-reverse dot blot hybridization specimens and genotyping of HPV DNA.
4. Combinded the state of HPV infection with the patient's clinical data and made correlation Analysis.
Results
1.28cases of hypopharyngeal squamous cell carcinoma of HPV infection were as follows:HPV positive specimens were7cases, total positive rate was25.0% (7/28). Five cases for a single type. HPV-16in three cases, HPV-18type in1case, HPV-52type in1case.2cases were two types of HPV infection at the same time, HPV-16, HPV-33in type1case, HPV-16,52in type1case.
2. Compared with HPV-positive and HPV-negative patients, less HPV-positive patients had heavy consumption of alcohol and/or tobacco use history (P<0.05), and there were no significant differences between age, gender, pathological type and tumor T stage (P>0.05).
Part two HPV-16E6-E7gene integration affect FaDu cells biological behavior
Methods
1. Preparation of recombinant HPV-16E6-E7lentiviral:Gene synthesis HPV-16E6-E7genes and addition HindⅢ and KpnI restriction endonuclease enzyme sites at both ends respectively. Then, connected with lentiviral vectors pLV-EGFP-C, packaging the reorganization of HPV-16E6-E7lentiviral, determining the lentiviral titer.
2. Packaging of Recombinant HPV-16E6-E7lentiviral
Prepared the packaging plasmid DNA:The lentivirus packaging vectors PHI, PH2, and recombinant lentivirus vectors HPV-16E6-E7in accordance with a certain ratio, mixed in500μL serum-free DMEM medium.
Prepared transfection reagent dilutions:A certain volume of transfection solution Polyfect-V was mixed with500μL serum-free DMEM medium.
Transfection reagent dilution was added into the packaging plasmid DNA solution, and immediately mixes thoroughly, this was transfection mixture. Incubated transfection mixture for15minutes, digestion HEK293T cells, per1ml transfection mixture was added10ml of cell suspension, and gently blowing and suction cell.The cell suspension was added to a10cm petri dish, and cultured for24hours in37℃. Removed the culture medium containing the transfection reagent, the medium was changed with10ml of virus medium. After48hours transfection, the virus particles were collected from the culture supernatan and virus titer was measured, stored at-80℃.
3. Recombinant HPV-16E6-E7lentiviral infection laryngeal pharyngeal squamous carcinoma FaDu cells:recoved FaDu cells, and infected with recombinant virus HPV-16E6-E7lentiviral particles liquid. Fluorescence expression was observed after48hours. DMEM high glucose medium containing0.5μg/mL puromycin was used for screening culture, and stably transfected cells were obtained after3weeks. PCR detected the HPV-16E6-E7DNA in FaDu cells, RT-PCR detected E6, E7mRNA, Western-blot detected E6-E7protein.
4. Cells were divided into three groups:the experimental group (the stable infection FaDu cells with recombinant HPV-16E6-E7lentiviral), lentiviral empty vector (stable FaDu cells infected by the vector control group) and blank control group (not infected with lentivirus FaDu cells).
5. CCK-8kit was used to detect the cell proliferation. It measured the absorbance values hole in the experimental group, vector control group and blank control group cells (A), draw the growth curve of the cells in each group.
6. Collected each group cells. Flow cytometer were used to determine the variation of cell cycles and apoptosis through PI and FITC-Annexin-V dying. The datas were analyzed by CellQuest acquistion and analysis program.
7. Experimental group, vector control group and blank control group were cllected, then, the enzyme-linked immunosorbent assay was used to detect cell Caspase-3, Caspase-9activity.
8. Transwell invasion assay was used to detect changes in vitro invasion capacity of the cells in each group. Matrigel glue was spread evenly on the surface of the polycarbonate membrane.100μL cell suspensions with three groups were added into the upper chamber.500μL DMEM complete medium was added into the lower chamber containing a chemokine. Incubated at37℃incubator for48h, and calculated invasion inhibition rate.
Results
1. The recombinant HPV-16E6-E7lentiviral sequencs were conformed to the design.
2. Lentiviral titer measurement results:reorganization of the HPV-16E6-E7 lentiviral titer was1.15x108MOI, and empty vector control lentiviral titer was2.09x108MOI.
3. The hypopharyngeal squamous cell carcinoma FaDu cells which recombinant HPV-16E6-E7lentiviral infection and screening results show that recombinant HPV-16E6-E7lentiviral liquid and empty vector control lentivirus infection after48hours with a fluorescent microscope view of the investigation, bright green fluorescent protein seen in cells infected with the virus. It showed that stably transfected with the recombinant HPV-16E6-E7Lentiviral FaDu cells stably transfected FaDu cells transfected with empty vector control lentivirus.
4. FaDu cells were collected after infection of recombinant lentivirus. PCR was used to identify the E6and E7genes.1.5%agarose gel electrophoresis showed a bright strip near at500bp and474bp consistent with the theoretical value of the HPV-16E6gene sequence; also have a bright strip near the250bp consistent with the theoretical value of297bp of the sequence of HPV-16E7.
5. RT-PCR confirmed that the lentiviral stability infected FaDu cell E6, E7mRNA were expressed at high levels. The experimental group E6, E7mRNA relative expression level (2.6±0.22and1.8±0.12) was significantly higher than the vector control group (0.003±0.0001and0.003±0.0002) and untransfected group (0.002±0.0002and0.005±0.0001). This lentiviral stable infection FaDu cell E6, E7showed a high level of expression (P<0.05).
6. Western blot analysis showed significantly E6, E7protein blot bands of the lentiviral stability infected FaDu cells. Consistent with the results of the RT-PCR. Preparation of recombinant HPV-16E6-E7infection of human pharyngeal squamous cell carcinoma cells FaDu cells capable of efficient express E6, E7protein.
7. CCK-8cell proliferation assay kit determined the absorbance value of the cells in each group hole (A). Experimental group in2d,3d,4d,5d absorbance values were0.6418±0.0391,1.0808±0.0878,1.3598±0.0464,1.5936±0.1107. Comparing with vector control group (0.5442±0.01490,0.8952±0.0358,1.2002±0.0549,1.3848±0.0388) and blank control group (0.5268±0.0320,0.888±0.03302,1.2162±0.0320,1.4228±0.0444), there is a significant difference (p <0.05).
8. Cell apoptosis was detected by flow cytometry (FCM). It showed that the experimental group (7.246±0.815) comparing with the vector control group (13.464±0.609) and blank control group (13.298±1.324) the apoptosis rate the were significantly reduced, which have a significant difference (p<0.01). Comparing with the blank control group and vector control group, the average rate of apoptosis was no significant difference (p>0.05). Pharyngeal squamous cell carcinoma FaDu cell genomic integrated with HPV E6-E7can inhibit pharyngeal squamous cell carcinoma FaDu cell apoptosis.
9. ELISA was used to detect the Caspase-3, Caspase-9activity:Caspase-3relative activity of the experimental group, vector control group and blank control group were1.398±0.106,2.538±0.24and2.587±0.19. Caspase-9relative activity were1.268±0.083,1.992±0.13and1.984±0.11. Compared with the experimental group and the control group (blank control group and vector control group), the Caspase-3, caspase-9relative activity was significantly reduced, there were significant differences (p<0.01).
10. Flow cytometric analysis of cell cycle:percentage of experimental group cell cycle:G0～G1phase, S phase and G2～M phase were53.816±1.665,28.836±1.013and17.348±0.878. Vector control group were62.284±1.609,22.292±0.970and15.424±1.236; blank control group were62.262±2.139,22.664±1.551and15.074±1.451. Blank control group and vector control group in S phase and G2/M phase and G0/G1, the difference was not significant (p>0.05). The experimental group compared with the control group (blank control group and vector control group), was increasing in S phase and G2/M phase, reducing the proportion of G0/G1phase, there was a significant difference (p<0.05). It suggested that Pharyngeal squamous cell carcinoma FaDu cells genomic integration of HPV E6-E7can increase the proportion of S phase and G2/M phase cells, reduce the proportion of cells in G0/G1phase, promote cell division and proliferation.
11. The Transwell cell experimental results showed:the average number of cells in vitro invasion assay within the field were:the experimental group was117.4± 14.8, and the vector control group and blank control group were68.4±8.2and72±8.1. Comparing with the experimental group and two control groups (vector control groupblank control group), the mean cell number of penetrate the Matrigel was increased significantly, and there were significant differences (p <0.01). It suggested that Pharyngeal squamous cell carcinoma FaDu cell genomic integration of HPV-16E6-E7can significantly increase pharyngeal squamous cell carcinoma FaDu cell.
Part three Regulation of HPV16infection squamous cell carcinoma of the hypopharynx and FaDu cell miRNA expression
Methods
1. Stable cultured the integration of HPV-16E6-E7FaDu cells and uninfected lentiviral FaDu cells. To confirm three only HPV-16infection and twenty-one HPV-negative fresh frozen hypopharynx squamous cell carcinoma specimen.
2. Extracted human hypopharyngeal squamous cell carcinoma tissue and cellular total RNA.
3. qRT-PCR was used to detect the total cellular RNA of miR-363, miR-15a and miR-155expression levels in each group of specimens and cell.
Results
1. Hypopharyngeal squamous cell carcinoma tissue Only infected with HPV-16express miR-363, miR-15a and miR-155. The expression levels were5.62±3.48,1.97±0.48and0.34±0.08. Hypopharyngeal squamous cell carcinoma of HPV-negative specimensin miRNA expression levels were0.90±1.53,1.09±1.31and1.92±1.47. Each miRNA expression levels in the experimental group were6.75±0.17,4.15±0.13and0.16±0.01. miRNA expression levels in the vector control group cells were0.09±0.01,0.20±0.01and7.01±1.04. And the blank control group cells miRNA expression levels were0.09±0.003,0.21±0.003and4.14±0.02.
2. In HPV-16positive human hypopharyngeal squamous cell carcinoma, the miR-363, miR-15a expression were significantly raised, while miR-155 expression was no significant change. Hypopharyngeal squamous cell carcinoma FaDu cells transfected with the HPV-16E6-E7genes, the miR-363, miR-15a expression were significantly raised. While the miR-155expression was no significant change. There was no significant difference between HPV positive and HPV-16E6-E7infected human hypopharyngeal squamous cell carcinoma FaDu cells in miR-363and miR-15a and miR-155expression levels. And slo, There was no significant difference between HPV negative human hypopharyngeal squamous cell carcinoma and FaDu cells in miR-363and miR-15a and miR-155expression levels.
Conclusions
1. Hypopharyngeal squamous cell carcinoma in the presence of HPV infection, HPV-positive specimens is25.0%(7/28), HPV-16is the most common infection type.
2. The subject includ28cases of hypopharyngeal squamous cell carcinoma patients. Compare with HPV-negative patients, less HPV-positive patients have heavy consumption of alcohol and/or tobacco use history. There are significant differences.
3. Human hypopharyngeal squamous cell carcinoma FaDu cell genome integrated with HPV-16E6-E7, the cell proliferation and invasiveness are enhanced, apoptosis is suppressed, the proportion of S phase and G2/M phase is increased, and G0/G1phase is reducded.
4. Comparing with HPV-16positive hypopharyngeal squamous cell carcinoma and FaDu cells transfected with the HPV-16E6-E7genes, there have significantly raised in miR-363, miR-15a expression, while there has no significant change in miR-155.
引文
[1]IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Human papillomaviruses. IARC Monogr Eval Carcinog Risks Hum.1995;64:1-378.
[2]Braakhuis BJ, Snijders PJ, Keune WJ, et al. Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus. J Natl Cancer Inst. 2004;96(13):998-1006.
[3]D'Souza G, Kreimer AR, Viscidi R, et al. Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med.2007;356(19):1944-56.
[4]Weinberger PM, Yu Z, Kountourakis P, et al. Defining molecular phenotypes of human papillomavirus-associated oropharyngeal squamous cell carcinoma:validation of three-class hypothesis. Otolaryngol Head Neck Surg.2009 Sep;141(3):382-9.
[5]Andl T, Kahn T, Pfuhl A, et al. Etiological involvement of oncogenic human papillomavirus in tonsillar squamous cell carcinomas lacking retinoblastoma cell cycle control. Cancer Res.1998;58(1):5-13.
[6]Frisch M, Biggar RJ. Aetiological parallel between tonsillar and anogenital squamous-cell carcinomas. Lancet.1999;354(9188):1442-3.
[7]Kreimer AR, Clifford GM, Boyle P, et al. Human papillomavirus types in head and neck squamous cell carcinomas worldwide:a systematic review. Cancer Epidemiol Biomarkers Prev 2005;14(2):467-75.
[8]Morris KV, Rossi JJ. Lentiviral-mediated delivery of siRNAs for antiviral therapy. Gene therapy 2006;13:553-8.
[9]D.P. Bartel. MicroRNAs:genomics, biogenesis, mechanism, and function. Cell 2004;116 (2) 281-97.
[10]Lu J, Getz G, Miska EA, et al. Nature.2005 Jun 9; 435(7043):834-8. Nature.2005 Jun 9; 435(7043):834-8.
[11]zur Hausen H. Papillomaviruses and cancer:from basic studies to clinical application. Nat Rev Cancer.2002;2(5):342-50.
[12]Gillison ML and Shah KV (2001) Human papillomavirusassociated head and neck squamous cell carcinoma:mounting evidence for an etiologic role for human papillomavirus in a subset of head and neck cancers. Curr Opin Oncol 13:183-8.
[13]Sedaghat AR, Zhang Z, Begum S, et al. Prognostic significance of human papillomavirus in oropharyngeal squamous cell carcinomas. Laryngoscope.2009;119(8):1542-9.
[14]Ragin CCR, Taioli E. Survival of squamous cell carcinoma of the head and neck in relation to human papillomavirus infection:review and meta-analysis. Int J Cancer 2007; 121:1813-20.
[15]Robinson M, Sloan P, Shaw R. Refining the diagnosis of oropharyngeal squamous cell carcinoma using human papillomavirus testing. Oral Oncol 2010; 46(7):492-6.
[16]Schlecht NF, Brandwein-Gensler M, Nuovo GJ, et al. A comparison of clinically utilized human papillomavirus detection methods in head and neck cancer. Mod Pathol. 2011;24(10):1295-305.
[17]Schwartz SM, Daling JR, Doody DR, et al. Oral cancer risk in relation to sexual history and evidence of human papillomavirus infection. J Natl Cancer Inst 1998; 90(21):1626-36.
[18]D'Souza G, Kreimer AR, Viscidi R, et al. Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med.2007 May 10;356(19):1944-56.
[19]Van Doornum GJ, Korse CM, Buning-Kager JC, et al. Reactivity to human papillomavirus type 16 LI virus-like particles in sera from patients with genital cancer and patients with carcinomas at five different extragenital sites. Br J Cancer 2003; 88(7):1095-100.
[20]Rosenquist K. Risk factors in oral and oropharyngeal squamous cell carcinoma:a population-based case-control study in southern Sweden. Swed Dent J Suppl.2005;(179): 1-66.
[21]Mork J, Lie AK, Glattre E, et al. Human papillomavirus infection as a risk factor for squamous-cell carcinoma of the head and neck. N Engl J Med 2001; 344(15):1125-31.
[22]Applebaum KM, Furniss CS, Zeka A, et al. Lack of association of alcohol and tobacco with HPV16-associated head and neck cancer. J Natl Cancer Inst 2007; 99(23):1801-10.
[23]Benard VB, Johnson CJ, Thompson TD, et al. Examining the association between socioeconomic status and potential human papillomavirusassociated cancers. Cancer 2008; 113(Suppl 10):2910-8.
[24]Ang KK, Harris J, Wheeler R, et al. Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med 2010; 363(1):24-35.
[25]Beachler DC, D'Souza G. Nuances in the changing epidemiology of head and neck cancer. Oncology 2010;24(10):924-6.
[1]Scheffner M, Werness BA, Huibregtse JM, et al. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 1990;63(6):1129-36.
[2]lingelhutz AJ, Foster SA, McDougall JK. Telomerase activation by the E6 gene product of human papillomavirus type 16. Nature 1996;380(6596):79-82.
[3]Dyson N, Howley PM, Munger K, et al. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 1989;243(4893):934-7.
[4]Longworth MS, Wilson R, Laimins LA. HPV31 E7 facilitates replication by activating E2F2 transcription through its interaction with HDACs. EMBO J 2005;24(10):1821-30.
[5]Zerfass-Thome K, Zwerschke W, Mannhardt B, et al. Inactivation of the cdk inhibitor p27KIP1 by the human papillomavirus type 16 E7 oncoprotein. Oncogene 1996;13 (11):2323-30.
[6]Funk JO, Waga S, Harry JB, et al. Inhibition of CDK activity and PCNAdependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein. Genes Dev 1997;11(16):2090-100.
[7]Morris KV, Rossi JJ. Lentiviral-mediated delivery of siRNAs for antiviral therapy. Gene therapy 2006; 13(6):553-8.
[8]Howley PM, Lowy DR. Papillomaviruses. In:Knipe DM, Howley PM, editors. Fields virology 2.5th edition. Philadelphia:Lippincott, Williams & Wilkins, Wolters Kluwer; 2007. p.2299-354.
[9]Pim D, Thomas M, Javier R, et al. HPV E6 targeted degradation of the discs large protein: evidence for the involvement of a novel ubiquitin ligase. Oncogene.2000;19(6):719-25.
[10]Werness BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990;248(4951):76-79.
[11]Traidej M, Chen L, Yu D, et al. The roles of E6-AP and MDM2 in p53 regulation in human papillomavirus-positive cervical cancer cells. Antisense Nucleic Acid Drug Dev.2000;10 (1):17-27.
[12]Zwerschke W, Jansen-Durr P. Cell transformation by the E7 oncoprotein of human papillomavirus type 16:interactions with nuclear and cytoplasmic target proteins. Adv Cancer Res 2000;78:1-29.
[13]Pei XF, Sherman L, Sun YH. HPV-16 E7 protein bypasses keratinocyte growth inhibition by serum and calcium. Carcinogenesis.1998;19(8):1481-6.
[14]Duensing S, Munger K. Mechanisms of genomic instability in human cancer:insights from studies with human papillomavirus oncoproteins. Int J Cancer.2004;109(2):157-62.
[15]Howie HL, Katzenellenbogen RA, Galloway DA. Papillomavirus E6 proteins. Virology. 2009;384(2):324-34.
[16]Wise-Draper TM, Wells SI. Papillomavirus E6 and E7 proteins and their cellular targets. Front Biosci.2008; 13:1003-17.
[17]Humeau LM, Binder GK, Lu X, et al. Efficient lentiviral vector-mediated control of HIV-1 replication in CD4 lymphocytes from diverse HIV+ infected patients grouped according to CD4 count and viral load. Mol Ther.2004;9(6):902-13.
[18]Frecha C, Szecsi J, Cosset FL, et al. Strategies for targeting lentiviral vectors. Curr Gene Ther.2008;8(6):449-60.
[19]Cohen GM. Caspases:the executioners of apoptosis. Biochem J.1997; 326 (Pt 1):1-16.
[20]Cryns V, Yuan J. Protease to die for. Genes Dev.1998; 12(11):1551-70.
1. Tran N, Rose BR, O'Brien CJ. Role of human papillomavirus in the etiology of head and neck cancer. Head Neck 2007;29(1):64-70.
2. Chaturvedi AK, Engels EA, Anderson WF, et al. Incidence trends for human papillomavirus-related and -unrelated oral squamous cell carcinomas in the United States. J Clin Oncol 2008;26(4):612-9.
3. Vidal L, Gillison ML. Human papillomavirus in HNSCC:recognition of a distinct disease type. Hematol Oncol Clin North Am 2008;22(6):1125-42.
4. Wienholds E, Kloosterman WP, Miska E, et al. MicroRNA expression in zebrafish embryonic development. Science.2005;309(5732):310-1.
5. Sempere LF, Freemantle S, Pitha-Rowe I, et al. Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol.2004;5(3):R13.
6. D.P. Bartel. MicroRNAs:genomics, biogenesis, mechanism, and function, Cell.2004;116 (2):281-97.
7. W. Filipowicz, S.N. Bhattacharyya, N. Sonenberg. Mechanisms of post-transcriptional regulation by microRNAs:are the answers in sight? Nat. Rev., Genet.2008;9(2):102-14.
8. Calin GA, Sevignani C, Dumitru CD, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U S A. 2004;101(9):2999-3004.
9. Lu J, Getz G, Miska EA, et al. Nature.2005 Jun 9;435(7043):834-8. Nature.2005;435 (7043):834-8.
10. Berkhout B, Haasnoot J. The interplay between virus infection and the cellular RNA interference machinery. FEBS Lett.2006;580(12):2896-902.
11. Jopling CL, Yi M, Lancaster AM, et al. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science.2005;309(5740):1577-81.
12. Nair V, Zavolan M. Virus-encoded microRNAs:novel regulators of gene expression. Trends Microbiol.2006;14(4):169-75.
13. Ramdas L, Giri U, Ashorn CL, et al. miRNA expression profiles in head and neck squamous cell carcinoma and adjacent normal tissue. Head Neck 2009;31(5):642-54.
14. Hui AB, Lenarduzzi M, Krushel T, et al. Comprehensive Micro-RNA profiling for head and neck squamous cell carcinomas. Clin Cancer Res 2010;16(4):1129-1139.
15. Childs G, Fazzari M, Kung G, et al. Low-level expression of microRNAs let-7d and miR-205 are prognostic markers of head and neck squamous cell carcinoma. Am J Pathol 2009;174(3):736-45.
16. Tran N, McLean T, Zhang X, et al. MicroRNA expression profiles in head and neck cancer cell lines. Biochem Biophys Res Commun 2007;358(1):12-17.
17. Wald Al, Hoskins EE, Wells SI, et al. Alteration of microRNA profiles in squamous cell carcinoma of the head and neck cell lines by human papillomavirus. Head Neck.2011;33 (4):504-12.
18. Lajer CB, Garnaes E, Friis-Hansen L, et al. The role of miRNAs in human papilloma virus (HPV)-associated cancers:bridging between HPV-related head and neck cancer and cervical cancer. Br J Cancer.2012;106(9):1526-34.
19. Lum AM, Wang BB, Li L, et al. Retroviral activation of the mir-106a microRNA cistron in T lymphoma. Retrovirology 2007;4:5.
20. Ng SB, Yan J, Huang G, et al. Dysregulated microRNAs affect pathways and targets of biologic relevance in nasal-type natural killer/T-cell lymphoma. Blood 2011;118(18): 4919-29.
21. Beltran AS, Russo A, Lara H, et al. Suppression of breast tumor growth and metastasis by an engineered transcription factor. PLoS ONE 2011;6(9):e24595.
22. Sun Q, Zhang J, Cao W, et al. Dysregulated miR-363 affects head and neck cancer invasion and metastasis by targeting podoplanin. Int J Biochem Cell Biol.2013;45(3):513-20.
23. Aqeilan RI, Calin GA, Croce CM. miR-15a and miR-16-1 in cancer:discovery, function and future perspectives. Cell Death Differ.2010;17(2):215-20.
[1]Ries LAG, Melbert D, Krapcho M, et al. SEER Cancer Statistics Review. Bethesda: National Cancer Institute; 2006.1975-2004.
[2]Saba NF, Goodman M, Ward K, et al. Gender and ethnic disparities in incidence and survival of squamous cell carcinoma of the oral tongue, base of tongue, and tonsils:a surveillance, epidemiology and end results program-based analysis. Oncology.2011;81 (1):12-20.
[3]Canto MT, Devesa SS. Oral cavity and pharynx cancer incidences rates in the United States,1975-1998. Oral Oncol 2002;38(6):610-7.
[4]Golas SM. Trends in palatine tonsillar cancer incidence and mortality rates in the United States. Community Dent Oral Epidemiol.2007;35(2):98-108.
[5]Gillison ML, Koch WM, Capone RB. Evidence for a casual association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst 2000;92(9): 709-20.
[6]Fei J, Hong A, Dobbins TA, et al. Prognostic significance of vascular endothelial growth factor in squamous cell carcinomas of the tonsil in relation to human papillomavirus status and epidermal growth factor receptor. Ann Surg Oncol.2009;16(10):2908-17.
[7]Hammarstead L, Lindquist D, Dahlstrand H, et al. Human papillomavirus as a risk factor for the increase in incidence of tonsillar cancer. Int J Cancer 2006;119(11):2620-3.
[8]Gillison ML, D'Souza G, Westra W, et al. Distinct risk factor profiles for human papillomavirus type 16-positive and human papillomavirus type 16-negative head and neck cancers. J Natl Cancer Inst.2008;100(6):407-20.
[9]Chaturvedi AK, Engels EA, Anderson WF, et al. Incidence trends for human papillomavirus -related and -unrelated oral squamous cell carcinomas in the United States. J Clin Oncol 2008;26(4):612-9.
[10]McKaig RG, Baric RS, Olshan AF. Human papillomavirus and head and neck cancer: epidemiology and molecular biology. Head Neck.1998;20(3):250-65.
[11]Werness BA, Levine AJ, Howley PM. Human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990;248(4951):76-9.
[12]Kehmeier E, Ruhl H, Voland B, et al. Cellular steady-state levels of "high risk" but not "low risk" human papillomavirus (HPV) E6 proteins are increased by inhibition of proteasome-dependent degradation independent of their p53-and E6AP-binding capabilities. Virology.2002;299(1):72-87.
[13]Boyer SN, Wazer DE, Band V. E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway. Cancer Res 1996;56(20):4620-4.
[14]Howley PM, Munger K, Werness BA, Molecular mechanisms of transformation by the human papillomaviruses. Princess Takamatsu Symp.1989;20:199-206.
[15]Fakhry C, Westra W, Li S, et al. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst 2008;100(4):261-9.
[16]Bernard HU, Calleja-Macias IE, Dunn ST. Genome variation of human papillomavirus types:phylogenetic and medical implications. Int J Cancer.2006;118(5):1071-6.
[17]Hazard K, Andersson K, Dillner J, et al. Human papillomavirus subtypes are not uncommon. Virology 2007;362(1):6-9.
[18]Bens G, Wieland U, Hofmann A, et al. Detection of new human papillomavirus sequences in skin lesions of a renal transplant recipient and characterization of one complete genome related to epidermodysplasia verruciformis-associated types. J Gen Virol.1998;79 (Pt 4):779-87.
[19]Majewski S, Jablonska S, Orth G. Epidermodysplasia verruciformis; immunological and nonimmunological surveillance mechanisms; role in tumor progression. Clin Dermatol 1997;15(3):321-34.
[20]Ergunay K, Misirlioglu M, Firat P, Detection and typing of human papilloma virus by polymerase chain reaction and hybridization assay in cervical samples with cytological abnormalities. Mikrobiyol Bul.2008;42(2):273-82.
[21]Zur Hausen H, de Villiers EM. Human papillomaviruses. Annu Rev Microbiol. 1994;48:427-47.
[22]Ishiji T. Molecular mechanism of carcinogenesis by human papillomavirus-16. J Dermatol. 2000;27(2):73-86.
[23]Bosch FX, Manos MM, Munoz N, et al. Prevalence of human papillomavirus in cervical cancer:a world perspective. J Natl Cancer Inst 1995;87:796-802:
[24]Kim YT, Thomas NF, Kessis TD, p53 mutations and clonality in vulvar carcinomas and squamous hyperplasias:evidence suggesting that squamous hyperplasias do not serve as direct precursors of human papillomavirus-negative vulvar carcinomas. Hum Pathol. 1996;27(4):389-95.
[25]Wiener JS, Effert PJ, Humphrey PA, et al. Prevalence of human papillomavirus type 16 and 18 in squamous cell carcinoma of the penis:a retrospective analysis of primary and metastatic lesions by differential polymerase chain reaction. Int J Cancer 1992;50(5):694-701.
[26]Beckmann AM, Daling JR, Sherman KJ, et al. Human papillomavirus infection and anal cancer. Int J Cancer.1989;43(6):1042-9.
[27]Howley PM, Lowy DR. Papillomaviruses. In:Knipe DM, Howley PM, editors. Fields virology 2.5th edition. Philadelphia:Lippincott, Williams & Wilkins, Wolters Kluwer; 2007. p.2299-354.
[28]Han R, Reed CA, Cladel NM, et al. Immunization of rabbits with cottontail rabbit papillomavirus E1 and E2 genes:protective immunity induced by gene gun-mediated intracutaneous delivery but not by intramuscular injection. Vaccine.2000;18(26):2937-44.
[29]Thierry F, Yaniv M. The BPVl-E2-trans-acting protein can be either an activator or repressor of the HPV18 regulatory region. EMBO J 1987;6:3391-7.
[30]Knight GL, Grainger JR, Gallimore PH, et al. Cooperation between different forms of the human papillomavirus type 1 E4 protein to block cell cycle progression and cellular DNA synthesis. J Virol.2004;78(24):13920-33.
[31]Nakahara T, Peh WL, Doorbar J, et al. Human papillomavirus type 16 El circumflex E4 contributes to multiple facets of the papillomavirus life cycle. J Virol.2005;79(20): 13150-65.
[32]Rodriguez MI, Finbow ME, Alonso A. Binding of human papillomavirus 16 E5 to the 16 kDa subunit c (proteolipid) of the vacuolar H+-ATPase can be dissociated from the E5-mediated epidermal growth factor receptor overaciovation. Oncogene.2000; 19(33): 3727-32.
[33]Crusius K, Auvinen A, Alonso A. Enhancement of EGF and PMA-mediated MAP kinase activation in cells expressing the human papillomavirus type 16 E5 protein. Oncogene 1997;15(12):1437-44.
[34]Flores ER, Allen-Hoffmann BL, Lee D, et al. The human papillomavirus type 16 E7 oncogene is required for the productive stage of the viral life cycle. J Virol.2000;74(14): 6622-31.
[35]Scheffner M, Werness BA, Huibregtse JM, et al. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 1990;63(6): 1129-36.
[36]Sprague DL, Phillips SL, Mitchell CJ, et al. Telomerase activation in cervical keratinocytes containing stably replicating human papillomavirus type 16 episomes. Virology.2002;301(2):247-54.
[37]Longworth MS, Wilson R, Laimins LA. HPV31 E7 facilitates replication by activating E2F2 transcription through its interaction with HDACs. EMBO J 2005;24(10):1821-30.
[38]Zerfass K, Schulze A, Spitkovsky D, et al. Sequential activation of cyclin E and cyclin A gene expression by human papillomavirus type 16 E7 through sequences necessary for transformation. J Virol.1995;69(10):6389-99.
[39]Funk JO, Waga S, Harry JB, et al. Inhibition of CDK activity and PCNAdependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein. Genes Dev 1997;11(6):2090-100.
[40]Nardelli-Haefliger D, Roden RB, Benyacoub J, et al. Human papillomavirus type 16 virus-like particles expressed in attenuated Salmonella typhimurium elicit mucosal and systemic neutralizing antibodies in mice. Infect Immun.1997;65(8):3328-36.
[41]Hagensee ME, Yaegashi N, Galloway DA. Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins. J Virol 1993;67(1):315-22.
[42]Becker KA, Florin L, Sapp C, et al. Dissection of human papillomavirus type 33 L2 domains involved in nuclear domains (ND) 10 homing and reorganization. Virology. 2003;314(1):161-7.
[43]Darshan MS, Lucchi J, Harding E, et al. The L2 minor capsid protein of human papillomavirus type 16 interacts with a network of nuclear import receptors. J Virol 2004;78(22):12179-88.
[44]Genther SM, Sterling S, Duensing S, et al. Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle. J Virol.2003;77(5): 2832-42.
[45]Evander M, Frazer IH, Payne E, et al. Identification of the alpha6 integrin as a candidate receptor for papillomaviruses. J Virol 1997;71(3):2449-56.
[46]Patterson NA, Smith JL, Ozbun MA. Human papillomavirus type 31b infection of human keratinocytes does not require heparan sulfate. J Virol.2005;79(11):6838-47.
[47]Cason J, Kaye J, Pakarin F, et al. HPV-16 transmission. Lancet 1995;345(8943):197-8.
[48]Rombaldi RL, Serafini EP, Mandelli J, et al. Perinatal transmission of human papilomavirus DNA. Virol J.2009;6:83.
[49]Rintala MA, Grenman SE, Puranen MH, et al. Transmission of high-risk human papillomavirus (HPV) between parents and infants:a prospective study of HPV in families in Finland. J Clin Microbiol.2005;43(1):376-81.
[50]Jenson AB, Kurman RJ, Lancaster WD. Tissue effects of and host response to human papillomavirus infection. Obstet Gynecol Clin North Am.1987;14(2):397-406.
[51]Schiffman M, Castle PE, Jeronimo J, et al. Human papillomavirus and cervical cancer. The Lancet.2007;370(9590):890-907.
[52]Rodriguez AC, Schiffman M, Herrero R, et al. Longitudinal study of human papillomavirus persistence and cervical intraepithelial neoplasia grade 2/3:critical role of duration of infection J Natl Cancer Inst.2010;102(5):315-24.
[53]Wentzensen N, Vinokurova S, von Knebel Doeberitz M. Systematic review of genomic integration sites of human papillomavirus genomes in epithelial dysplasia and invasive cancer of the female lower genital tract. Cancer Res.2004;64(11):3878-84.
[54]Park JS, Hwang ES, Park SN, et al. Physical status and expression of HPV genes in cervical cancers. Gynecol Oncol.1997;65(1):121-9.
[55]Mellin H, Dahlgren L, Munck-Wikland E, et al. Human papillomavirus type 16 is episomal and a high viral load may be correlated to better prognosis in tonsillar cancer. Int J Cancer 2002;102(2):152-8.
[56]Alani RM, Miinger K. Human papilloma viruses and associated malignancies. J Clin Oncol.1998;16(1):330-7.
[57]Lechner MS, Laimins LA. Inhibition of p53 DNA binding by human papillomavirus E6 proteins. J Virol.1994;68(7):4262-73.
[58]Helt AM, Galloway DA. Destabilization of the retinoblastoma tumor suppressor by human papillomavirus type 16 E7 is not sufficient to overcome cell cycle arrest in human keratinocytes. J Virol.2001;75(15):6737-47.
[59]Munger K,, Baldwin A, Edwards KM, et al. Mechanisms of human papillomavirus-induced oncogenesis. J Virol 2004;78(21):11451-60.
[60]Duensing S, Lee LY, Duensing A, et al. The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects andgenomic instability by uncoupling centrosome duplication from the cell division cycle. Proc Natl Acad Sci U S A.2000; 97(18):10002-7.
[61]IARC Working group on the Evaluation of Carcinogenic Risks to Humans. Human papillomaviruses. IARC Monogr Eval Carcinog Risks Hum 1995;64:1-378.
[62]Heck DV, Yee CL, Howley PM, et al. Efficiency of binding the retinoblastoma protein correlates with the transforming capacity of the E7 oncoproteins of the human papillomaviruses. Proc Natl Acad Sci U S A.1992;89(10):4442-6.
[63]Scheffner M, Munger K, Huibregtse JM, et al. Targeted degradation of the retinoblastoma protein by human papillomavirus E7-E6 fusion proteins. EMBO J.1992;11(7):2425-31.
[64]Syrjanen K, et al. Morphological and immunohistochemical evidence suggesting human papillomavirus (HPV) involvement in oral squamous cell carcinogenesis. Int J Oral Surg 1983;12(6):418-24.
[65]Chang F, Syrjanen S, Shen Q, et al. Human papillomavirus involvement in esophageal precancerous lesions and squamous cellcarcinomas as evidenced by microscopy and different DNA techniques. Scand J Gastroenterol.1992;27(7):553-63.
[66]Klussmann J, Weissenborn S, Wieland U, et al. Prevalence, distribution, and viral load of human papillomavirus 16 DNA in tonsillar carcinomas. Cancer 2001; 92(11):2875-84.
[67]Braakhuis BJ, Snijders PJ, Keune WJ, et al. Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus. J Natl Cancer Inst. 2004;96(13):998-1006.
[68]Snijders PJ, Scholes AG, Hart CA, et al. Prevalence of mucosotropic human papillomaviruses in squamous-cell carcinoma of the head and neck. Int J Cancer 1996;66 (4):464-9.
[69]Wazer DE, Liu XL, Chu Q, et al. Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 E6 or E7. Proc Natl Acad Sci U S A.1995;92(9): 3687-91.
[70]Halbert CL, Demers GW, Galloway DA. The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J Virol 1991;65(1):473-8.
[71]Li SL, Kim MS, Cherrick HM, Sequential combined tumorigenic effect of HPV-16 and chemical carcinogens. Carcinogenesis.1992;13(11):1981-7.
[72]Strati K, Lambert PR Role of Rb-dependent and Rb-independent functions of papillomavirus E7 oncogene in head and neck cancer. Cancer Res 2007;67(24):11585-93.
[73]Schaeffer AJ, Nguyen M, Liem A, et al. E6 and E7 oncoproteins induce distinct patterns of chromosomal aneuploidy in skin tumors fromtransgenic mice. Cancer Res.2004;64(2): 538-46.
[74]Hawley-Nelson P, Vousden KH, Hubbert NL, et al. HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J 1989;8(12):3905-10.
[75]International Collaboration of Epidemiological Studies of Cervical Cancer. Comparison of risk factors for invasive squamous cell carcinoma and adenocarcinoma of the cervix:collaborative reanalysis of individual data on 8,097 women with squamous cell carcinoma and 1,374 women with adenocarcinoma from 12 epidemiological studies. Int J Cancer.2007;120(4):885-91.
[76]Vaccarella S, Herrero R, Snijders PJ. Smoking and human papillomavirus infection: pooled analysis of the International agency for research on cancer HPV prevalence surveys. Int J Epidemiol 2008;37(3):536-46.
[77]Castle PE. Beyond human papillomavirus:the cervix, exogenous secondary factors, and the development of cervical precancer and cancer. J Low Genit Tract Dis.2004;8(3): 224-30
[78]Melbye M, Palefsky J, Gonzales J, et al. Immune status as a determinant of human papillomavirus detection and its association with anal epithelial abnormalities. Int J Cancer 1990;46(2):203-6.
[79]Melbye M, Palefsky J, Gonzales J, et al. Immune status as a determinant of human papillomavirus detection and its association with anal epithelial abnormalities. Int J Cancer.1990;46(2):203-6.
[80]Johnson JC, Burnett AF, Willet GD, et al. High frequency of latent and clinical human papillomavirus cervical infections in immunocompromised human immunodeficiency virus-infected women. Obstet Gynecol 1992;79(3):321-7.
[81]Yang R, Wheeler CM, Chen X, Papillomavirus capsid mutation to escape dendritic cell-dependent innate immunity in cervical cancer. J Virol.2005;79(11):6741-50.
[82]Crook T, Morgenstern JP, Crawford L, et al. Continued expression of HPV-16 E7 protein is required for maintenance of the transformed phenotype of cells cotransformed by HPV-16 plus EJ-ras. EMBO J 1989;8(2):513-9.
[83]Goldie SJ, Grima D, Kohli M, et al. A comprehensive natural history model of HPV infection and cervical cancer to estimate the clinical impact of a prophylactic HPV-16/18 vaccine. Int J Cancer.200;106(6):896-904.
[84]Mork L, Lie AX, Glattre E, et al. Human papillomavirus infection as a risk factor for squamous-cell carcinoma of the head and necl.NEngl JMed 2001;344(15):1125-31.
[85]Kreimer AR, Clifford GM, Snijders PJ, et al. HPV16 semiquantitative viral load and serologic biomarkers in oral and oropharyngeal squamous cell carcinomas. Int J Cancer. 2005;115(2):329-32.
[86]Kreimer AR, Clifford GM, Boyle P, et al. Human papillomavirus types in head and neck squamous cell carcinomas worldwide:a systematic review. Cancer Epidemiol Biomarkers Prev 2005;14(2):467-75.
[87]Li W, Thompson CH, O'Brien CJ, et al. Human papillomavirus positivity predicts favourable outcome for squamous carcinoma of the tonsil. Int J Cancer.2003; 106(4): 553-8.
[88]Paz IB, Cook N, Odom-Maryon T, et al. Human papillomavirus (HPV) in head and neck cancer:an association of HPV with squamous cell carcinoma of Waldeyer's tonsillar ring. Cancer 1997;79(3):595-604.
[89]Applebaum KM, Furniss CS, Zeka A, Lack of association of alcohol and tobacco with HPV16-associated head and neck cancer. J Natl Cancer Inst.2007;99(23):1801-10.
[90]Li W, Thompson CH, Xin D, et al. Absence of human papillomavirus in tonsillar squamous cell carcinomas from Chinese patients. Am J Pathol 2003;163(6):2185-9.
[91]Hafkamp HC, Mooren JJ, Claessen SM, et al. P21 Cipl/WAF1 expression is strongly associated with HPV-positive tonsillar carcinoma and a favorable prognosis. Mod Pathol.2009;22(5):686-98.
[92]Miller CS, White DK. Human papillomavirus expression in oral mucosa, premalignant conditions, and squamous cell carcinoma; a retrospective review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996;82(1):57-68.
[93]Fuessel Haws AL,He Q, Rady PL,et al. Nested PCR with the PGMY09/11 and GP5(+)/6(+) primer sets improves detection of HPV DNA in cervical samples. J Virol Methods.200;122(1):87-93.
[94]Miller CS, Johnstone BM. Human papillomavirus as a risk factor for oral squamous cell carcinoma:a meta-analysis,1982-1997. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91(6):622-35.
[95]Kansky AA, Poljak M, Seme K, et al. Human papillomavirus DNA in oral squamous cell carcinomas and normal oral mucosa. Acta Virol.2003;47(1):11-6
[96]Balaram P, Nalinkumari KR, Abraham E, et al. Human papillomaviruses in 91 oral cancers from India betel quid chewers:high prevalence and multiplicity of infections. Int J Cancer 1995;61(4):450-4.
[97]Greer RO Jr, Eversole LR, Crosby LK. Detection of human papillomavirus-genomic DNA in oral epithelial dysplasias, oral smokeless tobacco-associated leukoplakias, and epithelial malignancies. J Oral Maxillofac Surg.1990;48(11):1201-5.
[98]Garcia-Millan R, Hernandez H, Panade L, et al. Detection and typing of human papillomavirus DNA in benign and malignant tumors of laryngeal epithelium. Acta Otolaryngol 1998;118(5):754-8.
[99]Smith EM, Ritchie JM, Summersgill KF, et al. Human papillomavirus in oral exfoliated cells and risk of head and neck cancer. J Natl Cancer Inst.2004;96(6):449-55.
[100]McKaig RG, Baric RS, Olshan AF. Human papillomavirus and head and neck cancer: epidemiology and molecular biology. Head and Neck 1998;20(3):250-65.
[101]Ergunay K, Misirlioglu M, Firat P, Detection and typing of human papilloma virus by polymerase chain reaction and hybridization assay in cervical samples with cytological abnormalities. Mikrobiyol Bul.2008;42(2):273-82.
[102]Gillison ML, D'Souza G, Westra W, et al. Distinct risk factor profiles for human papillomavirus type 16-positive and human papillomavirus type 16-negative head and neck cancers. J Natl Cancer Inst 2008;100(6):407-20.
[103]Plummer TB, Sperry AC, Xu HS, et al. In situ hybridization detection of low copy nucleic acid sequences using catalyzed reporter deposition and its usefulness in clinical human papillomavirus typing. Diagn Mol Pathol.1998;7(2):76-84.
[104]Begum S, Gillison ML, Nicol TL, et al. Detection of human papillomavirus-16 in fine-needle aspirates to determine tumor origin in patients with metastatic squamous cell carcinoma of the head and neck. Clin Cancer Res 2007;13(4):1186-91.
[105]Gaffey MJ, Frierson HF, Weiss LM, et al. Human papillomavirus and Epstein-Barr virus in sinonasal Schneiderian papillomas. An in situ hybridization and polymerase chain reaction study. Am J Clin Pathol.1996;106(4):475-82.
[106]Adams V, Schmid S, Zariwala M, et al. Prevalence of human papillomavirus DNA in head and neck cancer carrying wild type or mutant p53 tumor suppressor genes. Anticancer Res 1999; 19(4):1-6.
[107]Torrente MC,Ampuero S,Abud M, et al. Molecular detection and typing of human papillomavirus in laryngeal carcinoma specimens. Acta Otolaryngol.2005;125(8):888-93.
[108]Zang ZY, Sdek P, Cao J, et al. Human papillomavirus type 16 and 18 DNA in oral squamous cell carcinoma and normal mucosa. Int J Oral Maxillofac Surg 2004; 33(1):71-4.
[109]Paz IB, Cook N, Odom-Maryon T, et al. Human papillomavirus (HPV) in head and neck cancer. An association of HPV 16 with squamous cell carcinoma of Waldeyer's tonsillar ring. Cancer.1997;79(3):595-604.
[110]Braakhuis BJ, Snijders PJ, Keune WJ, et al. Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus. J Natl Cancer Inst.2004;96(13):998-1006.
[111]Westra WH, Taube JM, Poeta ML, et al. Inverse relationship between human papillomavirus-16 infection and disruptive p53 gene mutations in squamous cell carcinoma of the head and neck. Clin Cancer Res 2008;14(2):366-9.
[112]Badaracco G,Venuti A. Physical status of HPV types 16 and 18 in topographically different areas of genital tumours and in paired tumour-free mucosa. Int J Oncol.2005 Jul;27(1):161-7.
[113]Ragin CC, Modugno F, Gollin SM. The epidemiology and risk factors of head and neck cancer, a focus on human papillomavirus. J Dent Res 2007;86(2):101-14.
[114]Zumbach K, Kisseljov F, Sacharova O, et al. Antibodies against oncoproteins E6 and E7 of human papillomavirus types 16 and 18 in cervical-carcinoma patients from Russia, nt J Cancer.2000;85(3):313-8.
[115]Schwartz SM, Daling JR, Dr Doody, et al. Oral cancer risk in relation to sexual history and evidence of human papillomavirus infection. J Natl Cancer Inst 1998;90:1626-36.
[116]Rosenquist K. Risk factors in oral and oropharyngeal squamous cell carcinoma:a population-based case-control study in southern Sweden. Swed Dent J Suppl. 2005;(179):1-66.
[117]Smith EM, Ritchie JM, Summersgill KF, et al. Age, sexual behavior and human papillomavirus infection in oral cavity and oropharyngeal cancers. Int J Cancer 2004;108(5):766-72.
[118]Braakhuis BJ, Snijders PJ, Keune WJ, et al. Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus. J Natl Cancer Inst.2004;96(13):998-1006.
[119]Hafkamp HC, Speel EJ, Haesevoets A, et al. A subset of head and neck squamous cell carcinomas exhibits integration of HPV16/18 DNA and overexpression of p161NK4A and p53 in the absence of mutations in p53 exons 5-8. Int J Cancer 2003;107(3):394-400.
[120]Ezhevsky SA, Ho A, Becker-Hapak M, et al. Differential regulation of retinoblastoma tumor suppressor protein by G(1) cyclin-dependent kinase complexes in vivo. Mol Cell Biol.2001;21(14):4773-84.
[121]Olshan AF, Weisller MC, Pei H. Alterations of the pl6 gene in head and neck cancer: frequency and association with p53, PRAD-1 and HPV. Oncogene 1997;14(7):811-8.
[122]Klussmann JP, Mooren JJ, Lehnen M, et al. Genetic signatures of HPV-related and unrelated oropharyngeal carcinoma and their prognostic implications. Clin Cancer Res.2009;15(5):1779-86
[123]Kumar B, Cordell KG, Lee JS, et al. Response to therapy and outcomes in oropharyngeal cancer are associated with biomarkers including human papillomavirus, epidermal growth factor receptor, gender and smoking. Int J Radiat Oncol Biol Phys 2001;69(2 suppl):S109-11.
[124]Campisi G, Di Fede O, Giovannelli L,et al. Use of fuzzy neural networks in modeling relationships of HPV infection with apoptotic and proliferation markers in potentially malignant oral lesions. Oral Oncol.2005;41(10):994-1004.
[125]Lowy D, Gillison ML. New link between Fanconi anemia and human papillomavirus associated malignancies. J Natl Cancer Inst.2003;95:1648-50.
[126]Chaturvedi AK, Madeleine MM, Biggar RJ, et al. Risk of human papillomavirus-associated cancers among persons with AIDS. J Natl Cancer Inst.2009;101(16):1120-30.
[127]Strome SE, Savva A, Brissett AE, et al. Squamous cell carcinoma of the tonsils:a molecular analysis of HPV associations. Clin Cancer Res 2002;8(4):1093-100.
[128]Zhang ZY, Sdek P, Cao J, Human papillomavirus type 16 and 18 DNA in oral squamous cell carcinoma and normal mucosa. Int J Oral Maxillofac Surg.2004;33(1):71-4.
[129]Ritchie JM, Smith EM, Summersgill KF, et al. Human papillomavirus infection as a prognostic factor in carcinomas of the oral cavity and oropharynx. Int J Cancer 2003;104(3):336-44.
[130]Gillison ML, Koch WM, Capone RB, Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst.2000; 92(9):709-20.
[131]Poetsch M, Lorenz G, Banku A, et al. Basaloid in contrast to non-basaloid head and neck squamous cell carcinomas display aberrations especially in cell cycle control genes. Head and Neck 2003;25:904-10.
[132]Brachman DG, Graves D, Vokes E, et al. Occurrence of p53 gene deletions and human papilloma virus infection in human head and neck cancer. Cancer Res.1992;52(17): 4832-6.
[133]Hafkamp HC, Manni J, Haesevoets A, et al. Marked differences in survival rate between smokers and nonsmokers with HPV 16-associated tonsillar carcinomas. Int J Cancer 2008;122(12):2656-64.
[134]Fouret P, Monceaux G, Temam S, et al. Human papillomavirus in head and neck squamous cell carcinomas in nonsmokers. Arch Otolaryngol Head Neck Surg. 1997;123(5):513-6.
[135]Goldenberg D, Begum S, Westra WH. Cystic lymph node metastasis in patients with head and neck cancer:an HPV-associated phenomenon. Head Neck 2008; 30(7):898-903.
[136]Fakhry C, Westra WH, Li S, et al. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst.2008;100(4):261-9.
[2]Braakhuis BJ, Snijders PJ, Keune WJ, et al. Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus. J Natl Cancer Inst. 2004;96(13):998-1006.
[3]D'Souza G, Kreimer AR, Viscidi R, et al. Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med.2007;356(19):1944-56.
[4]Weinberger PM, Yu Z, Kountourakis P, et al. Defining molecular phenotypes of human papillomavirus-associated oropharyngeal squamous cell carcinoma:validation of three-class hypothesis. Otolaryngol Head Neck Surg.2009 Sep;141(3):382-9.
[5]Andl T, Kahn T, Pfuhl A, et al. Etiological involvement of oncogenic human papillomavirus in tonsillar squamous cell carcinomas lacking retinoblastoma cell cycle control. Cancer Res.1998;58(1):5-13.
[6]Frisch M, Biggar RJ. Aetiological parallel between tonsillar and anogenital squamous-cell carcinomas. Lancet.1999;354(9188):1442-3.
[7]Kreimer AR, Clifford GM, Boyle P, et al. Human papillomavirus types in head and neck squamous cell carcinomas worldwide:a systematic review. Cancer Epidemiol Biomarkers Prev 2005;14(2):467-75.
[8]Morris KV, Rossi JJ. Lentiviral-mediated delivery of siRNAs for antiviral therapy. Gene therapy 2006;13:553-8.
[9]D.P. Bartel. MicroRNAs:genomics, biogenesis, mechanism, and function. Cell 2004;116 (2) 281-97.
[10]Lu J, Getz G, Miska EA, et al. Nature.2005 Jun 9; 435(7043):834-8. Nature.2005 Jun 9; 435(7043):834-8.
[11]zur Hausen H. Papillomaviruses and cancer:from basic studies to clinical application. Nat Rev Cancer.2002;2(5):342-50.
[12]Gillison ML and Shah KV (2001) Human papillomavirusassociated head and neck squamous cell carcinoma:mounting evidence for an etiologic role for human papillomavirus in a subset of head and neck cancers. Curr Opin Oncol 13:183-8.
[13]Sedaghat AR, Zhang Z, Begum S, et al. Prognostic significance of human papillomavirus in oropharyngeal squamous cell carcinomas. Laryngoscope.2009;119(8):1542-9.
[14]Ragin CCR, Taioli E. Survival of squamous cell carcinoma of the head and neck in relation to human papillomavirus infection:review and meta-analysis. Int J Cancer 2007; 121:1813-20.
[15]Robinson M, Sloan P, Shaw R. Refining the diagnosis of oropharyngeal squamous cell carcinoma using human papillomavirus testing. Oral Oncol 2010; 46(7):492-6.
[16]Schlecht NF, Brandwein-Gensler M, Nuovo GJ, et al. A comparison of clinically utilized human papillomavirus detection methods in head and neck cancer. Mod Pathol. 2011;24(10):1295-305.
[17]Schwartz SM, Daling JR, Doody DR, et al. Oral cancer risk in relation to sexual history and evidence of human papillomavirus infection. J Natl Cancer Inst 1998; 90(21):1626-36.
[18]D'Souza G, Kreimer AR, Viscidi R, et al. Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med.2007 May 10;356(19):1944-56.
[19]Van Doornum GJ, Korse CM, Buning-Kager JC, et al. Reactivity to human papillomavirus type 16 LI virus-like particles in sera from patients with genital cancer and patients with carcinomas at five different extragenital sites. Br J Cancer 2003; 88(7):1095-100.
[20]Rosenquist K. Risk factors in oral and oropharyngeal squamous cell carcinoma:a population-based case-control study in southern Sweden. Swed Dent J Suppl.2005;(179): 1-66.
[21]Mork J, Lie AK, Glattre E, et al. Human papillomavirus infection as a risk factor for squamous-cell carcinoma of the head and neck. N Engl J Med 2001; 344(15):1125-31.
[22]Applebaum KM, Furniss CS, Zeka A, et al. Lack of association of alcohol and tobacco with HPV16-associated head and neck cancer. J Natl Cancer Inst 2007; 99(23):1801-10.
[23]Benard VB, Johnson CJ, Thompson TD, et al. Examining the association between socioeconomic status and potential human papillomavirusassociated cancers. Cancer 2008; 113(Suppl 10):2910-8.
[24]Ang KK, Harris J, Wheeler R, et al. Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med 2010; 363(1):24-35.
[25]Beachler DC, D'Souza G. Nuances in the changing epidemiology of head and neck cancer. Oncology 2010;24(10):924-6.
[1]Scheffner M, Werness BA, Huibregtse JM, et al. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 1990;63(6):1129-36.
[2]lingelhutz AJ, Foster SA, McDougall JK. Telomerase activation by the E6 gene product of human papillomavirus type 16. Nature 1996;380(6596):79-82.
[3]Dyson N, Howley PM, Munger K, et al. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 1989;243(4893):934-7.
[4]Longworth MS, Wilson R, Laimins LA. HPV31 E7 facilitates replication by activating E2F2 transcription through its interaction with HDACs. EMBO J 2005;24(10):1821-30.
[5]Zerfass-Thome K, Zwerschke W, Mannhardt B, et al. Inactivation of the cdk inhibitor p27KIP1 by the human papillomavirus type 16 E7 oncoprotein. Oncogene 1996;13 (11):2323-30.
[6]Funk JO, Waga S, Harry JB, et al. Inhibition of CDK activity and PCNAdependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein. Genes Dev 1997;11(16):2090-100.
[7]Morris KV, Rossi JJ. Lentiviral-mediated delivery of siRNAs for antiviral therapy. Gene therapy 2006; 13(6):553-8.
[8]Howley PM, Lowy DR. Papillomaviruses. In:Knipe DM, Howley PM, editors. Fields virology 2.5th edition. Philadelphia:Lippincott, Williams & Wilkins, Wolters Kluwer; 2007. p.2299-354.
[9]Pim D, Thomas M, Javier R, et al. HPV E6 targeted degradation of the discs large protein: evidence for the involvement of a novel ubiquitin ligase. Oncogene.2000;19(6):719-25.
[10]Werness BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990;248(4951):76-79.
[11]Traidej M, Chen L, Yu D, et al. The roles of E6-AP and MDM2 in p53 regulation in human papillomavirus-positive cervical cancer cells. Antisense Nucleic Acid Drug Dev.2000;10 (1):17-27.
[12]Zwerschke W, Jansen-Durr P. Cell transformation by the E7 oncoprotein of human papillomavirus type 16:interactions with nuclear and cytoplasmic target proteins. Adv Cancer Res 2000;78:1-29.
[13]Pei XF, Sherman L, Sun YH. HPV-16 E7 protein bypasses keratinocyte growth inhibition by serum and calcium. Carcinogenesis.1998;19(8):1481-6.
[14]Duensing S, Munger K. Mechanisms of genomic instability in human cancer:insights from studies with human papillomavirus oncoproteins. Int J Cancer.2004;109(2):157-62.
[15]Howie HL, Katzenellenbogen RA, Galloway DA. Papillomavirus E6 proteins. Virology. 2009;384(2):324-34.
[16]Wise-Draper TM, Wells SI. Papillomavirus E6 and E7 proteins and their cellular targets. Front Biosci.2008; 13:1003-17.
[17]Humeau LM, Binder GK, Lu X, et al. Efficient lentiviral vector-mediated control of HIV-1 replication in CD4 lymphocytes from diverse HIV+ infected patients grouped according to CD4 count and viral load. Mol Ther.2004;9(6):902-13.
[18]Frecha C, Szecsi J, Cosset FL, et al. Strategies for targeting lentiviral vectors. Curr Gene Ther.2008;8(6):449-60.
[19]Cohen GM. Caspases:the executioners of apoptosis. Biochem J.1997; 326 (Pt 1):1-16.
[20]Cryns V, Yuan J. Protease to die for. Genes Dev.1998; 12(11):1551-70.
1. Tran N, Rose BR, O'Brien CJ. Role of human papillomavirus in the etiology of head and neck cancer. Head Neck 2007;29(1):64-70.
2. Chaturvedi AK, Engels EA, Anderson WF, et al. Incidence trends for human papillomavirus-related and -unrelated oral squamous cell carcinomas in the United States. J Clin Oncol 2008;26(4):612-9.
3. Vidal L, Gillison ML. Human papillomavirus in HNSCC:recognition of a distinct disease type. Hematol Oncol Clin North Am 2008;22(6):1125-42.
4. Wienholds E, Kloosterman WP, Miska E, et al. MicroRNA expression in zebrafish embryonic development. Science.2005;309(5732):310-1.
5. Sempere LF, Freemantle S, Pitha-Rowe I, et al. Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol.2004;5(3):R13.
6. D.P. Bartel. MicroRNAs:genomics, biogenesis, mechanism, and function, Cell.2004;116 (2):281-97.
7. W. Filipowicz, S.N. Bhattacharyya, N. Sonenberg. Mechanisms of post-transcriptional regulation by microRNAs:are the answers in sight? Nat. Rev., Genet.2008;9(2):102-14.
8. Calin GA, Sevignani C, Dumitru CD, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U S A. 2004;101(9):2999-3004.
9. Lu J, Getz G, Miska EA, et al. Nature.2005 Jun 9;435(7043):834-8. Nature.2005;435 (7043):834-8.
10. Berkhout B, Haasnoot J. The interplay between virus infection and the cellular RNA interference machinery. FEBS Lett.2006;580(12):2896-902.
11. Jopling CL, Yi M, Lancaster AM, et al. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science.2005;309(5740):1577-81.
12. Nair V, Zavolan M. Virus-encoded microRNAs:novel regulators of gene expression. Trends Microbiol.2006;14(4):169-75.
13. Ramdas L, Giri U, Ashorn CL, et al. miRNA expression profiles in head and neck squamous cell carcinoma and adjacent normal tissue. Head Neck 2009;31(5):642-54.
14. Hui AB, Lenarduzzi M, Krushel T, et al. Comprehensive Micro-RNA profiling for head and neck squamous cell carcinomas. Clin Cancer Res 2010;16(4):1129-1139.
15. Childs G, Fazzari M, Kung G, et al. Low-level expression of microRNAs let-7d and miR-205 are prognostic markers of head and neck squamous cell carcinoma. Am J Pathol 2009;174(3):736-45.
16. Tran N, McLean T, Zhang X, et al. MicroRNA expression profiles in head and neck cancer cell lines. Biochem Biophys Res Commun 2007;358(1):12-17.
17. Wald Al, Hoskins EE, Wells SI, et al. Alteration of microRNA profiles in squamous cell carcinoma of the head and neck cell lines by human papillomavirus. Head Neck.2011;33 (4):504-12.
18. Lajer CB, Garnaes E, Friis-Hansen L, et al. The role of miRNAs in human papilloma virus (HPV)-associated cancers:bridging between HPV-related head and neck cancer and cervical cancer. Br J Cancer.2012;106(9):1526-34.
19. Lum AM, Wang BB, Li L, et al. Retroviral activation of the mir-106a microRNA cistron in T lymphoma. Retrovirology 2007;4:5.
20. Ng SB, Yan J, Huang G, et al. Dysregulated microRNAs affect pathways and targets of biologic relevance in nasal-type natural killer/T-cell lymphoma. Blood 2011;118(18): 4919-29.
21. Beltran AS, Russo A, Lara H, et al. Suppression of breast tumor growth and metastasis by an engineered transcription factor. PLoS ONE 2011;6(9):e24595.
22. Sun Q, Zhang J, Cao W, et al. Dysregulated miR-363 affects head and neck cancer invasion and metastasis by targeting podoplanin. Int J Biochem Cell Biol.2013;45(3):513-20.
23. Aqeilan RI, Calin GA, Croce CM. miR-15a and miR-16-1 in cancer:discovery, function and future perspectives. Cell Death Differ.2010;17(2):215-20.
[1]Ries LAG, Melbert D, Krapcho M, et al. SEER Cancer Statistics Review. Bethesda: National Cancer Institute; 2006.1975-2004.
[2]Saba NF, Goodman M, Ward K, et al. Gender and ethnic disparities in incidence and survival of squamous cell carcinoma of the oral tongue, base of tongue, and tonsils:a surveillance, epidemiology and end results program-based analysis. Oncology.2011;81 (1):12-20.
[3]Canto MT, Devesa SS. Oral cavity and pharynx cancer incidences rates in the United States,1975-1998. Oral Oncol 2002;38(6):610-7.
[4]Golas SM. Trends in palatine tonsillar cancer incidence and mortality rates in the United States. Community Dent Oral Epidemiol.2007;35(2):98-108.
[5]Gillison ML, Koch WM, Capone RB. Evidence for a casual association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst 2000;92(9): 709-20.
[6]Fei J, Hong A, Dobbins TA, et al. Prognostic significance of vascular endothelial growth factor in squamous cell carcinomas of the tonsil in relation to human papillomavirus status and epidermal growth factor receptor. Ann Surg Oncol.2009;16(10):2908-17.
[7]Hammarstead L, Lindquist D, Dahlstrand H, et al. Human papillomavirus as a risk factor for the increase in incidence of tonsillar cancer. Int J Cancer 2006;119(11):2620-3.
[8]Gillison ML, D'Souza G, Westra W, et al. Distinct risk factor profiles for human papillomavirus type 16-positive and human papillomavirus type 16-negative head and neck cancers. J Natl Cancer Inst.2008;100(6):407-20.
[9]Chaturvedi AK, Engels EA, Anderson WF, et al. Incidence trends for human papillomavirus -related and -unrelated oral squamous cell carcinomas in the United States. J Clin Oncol 2008;26(4):612-9.
[10]McKaig RG, Baric RS, Olshan AF. Human papillomavirus and head and neck cancer: epidemiology and molecular biology. Head Neck.1998;20(3):250-65.
[11]Werness BA, Levine AJ, Howley PM. Human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990;248(4951):76-9.
[12]Kehmeier E, Ruhl H, Voland B, et al. Cellular steady-state levels of "high risk" but not "low risk" human papillomavirus (HPV) E6 proteins are increased by inhibition of proteasome-dependent degradation independent of their p53-and E6AP-binding capabilities. Virology.2002;299(1):72-87.
[13]Boyer SN, Wazer DE, Band V. E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway. Cancer Res 1996;56(20):4620-4.
[14]Howley PM, Munger K, Werness BA, Molecular mechanisms of transformation by the human papillomaviruses. Princess Takamatsu Symp.1989;20:199-206.
[15]Fakhry C, Westra W, Li S, et al. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst 2008;100(4):261-9.
[16]Bernard HU, Calleja-Macias IE, Dunn ST. Genome variation of human papillomavirus types:phylogenetic and medical implications. Int J Cancer.2006;118(5):1071-6.
[17]Hazard K, Andersson K, Dillner J, et al. Human papillomavirus subtypes are not uncommon. Virology 2007;362(1):6-9.
[18]Bens G, Wieland U, Hofmann A, et al. Detection of new human papillomavirus sequences in skin lesions of a renal transplant recipient and characterization of one complete genome related to epidermodysplasia verruciformis-associated types. J Gen Virol.1998;79 (Pt 4):779-87.
[19]Majewski S, Jablonska S, Orth G. Epidermodysplasia verruciformis; immunological and nonimmunological surveillance mechanisms; role in tumor progression. Clin Dermatol 1997;15(3):321-34.
[20]Ergunay K, Misirlioglu M, Firat P, Detection and typing of human papilloma virus by polymerase chain reaction and hybridization assay in cervical samples with cytological abnormalities. Mikrobiyol Bul.2008;42(2):273-82.
[21]Zur Hausen H, de Villiers EM. Human papillomaviruses. Annu Rev Microbiol. 1994;48:427-47.
[22]Ishiji T. Molecular mechanism of carcinogenesis by human papillomavirus-16. J Dermatol. 2000;27(2):73-86.
[23]Bosch FX, Manos MM, Munoz N, et al. Prevalence of human papillomavirus in cervical cancer:a world perspective. J Natl Cancer Inst 1995;87:796-802:
[24]Kim YT, Thomas NF, Kessis TD, p53 mutations and clonality in vulvar carcinomas and squamous hyperplasias:evidence suggesting that squamous hyperplasias do not serve as direct precursors of human papillomavirus-negative vulvar carcinomas. Hum Pathol. 1996;27(4):389-95.
[25]Wiener JS, Effert PJ, Humphrey PA, et al. Prevalence of human papillomavirus type 16 and 18 in squamous cell carcinoma of the penis:a retrospective analysis of primary and metastatic lesions by differential polymerase chain reaction. Int J Cancer 1992;50(5):694-701.
[26]Beckmann AM, Daling JR, Sherman KJ, et al. Human papillomavirus infection and anal cancer. Int J Cancer.1989;43(6):1042-9.
[27]Howley PM, Lowy DR. Papillomaviruses. In:Knipe DM, Howley PM, editors. Fields virology 2.5th edition. Philadelphia:Lippincott, Williams & Wilkins, Wolters Kluwer; 2007. p.2299-354.
[28]Han R, Reed CA, Cladel NM, et al. Immunization of rabbits with cottontail rabbit papillomavirus E1 and E2 genes:protective immunity induced by gene gun-mediated intracutaneous delivery but not by intramuscular injection. Vaccine.2000;18(26):2937-44.
[29]Thierry F, Yaniv M. The BPVl-E2-trans-acting protein can be either an activator or repressor of the HPV18 regulatory region. EMBO J 1987;6:3391-7.
[30]Knight GL, Grainger JR, Gallimore PH, et al. Cooperation between different forms of the human papillomavirus type 1 E4 protein to block cell cycle progression and cellular DNA synthesis. J Virol.2004;78(24):13920-33.
[31]Nakahara T, Peh WL, Doorbar J, et al. Human papillomavirus type 16 El circumflex E4 contributes to multiple facets of the papillomavirus life cycle. J Virol.2005;79(20): 13150-65.
[32]Rodriguez MI, Finbow ME, Alonso A. Binding of human papillomavirus 16 E5 to the 16 kDa subunit c (proteolipid) of the vacuolar H+-ATPase can be dissociated from the E5-mediated epidermal growth factor receptor overaciovation. Oncogene.2000; 19(33): 3727-32.
[33]Crusius K, Auvinen A, Alonso A. Enhancement of EGF and PMA-mediated MAP kinase activation in cells expressing the human papillomavirus type 16 E5 protein. Oncogene 1997;15(12):1437-44.
[34]Flores ER, Allen-Hoffmann BL, Lee D, et al. The human papillomavirus type 16 E7 oncogene is required for the productive stage of the viral life cycle. J Virol.2000;74(14): 6622-31.
[35]Scheffner M, Werness BA, Huibregtse JM, et al. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 1990;63(6): 1129-36.
[36]Sprague DL, Phillips SL, Mitchell CJ, et al. Telomerase activation in cervical keratinocytes containing stably replicating human papillomavirus type 16 episomes. Virology.2002;301(2):247-54.
[37]Longworth MS, Wilson R, Laimins LA. HPV31 E7 facilitates replication by activating E2F2 transcription through its interaction with HDACs. EMBO J 2005;24(10):1821-30.
[38]Zerfass K, Schulze A, Spitkovsky D, et al. Sequential activation of cyclin E and cyclin A gene expression by human papillomavirus type 16 E7 through sequences necessary for transformation. J Virol.1995;69(10):6389-99.
[39]Funk JO, Waga S, Harry JB, et al. Inhibition of CDK activity and PCNAdependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein. Genes Dev 1997;11(6):2090-100.
[40]Nardelli-Haefliger D, Roden RB, Benyacoub J, et al. Human papillomavirus type 16 virus-like particles expressed in attenuated Salmonella typhimurium elicit mucosal and systemic neutralizing antibodies in mice. Infect Immun.1997;65(8):3328-36.
[41]Hagensee ME, Yaegashi N, Galloway DA. Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins. J Virol 1993;67(1):315-22.
[42]Becker KA, Florin L, Sapp C, et al. Dissection of human papillomavirus type 33 L2 domains involved in nuclear domains (ND) 10 homing and reorganization. Virology. 2003;314(1):161-7.
[43]Darshan MS, Lucchi J, Harding E, et al. The L2 minor capsid protein of human papillomavirus type 16 interacts with a network of nuclear import receptors. J Virol 2004;78(22):12179-88.
[44]Genther SM, Sterling S, Duensing S, et al. Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle. J Virol.2003;77(5): 2832-42.
[45]Evander M, Frazer IH, Payne E, et al. Identification of the alpha6 integrin as a candidate receptor for papillomaviruses. J Virol 1997;71(3):2449-56.
[46]Patterson NA, Smith JL, Ozbun MA. Human papillomavirus type 31b infection of human keratinocytes does not require heparan sulfate. J Virol.2005;79(11):6838-47.
[47]Cason J, Kaye J, Pakarin F, et al. HPV-16 transmission. Lancet 1995;345(8943):197-8.
[48]Rombaldi RL, Serafini EP, Mandelli J, et al. Perinatal transmission of human papilomavirus DNA. Virol J.2009;6:83.
[49]Rintala MA, Grenman SE, Puranen MH, et al. Transmission of high-risk human papillomavirus (HPV) between parents and infants:a prospective study of HPV in families in Finland. J Clin Microbiol.2005;43(1):376-81.
[50]Jenson AB, Kurman RJ, Lancaster WD. Tissue effects of and host response to human papillomavirus infection. Obstet Gynecol Clin North Am.1987;14(2):397-406.
[51]Schiffman M, Castle PE, Jeronimo J, et al. Human papillomavirus and cervical cancer. The Lancet.2007;370(9590):890-907.
[52]Rodriguez AC, Schiffman M, Herrero R, et al. Longitudinal study of human papillomavirus persistence and cervical intraepithelial neoplasia grade 2/3:critical role of duration of infection J Natl Cancer Inst.2010;102(5):315-24.
[53]Wentzensen N, Vinokurova S, von Knebel Doeberitz M. Systematic review of genomic integration sites of human papillomavirus genomes in epithelial dysplasia and invasive cancer of the female lower genital tract. Cancer Res.2004;64(11):3878-84.
[54]Park JS, Hwang ES, Park SN, et al. Physical status and expression of HPV genes in cervical cancers. Gynecol Oncol.1997;65(1):121-9.
[55]Mellin H, Dahlgren L, Munck-Wikland E, et al. Human papillomavirus type 16 is episomal and a high viral load may be correlated to better prognosis in tonsillar cancer. Int J Cancer 2002;102(2):152-8.
[56]Alani RM, Miinger K. Human papilloma viruses and associated malignancies. J Clin Oncol.1998;16(1):330-7.
[57]Lechner MS, Laimins LA. Inhibition of p53 DNA binding by human papillomavirus E6 proteins. J Virol.1994;68(7):4262-73.
[58]Helt AM, Galloway DA. Destabilization of the retinoblastoma tumor suppressor by human papillomavirus type 16 E7 is not sufficient to overcome cell cycle arrest in human keratinocytes. J Virol.2001;75(15):6737-47.
[59]Munger K,, Baldwin A, Edwards KM, et al. Mechanisms of human papillomavirus-induced oncogenesis. J Virol 2004;78(21):11451-60.
[60]Duensing S, Lee LY, Duensing A, et al. The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects andgenomic instability by uncoupling centrosome duplication from the cell division cycle. Proc Natl Acad Sci U S A.2000; 97(18):10002-7.
[61]IARC Working group on the Evaluation of Carcinogenic Risks to Humans. Human papillomaviruses. IARC Monogr Eval Carcinog Risks Hum 1995;64:1-378.
[62]Heck DV, Yee CL, Howley PM, et al. Efficiency of binding the retinoblastoma protein correlates with the transforming capacity of the E7 oncoproteins of the human papillomaviruses. Proc Natl Acad Sci U S A.1992;89(10):4442-6.
[63]Scheffner M, Munger K, Huibregtse JM, et al. Targeted degradation of the retinoblastoma protein by human papillomavirus E7-E6 fusion proteins. EMBO J.1992;11(7):2425-31.
[64]Syrjanen K, et al. Morphological and immunohistochemical evidence suggesting human papillomavirus (HPV) involvement in oral squamous cell carcinogenesis. Int J Oral Surg 1983;12(6):418-24.
[65]Chang F, Syrjanen S, Shen Q, et al. Human papillomavirus involvement in esophageal precancerous lesions and squamous cellcarcinomas as evidenced by microscopy and different DNA techniques. Scand J Gastroenterol.1992;27(7):553-63.
[66]Klussmann J, Weissenborn S, Wieland U, et al. Prevalence, distribution, and viral load of human papillomavirus 16 DNA in tonsillar carcinomas. Cancer 2001; 92(11):2875-84.
[67]Braakhuis BJ, Snijders PJ, Keune WJ, et al. Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus. J Natl Cancer Inst. 2004;96(13):998-1006.
[68]Snijders PJ, Scholes AG, Hart CA, et al. Prevalence of mucosotropic human papillomaviruses in squamous-cell carcinoma of the head and neck. Int J Cancer 1996;66 (4):464-9.
[69]Wazer DE, Liu XL, Chu Q, et al. Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 E6 or E7. Proc Natl Acad Sci U S A.1995;92(9): 3687-91.
[70]Halbert CL, Demers GW, Galloway DA. The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J Virol 1991;65(1):473-8.
[71]Li SL, Kim MS, Cherrick HM, Sequential combined tumorigenic effect of HPV-16 and chemical carcinogens. Carcinogenesis.1992;13(11):1981-7.
[72]Strati K, Lambert PR Role of Rb-dependent and Rb-independent functions of papillomavirus E7 oncogene in head and neck cancer. Cancer Res 2007;67(24):11585-93.
[73]Schaeffer AJ, Nguyen M, Liem A, et al. E6 and E7 oncoproteins induce distinct patterns of chromosomal aneuploidy in skin tumors fromtransgenic mice. Cancer Res.2004;64(2): 538-46.
[74]Hawley-Nelson P, Vousden KH, Hubbert NL, et al. HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J 1989;8(12):3905-10.
[75]International Collaboration of Epidemiological Studies of Cervical Cancer. Comparison of risk factors for invasive squamous cell carcinoma and adenocarcinoma of the cervix:collaborative reanalysis of individual data on 8,097 women with squamous cell carcinoma and 1,374 women with adenocarcinoma from 12 epidemiological studies. Int J Cancer.2007;120(4):885-91.
[76]Vaccarella S, Herrero R, Snijders PJ. Smoking and human papillomavirus infection: pooled analysis of the International agency for research on cancer HPV prevalence surveys. Int J Epidemiol 2008;37(3):536-46.
[77]Castle PE. Beyond human papillomavirus:the cervix, exogenous secondary factors, and the development of cervical precancer and cancer. J Low Genit Tract Dis.2004;8(3): 224-30
[78]Melbye M, Palefsky J, Gonzales J, et al. Immune status as a determinant of human papillomavirus detection and its association with anal epithelial abnormalities. Int J Cancer 1990;46(2):203-6.
[79]Melbye M, Palefsky J, Gonzales J, et al. Immune status as a determinant of human papillomavirus detection and its association with anal epithelial abnormalities. Int J Cancer.1990;46(2):203-6.
[80]Johnson JC, Burnett AF, Willet GD, et al. High frequency of latent and clinical human papillomavirus cervical infections in immunocompromised human immunodeficiency virus-infected women. Obstet Gynecol 1992;79(3):321-7.
[81]Yang R, Wheeler CM, Chen X, Papillomavirus capsid mutation to escape dendritic cell-dependent innate immunity in cervical cancer. J Virol.2005;79(11):6741-50.
[82]Crook T, Morgenstern JP, Crawford L, et al. Continued expression of HPV-16 E7 protein is required for maintenance of the transformed phenotype of cells cotransformed by HPV-16 plus EJ-ras. EMBO J 1989;8(2):513-9.
[83]Goldie SJ, Grima D, Kohli M, et al. A comprehensive natural history model of HPV infection and cervical cancer to estimate the clinical impact of a prophylactic HPV-16/18 vaccine. Int J Cancer.200;106(6):896-904.
[84]Mork L, Lie AX, Glattre E, et al. Human papillomavirus infection as a risk factor for squamous-cell carcinoma of the head and necl.NEngl JMed 2001;344(15):1125-31.
[85]Kreimer AR, Clifford GM, Snijders PJ, et al. HPV16 semiquantitative viral load and serologic biomarkers in oral and oropharyngeal squamous cell carcinomas. Int J Cancer. 2005;115(2):329-32.
[86]Kreimer AR, Clifford GM, Boyle P, et al. Human papillomavirus types in head and neck squamous cell carcinomas worldwide:a systematic review. Cancer Epidemiol Biomarkers Prev 2005;14(2):467-75.
[87]Li W, Thompson CH, O'Brien CJ, et al. Human papillomavirus positivity predicts favourable outcome for squamous carcinoma of the tonsil. Int J Cancer.2003; 106(4): 553-8.
[88]Paz IB, Cook N, Odom-Maryon T, et al. Human papillomavirus (HPV) in head and neck cancer:an association of HPV with squamous cell carcinoma of Waldeyer's tonsillar ring. Cancer 1997;79(3):595-604.
[89]Applebaum KM, Furniss CS, Zeka A, Lack of association of alcohol and tobacco with HPV16-associated head and neck cancer. J Natl Cancer Inst.2007;99(23):1801-10.
[90]Li W, Thompson CH, Xin D, et al. Absence of human papillomavirus in tonsillar squamous cell carcinomas from Chinese patients. Am J Pathol 2003;163(6):2185-9.
[91]Hafkamp HC, Mooren JJ, Claessen SM, et al. P21 Cipl/WAF1 expression is strongly associated with HPV-positive tonsillar carcinoma and a favorable prognosis. Mod Pathol.2009;22(5):686-98.
[92]Miller CS, White DK. Human papillomavirus expression in oral mucosa, premalignant conditions, and squamous cell carcinoma; a retrospective review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996;82(1):57-68.
[93]Fuessel Haws AL,He Q, Rady PL,et al. Nested PCR with the PGMY09/11 and GP5(+)/6(+) primer sets improves detection of HPV DNA in cervical samples. J Virol Methods.200;122(1):87-93.
[94]Miller CS, Johnstone BM. Human papillomavirus as a risk factor for oral squamous cell carcinoma:a meta-analysis,1982-1997. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91(6):622-35.
[95]Kansky AA, Poljak M, Seme K, et al. Human papillomavirus DNA in oral squamous cell carcinomas and normal oral mucosa. Acta Virol.2003;47(1):11-6
[96]Balaram P, Nalinkumari KR, Abraham E, et al. Human papillomaviruses in 91 oral cancers from India betel quid chewers:high prevalence and multiplicity of infections. Int J Cancer 1995;61(4):450-4.
[97]Greer RO Jr, Eversole LR, Crosby LK. Detection of human papillomavirus-genomic DNA in oral epithelial dysplasias, oral smokeless tobacco-associated leukoplakias, and epithelial malignancies. J Oral Maxillofac Surg.1990;48(11):1201-5.
[98]Garcia-Millan R, Hernandez H, Panade L, et al. Detection and typing of human papillomavirus DNA in benign and malignant tumors of laryngeal epithelium. Acta Otolaryngol 1998;118(5):754-8.
[99]Smith EM, Ritchie JM, Summersgill KF, et al. Human papillomavirus in oral exfoliated cells and risk of head and neck cancer. J Natl Cancer Inst.2004;96(6):449-55.
[100]McKaig RG, Baric RS, Olshan AF. Human papillomavirus and head and neck cancer: epidemiology and molecular biology. Head and Neck 1998;20(3):250-65.
[101]Ergunay K, Misirlioglu M, Firat P, Detection and typing of human papilloma virus by polymerase chain reaction and hybridization assay in cervical samples with cytological abnormalities. Mikrobiyol Bul.2008;42(2):273-82.
[102]Gillison ML, D'Souza G, Westra W, et al. Distinct risk factor profiles for human papillomavirus type 16-positive and human papillomavirus type 16-negative head and neck cancers. J Natl Cancer Inst 2008;100(6):407-20.
[103]Plummer TB, Sperry AC, Xu HS, et al. In situ hybridization detection of low copy nucleic acid sequences using catalyzed reporter deposition and its usefulness in clinical human papillomavirus typing. Diagn Mol Pathol.1998;7(2):76-84.
[104]Begum S, Gillison ML, Nicol TL, et al. Detection of human papillomavirus-16 in fine-needle aspirates to determine tumor origin in patients with metastatic squamous cell carcinoma of the head and neck. Clin Cancer Res 2007;13(4):1186-91.
[105]Gaffey MJ, Frierson HF, Weiss LM, et al. Human papillomavirus and Epstein-Barr virus in sinonasal Schneiderian papillomas. An in situ hybridization and polymerase chain reaction study. Am J Clin Pathol.1996;106(4):475-82.
[106]Adams V, Schmid S, Zariwala M, et al. Prevalence of human papillomavirus DNA in head and neck cancer carrying wild type or mutant p53 tumor suppressor genes. Anticancer Res 1999; 19(4):1-6.
[107]Torrente MC,Ampuero S,Abud M, et al. Molecular detection and typing of human papillomavirus in laryngeal carcinoma specimens. Acta Otolaryngol.2005;125(8):888-93.
[108]Zang ZY, Sdek P, Cao J, et al. Human papillomavirus type 16 and 18 DNA in oral squamous cell carcinoma and normal mucosa. Int J Oral Maxillofac Surg 2004; 33(1):71-4.
[109]Paz IB, Cook N, Odom-Maryon T, et al. Human papillomavirus (HPV) in head and neck cancer. An association of HPV 16 with squamous cell carcinoma of Waldeyer's tonsillar ring. Cancer.1997;79(3):595-604.
[110]Braakhuis BJ, Snijders PJ, Keune WJ, et al. Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus. J Natl Cancer Inst.2004;96(13):998-1006.
[111]Westra WH, Taube JM, Poeta ML, et al. Inverse relationship between human papillomavirus-16 infection and disruptive p53 gene mutations in squamous cell carcinoma of the head and neck. Clin Cancer Res 2008;14(2):366-9.
[112]Badaracco G,Venuti A. Physical status of HPV types 16 and 18 in topographically different areas of genital tumours and in paired tumour-free mucosa. Int J Oncol.2005 Jul;27(1):161-7.
[113]Ragin CC, Modugno F, Gollin SM. The epidemiology and risk factors of head and neck cancer, a focus on human papillomavirus. J Dent Res 2007;86(2):101-14.
[114]Zumbach K, Kisseljov F, Sacharova O, et al. Antibodies against oncoproteins E6 and E7 of human papillomavirus types 16 and 18 in cervical-carcinoma patients from Russia, nt J Cancer.2000;85(3):313-8.
[115]Schwartz SM, Daling JR, Dr Doody, et al. Oral cancer risk in relation to sexual history and evidence of human papillomavirus infection. J Natl Cancer Inst 1998;90:1626-36.
[116]Rosenquist K. Risk factors in oral and oropharyngeal squamous cell carcinoma:a population-based case-control study in southern Sweden. Swed Dent J Suppl. 2005;(179):1-66.
[117]Smith EM, Ritchie JM, Summersgill KF, et al. Age, sexual behavior and human papillomavirus infection in oral cavity and oropharyngeal cancers. Int J Cancer 2004;108(5):766-72.
[118]Braakhuis BJ, Snijders PJ, Keune WJ, et al. Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus. J Natl Cancer Inst.2004;96(13):998-1006.
[119]Hafkamp HC, Speel EJ, Haesevoets A, et al. A subset of head and neck squamous cell carcinomas exhibits integration of HPV16/18 DNA and overexpression of p161NK4A and p53 in the absence of mutations in p53 exons 5-8. Int J Cancer 2003;107(3):394-400.
[120]Ezhevsky SA, Ho A, Becker-Hapak M, et al. Differential regulation of retinoblastoma tumor suppressor protein by G(1) cyclin-dependent kinase complexes in vivo. Mol Cell Biol.2001;21(14):4773-84.
[121]Olshan AF, Weisller MC, Pei H. Alterations of the pl6 gene in head and neck cancer: frequency and association with p53, PRAD-1 and HPV. Oncogene 1997;14(7):811-8.
[122]Klussmann JP, Mooren JJ, Lehnen M, et al. Genetic signatures of HPV-related and unrelated oropharyngeal carcinoma and their prognostic implications. Clin Cancer Res.2009;15(5):1779-86
[123]Kumar B, Cordell KG, Lee JS, et al. Response to therapy and outcomes in oropharyngeal cancer are associated with biomarkers including human papillomavirus, epidermal growth factor receptor, gender and smoking. Int J Radiat Oncol Biol Phys 2001;69(2 suppl):S109-11.
[124]Campisi G, Di Fede O, Giovannelli L,et al. Use of fuzzy neural networks in modeling relationships of HPV infection with apoptotic and proliferation markers in potentially malignant oral lesions. Oral Oncol.2005;41(10):994-1004.
[125]Lowy D, Gillison ML. New link between Fanconi anemia and human papillomavirus associated malignancies. J Natl Cancer Inst.2003;95:1648-50.
[126]Chaturvedi AK, Madeleine MM, Biggar RJ, et al. Risk of human papillomavirus-associated cancers among persons with AIDS. J Natl Cancer Inst.2009;101(16):1120-30.
[127]Strome SE, Savva A, Brissett AE, et al. Squamous cell carcinoma of the tonsils:a molecular analysis of HPV associations. Clin Cancer Res 2002;8(4):1093-100.
[128]Zhang ZY, Sdek P, Cao J, Human papillomavirus type 16 and 18 DNA in oral squamous cell carcinoma and normal mucosa. Int J Oral Maxillofac Surg.2004;33(1):71-4.
[129]Ritchie JM, Smith EM, Summersgill KF, et al. Human papillomavirus infection as a prognostic factor in carcinomas of the oral cavity and oropharynx. Int J Cancer 2003;104(3):336-44.
[130]Gillison ML, Koch WM, Capone RB, Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst.2000; 92(9):709-20.
[131]Poetsch M, Lorenz G, Banku A, et al. Basaloid in contrast to non-basaloid head and neck squamous cell carcinomas display aberrations especially in cell cycle control genes. Head and Neck 2003;25:904-10.
[132]Brachman DG, Graves D, Vokes E, et al. Occurrence of p53 gene deletions and human papilloma virus infection in human head and neck cancer. Cancer Res.1992;52(17): 4832-6.
[133]Hafkamp HC, Manni J, Haesevoets A, et al. Marked differences in survival rate between smokers and nonsmokers with HPV 16-associated tonsillar carcinomas. Int J Cancer 2008;122(12):2656-64.
[134]Fouret P, Monceaux G, Temam S, et al. Human papillomavirus in head and neck squamous cell carcinomas in nonsmokers. Arch Otolaryngol Head Neck Surg. 1997;123(5):513-6.
[135]Goldenberg D, Begum S, Westra WH. Cystic lymph node metastasis in patients with head and neck cancer:an HPV-associated phenomenon. Head Neck 2008; 30(7):898-903.
[136]Fakhry C, Westra WH, Li S, et al. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst.2008;100(4):261-9.