头颈鳞状细胞癌中MiR-21的表达及常见microRNA单核甘酸多态性的初步研究
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摘要
头颈鳞状细胞癌(Head and Neck Squamous Cell Carcinoma, HNSCC)是一类发生于口腔、口咽部、喉咽部及喉部的鳞状细胞癌,其组织来源上呼吸消化道的粘膜上皮细胞。头颈鳞状细胞癌在世界范围内是第5大常见恶性肿瘤。吸烟饮酒被认为是头颈肿瘤最重要的危险因子。除烟酒外,人乳头状瘤病毒(human papillomavirus,HPV)感染可能是另外一种重要的病因因子。尽管近些年来,目前包括手术,放疗及化疗的综合治疗被运用以改善HNSCC的无病生存率,但进展期的HNSCC患者的5年生存率仅仅徘徊在30%左右。因此,很有必要进一步了解HNSCC的生物机制并识别预测分子标记物,这将有助于对疾病的早期诊断,个体化治疗及引导新的治疗方案的发展和评估。在过去的几十年里,肿瘤研究者主要集中在对蛋白编码基因的研究,包括癌基因和抑癌基因。但最近,作为非编码RNA基因产物microRNA已经引起了研究者的重视。
MicroRNAs是一种内源性的遗传上高度保守的大约22个碱基长度的单链非编码小分子RNA分子。通过与其靶点信使RNA的3’-端非编码区的碱基完全配对或者不完全配对结合,导致目的信使RNA的切除或者转录抑制,从而实现对基因表达的调控。MicroRNAs作为一类新发现基因调节因子,在广泛的生理病理活动中扮演着重要的角色,例如细胞增生、分化、凋亡、免疫／炎症应答系统、应急耐受和代谢等。尽管microRNA准确的生物作用机制依然未被完全揭示,但大量研究已经证实microRNAs可能作为肿瘤癌基因或抑癌基因通过影响细胞的生长发展参与人类肿瘤的形成,同时microRNA表达的改变与HNSCC等很多肿瘤病因学,诊断、发生发展及预后密切相关。
MiR-21是癌症研究中最常见的microRNAs之一,它在很多实体肿瘤中表达上调。例如乳腺癌、肺癌、胃癌、胶质母细胞瘤、胰腺癌、胆管癌和胰腺内分泌肿瘤等。MiR-21高表达在头颈肿瘤中也被观察到。因为microRNA表达具有组织特异性,因此它的表达情况能潜在地暗示HNSCC亚型之间的生物学通路/机制的不同。磷酸酶张力蛋白同源物(Phosphatase and tensin homologue, PTEN)在肿瘤的形成过程中扮演着重要角色,同时PTEN表达降低与肿瘤淋巴结转移、肿瘤分级、肿瘤的TNM分期及微血管密度密切相关。一些研究报道显示PTEN参与调节肿瘤的放疗敏感性及化疗敏感性。更重要的是,最近有研究显示PTEN在肝癌细胞株及一些肿瘤组织中可能是MiR-21的一种靶基因。但在喉鳞状细胞癌(Laryngeal Squamous Cell Carcinoma, LSCC)和喉咽鳞状细胞癌(Hypopharyngeal Squamous Cell Carcinoma, HSCC)中MiR-21的表达与PTEN表达之间的关系尚未报道。在本研究研究中,我们首次研究了在LSCC和HSCC中MiR-21和PTEN的表达以及他们之间关系,从而使我们更进一步了解MiR-21在LSCC和HSCC形成及发展过程中的作用。
研究证实HPV感染是HNSCC一个重要的发病因子,尤其是在口-口咽鳞状细胞癌(Oral Squamous Cell Carcinoma, OSCC)中。更重要的是,在对感染的固定免疫应答及适应性免疫应答过程中,MicroRNAs参与调节免疫/炎症应答系统及凋亡通路。值得注意的是,免疫/炎症应答系统及凋亡通路在HPV的清除及免疫逃逸过程中伴有重要角色。因此,microRNAs的某种基因改变可能影响HPV状态和对OSCC的易感性。
在microRNAs分子内部或者他们的链接位点上当单核甘酸多态性(Single Nucleotide Polymorphism, SNP)可能对pri-microRNA模板的转录过程,microRNA前体加工成熟microRNA的过程或microRNA与其靶点基因的相互作用过程产生影响。据我们所知,在microRNA分子内部及与其相结合的靶点基因上的一些microRNA单核苷酸多态性对microRNA的功能和他们的靶点基因的表达产生重要的影响,从而导致肿瘤易感性。最近的研究已经研究了包括miR146rs2910164,miR149rs2292832,miR196rs11614913,and miR499rs3746444在内的一些microRNA SNPs同HNSCC发病风险之间的关系。考虑到HPV在OSCC发生过程中的作用,我们研究血清HPV16状态和这四种常见microRNA SNPs的共同作用对OSCC发病风险的影响。
第一部分MiR-21和PTEN在喉及喉咽鳞状细胞癌中的表达及临床意义
试验目的：
研究MiR-21和PTEN在喉及喉咽鳞状细胞癌中的表达,并分析它们的表达情况同患者临床特征之间的关系以及它们的表达情况两者之间的关系。
试验方法：
1.收集60例原发性HNSCC肿瘤组织(30例LSCC和30例HSCC),并收集距离肿瘤切缘2cm以上的形态学正常的临近正常粘膜作为对照。
2. TRIzol法分别提取肿瘤组织和正常粘膜组织中的富含小RNA的总RNA。
3.用Taqman microRNA试剂盒及ABI7900HTSequence Detection PCR仪器通过Real-time RT-PCR方法检测MiR-21在肿瘤组织和临近正常粘膜组织中的表达情况。
4.免疫组织化学染色被用于检测PTEN在60例肿瘤组织和60例临近正常粘膜组织中的表达情况。
5.统计分析用SPSS13.0统计学软件。MiR-21的相对表达用均数(mean)±标准差(SD)表示。在肿瘤组织中和临近正常粘膜之间的MiR-21和PTEN的不同表达分别用配对t检验和Wilcoxon符号秩检验。两组样本间的比较分别采用独立样本t检验或Mann-Whitney U检验。多组间的比较采用单向方差分析或Kruskall-Wallis检验。所有统计分析均为双侧,当P<0.05时,认为有统计学意义。
实验结果：
1.在LSCC和HSCC肿瘤组织中MiR-21的表达水平明显高于非肿瘤组织(P<0.05)。上调表达的MiR-21同临床分期(P=0.001),肿瘤大小(P=0.007),病理特征(P=0.025)及淋巴结转移(P=0.002)相关。
2.在LSCC和HSCC肿瘤组织中PTEN的免疫组化染色明显弱于其临近的正常粘膜组织(P<0.05),其下调表达的PTEN同肿瘤分期(P=0.025),肿瘤大小(P=0.017),淋巴结转移(P=0.040)相关。
3.在LSCC和HSCC肿瘤组织中的MiR-21的表达与PTEN的表达成负性相关。
实验结论：
1.MiR-21可能在LSCC和HSCC的发生、发展过程中扮演重要的角色。
2. PTEN在LSCC和HSCC中表达下调。
3.在LSCC和HSCC肿瘤组织中的MiR-21的表达与PTEN的表达成负性相关。
第二部分MicroRNA单核苷酸多态性增加HPV16相关的口咽鳞状细胞癌的发病风险
实验目的：
证实microRNA单核甘酸多态性可能改变血清HPV16状态同OSCC发病风险之间的关系的假说。
实验方法：
1.按照频率配对原则,325例原发OSCC的非西班牙裔白人患者及335例非癌症正常对照被招募。所有研究参与候选人签订知情同意书并完成一份关于人口资料及包括吸烟饮酒状态在内的肿瘤危险因子的流行病问卷调查表。
2.通过标准的ELISA检测研究参与者的血清HPV16的状态。
3.血液DNA提取试剂盒从3m1全血中提取基因组DNA。
4.应用限制性片段长度多态性聚合酶链反应(Polymerase chain reaction-restriction fragment-length polymorphism, PCR-RFLP)扩增包括miR146rs2910164,miRl49rs2292832,miR196rs11614913,and miR499rs3746444.的多态性在内的片段。
5.用卡方检验对选定的人口变量、吸烟、饮酒、microRNA基因频率及血清HPV16不同状态在病例组合对照组之间的分布差异进行评估。用单因素和多因素logistic回归分析计算其比值比(Odds ratios, ORs)和95%的置信区间(Confidence interval, CIs)来评估血清HPV16状态和microRNA基因型对OSCC发病风险的影响。我们也评估血清HPV16状态和microRNA的基因型对OSCC发病风险的联合作用,同时根据烟酒状态及肿瘤位点对这种作用进行分层分析。应用SAS9.1进行所有的统计分析。所有统计分析均为双侧,当P<0.05时,认为有统计学意义。
实验结果：
1.在OSCC患者中的血清HPV16阳性比对照组中常见。
2.总体上,没有发现这四种常见microRNA多态性同OSCC发病风险之间存在有统计意义的关系。
3.同携带miR499rs3746444TT基因型同时血清HPV16阴性的个体相比,我们发现携带miR499rs3746444CT或CC基因型同时血清HPV16阴性的个体的OSCC发病风险增高(OR=I.4,95%CI=1.0-2.0),我们发现携带miR499rs3746444TT基因型同时血清HPV16阳性的个体的OSCC发病风险增高(OR=3.4,95%CI=2.0-5.6),携带miR499rs3746444CT或CC基因型同时血清HPV16阳性的个体明显增加OSCC发病风险(OR=4.1,95%CI=2.1-8.0)。类似的结果对于miR146rs2910164,miR149rs2292832和miR196rs11614913的多态性也存在。
4.按照吸烟饮酒状态进行进一步的分层分析,我们发现我们研究的每种microRNA多态性改变HPV16感染相关的OSCC的发病风险,同时这种改变作用在从不吸烟人群中尤其明细；同口腔鳞状细胞癌(Oral Cavity Squamous Cell Carcinoma, OCSCC)相比,口咽部鳞状细胞癌(Oropharynx Squamous CellCarcinoma, OPSCC)的这种作用更为明显。
实验结论：
与血清HPV16阳性相关的口咽鳞状细胞癌的发病风险受microRNA多态性影响而发生改变。需要更多大样本量的研究去进一步证实我们的发现。
Head and neck squamous cell carcinoma (HNSCC) is a type of tumor which derives from the epithelium mucosa cell of oral cavity, oropharynx, laryngopharynx and larynx. And HNSCC is the fifth common malignant tumor worldwide. Tobacco use and alcohol consumption are considered as the most important risk factors of HNSCC. In addition, human papillomavirus (HPV) infection might be another important etiologic factor. Although comprehensive therapies including surgery, radiotherapy and chemotherapy was applied to increase the disease-free survival rate in recent years, patients with locally advanced HNSCC have a5-year overall survival rate hovering approximately30%. Therefore, there is a need to acquire deeper understanding of HNSCC biology and to identify predictive molecular markers, which might improve early diagnosis and patient selection for appropriate treatment and guide the development and evaluation of new therapies. In the past decades, researchers have mainly focused on the protein-coding genes including oncogenes and tumor suppressor genes, however, in recent years microRNAs as non-coding RNA gene products, have drawn much more attentions from researchers.
MicroRNAs ware evolutionarily highly conserved, singled-stranded, short non-coding RNAs of about22nucleotides that mediate gene expression through completely or partially base pairing with target mRNA at the3'-untranslated region, leading to mRNA cleavage or translational repression. MicroRNAs, as a new family of gene regulators, play central roles in a broad range of physiologic and pathologic processes including cell proliferation, differentiation, apoptosis, immune/inflammation response system, stress resistance and metabolism. Although their precise biological mechanisms largely remain unclear, a great many studies have demonstrated that microRNAs might be involved in human tumorigenesis as tumor suppressors or oncogenes by affecting cell growth and development and altered expressions of microRNAs are associated with the etiology, diagnosis, progress and prognosis of numerous cancers including HNSCC.
MiR-21is one of the most studied microRNAs in cancers and is upregulated in many solid tumors, including breast cancer, lung cancer, stomach cancer, glioblastoma, cholangiocarcinoma and pancreatic endocrine tumor. In head and neck cancer, high expression of MiR-21is also observed in tumor tissues. As microRNA expression is tissue-specific, the expression signatures could potentially suggest biological pathways/mechanisms that differ between the HNSCC subtypes. Phosphatase and tensin homologue (PTEN) plays an important role in tumorigenesis and decreased PTEN expression is associated with lymph node metastasis, tumor grade, tumor-node-metastasis (TNM) stage, and microvessel density. And several studies indicated that PTEN participated in mediating radiosensitivity and chemosensitivity in head and neck cancers. Most importantly, PTEN might be considered as one of target genes of MiR-21in HCC cell lines and some tumors. However, the association between MiR-21expression and PTEN expression has not been reported in Laryngeal squamous cell carcinoma (LSCC) and Hypopharyngeal squamous cell carcinoma (HSCC). Hence, in the present study we first investigated the expression of both MiR-21and PTEN and evaluated the association of MiR-21expression with PTEN expression in LSCC and HSCC to help us understand both function of MiR-21in tumorigenesis and its clinical significance.
It has been demonstrated that HPV infection is an important etiologic factor of HNSCC, especially oral squamous cell carcinoma (OSCC). Most importantly, in response to infection during both the innate and acquired immune response, microRNAs may be involved in the regulation of immune/inflammation response system and apoptosis pathways. It is worth noting that both immune/inflammation response system and apoptosis pathways play key roles in the HPV clearance and escape of immune surveillance. Therefore, certain genetic alterations of microRNAs might have influence on the HPV status and the susceptibility to OSCC.
A single nucleotide polymorphism (SNP) in microRNAs or their binding sites might affect the transcript of pri-microRNA transcripants, the processing of microRNA precursors to mature microRNAs, or microRNA-target interactions. To our knowledge, several SNPs in microRNAs and within their targets may have an effect on microRNA functions and their target gene expressions and thus may contribute to cancer susceptibility. Recent studies have investigated the association between HNSCC risk and several mircoRNA SNPs including miR146rs2910164, miR149rs2292832, miR196rs11614913, and miR499rs3746444polymorphisms. However, these studies show conflicting results. Considering the important roles of HPV in the development of OSCC, we evaluated the joint effects of HPV16serology status and these four common microRNA SNPs on risk of OSCC.
Part1Altered expressions of MiR-21and PTEN in human laryngeal and hypopharyngeal squamous cell carcinomas
Objective:
To examine the expressions of MiR-21and PTEN in laryngeal squamous cell carcinoma (LSCC) and hypopharyngeal squamous cell carcinoma (HSCC), and assess the associations of their expressions with clinical characteristics of patients as well as the association between the expressions of MiR-21and PTEN.
Methods:
1. We collected60cases of primary HNSCC tumor tissue including30LSCC and HSCC, and morphologically normal mucosa epithelium tissue adjacent to carcinomas (at least2cm from tumor margin) as control.
2. Total RNA enriched with small RNA was isolated from fresh frozen tumor tissue and adjacent normal mucosal epithelium tissue.
3. Real-time RT-PCR for MiR-21expression was analyzed by using Taqman microRNA assays and ABI7900HT Sequence Detection.
4. Immunohistochemistry Staining was carried out to study altered PTEN protein expression in60tumor tissues and60matched adjacent normal mucosa epithelium tissues.
5. Statistical analysis was performed using SPSS software (version13.0, SPSS Inc., Chicago, IL, USA). The relative expression of MiR-21was presented as mean±standard deviation (SD). The differences of MiR-21expressions between tumor and matched adjacent normal tissue were assessed by the Paired-Student's t test and the differences of PTEN expressions were analyzed by Wilcoxon Singed Ranks Test. Differences between two groups were examined by the Independent Samples t Test or Mann-Whitney U test. Multiple group comparisons were analyzed using One-way ANOVA or Kruskall-Wallis test. The criterion for statistical significance was set at P<0.05.
Results:
1. MiR-21was up-regulated in LSCC and HSCC compared to adjacent non-tumor tissues (P<0.05), and the up-regulated expression of MiR-21was associated with clinical stage (P=0.001), T classification (P=0.007), pathologic differentiation (P=0.025), and lymph node positivity (P=0.002).
2. PTEN IHC staining was notably weaker in tumor tissues than that in matched non-tumor tissues (P<0.05), and the down-regulated expression of PTEN was correlated with tumor staging (P=0.025), the extent of tumor (P=0.017), and lymph node positivity (P=0.040).
3. The level of MiR-21was reversely correlated with PTEN expression (P=0.006).
Conclusion:
1. MiR-21might play a central role in the progression of LSCC and HSCC.
2. PTEN was down-regulated in LSCC and HSCC.
3. The expression of MiR-21was negatively associated with the expression of PTEN in LSCC and HSCC.
Part2MicroRNA Variants Increase the Risk of Squamous Cell Carcinoma of Oropharynx Associated with HPV16
Objectives:
To test the hypothesis that microRNA polymorphisms might modify the association of HPV16seropositivity with OSCC risk.
Methods:
1.325non-Hispanic white patients with primary squamous cell carcinoma of oral and335cancer-free control individuals were selected by frequency matching. And all study participants signed an informed consent forms and completed an epidemiological questionnaire regarding demographic and risk factors including smoking and drinking status.
2. HPV16serologic detection was tested in the plasma of study participants by using a standard ELISA.
3. Genomic DNA was extracted from3ml of whole blood with the DNA Blood Mini Kit.
4. A polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) assay was applied to amplify the fragments that contain polymorphisms of miR146rs2910164, miR149rs2292832, miR196rs11614913, and miR499rs3746444.
5. The differences of the distributions of selected demographic variables, tobacco smoking, alcohol consumption, and microRNA genotypes frequencies between HPV16seropositivity and HPV16seronegative cases were evaluated using the chi-square test of association. To evaluate the association of serologic HPV16status and microRNA genotypes with OSCC risk, the odds ratios (ORs) and95%confidence interval (CIs) were calculated using both univariate and multivariate logistic regression analyses. We also assessed the joint effects of HPV16serology and microRNA genotypes on OSCC risk, and the joint effects were further stratified by smoking status and tumor site. Statistical Analysis System software (Version9.1; SAS Institute, Cary, NC) was used for all the statistical analyses. All tests were two-sided, and a p<0.05was considered the cutoff for statistical significance.
Results:
1. HPV16seropositivity was significantly more common in patients than that in controls.
2. Overall, no significant association of these four microRNA polymorphisms with the risk of OSCC was observed
3. Compared with individuals with miR499rs3746444TT genotype and HPV16seronegativity, an increased risk of OSCC was found among those with TT or CC genotypes and HPV16seronegativity (OR=1.4,95%CI=1.0-2.0), TT genotype and HPV16seropositivity (OR=3.4,95%CI=2.0-5.6) and CT or CC genotypes and HPV16seropositivity (OR=4.1,95%CI=2.1-8.0), respectively. Similar results were found for miR146rs2910164, miR149rs2292832and miR196rs11614913polymorphisms.
4. With further analysis stratified by smoking status and tumor site, we observed that each microRNA polymorphism altered risk of HPV16-associated OSCC, and such effect modification was particularly prominent in the never smokers, and for oropharyngeal cancer as opposed to oral cavity cancer.
Conclusion:
The risk of oropharynx squamous cell carcinoma (OPSCC) associated with HPV16seropositivity could be modified by microRNA polymorphisms. Larger studies are needed to verify our findings.
MicroRNAs是一种内源性的遗传上高度保守的大约22个碱基长度的单链非编码小分子RNA分子。通过与其靶点信使RNA的3’-端非编码区的碱基完全配对或者不完全配对结合,导致目的信使RNA的切除或者转录抑制,从而实现对基因表达的调控。MicroRNAs作为一类新发现基因调节因子,在广泛的生理病理活动中扮演着重要的角色,例如细胞增生、分化、凋亡、免疫／炎症应答系统、应急耐受和代谢等。尽管microRNA准确的生物作用机制依然未被完全揭示,但大量研究已经证实microRNAs可能作为肿瘤癌基因或抑癌基因通过影响细胞的生长发展参与人类肿瘤的形成,同时microRNA表达的改变与HNSCC等很多肿瘤病因学,诊断、发生发展及预后密切相关。
MiR-21是癌症研究中最常见的microRNAs之一,它在很多实体肿瘤中表达上调。例如乳腺癌、肺癌、胃癌、胶质母细胞瘤、胰腺癌、胆管癌和胰腺内分泌肿瘤等。MiR-21高表达在头颈肿瘤中也被观察到。因为microRNA表达具有组织特异性,因此它的表达情况能潜在地暗示HNSCC亚型之间的生物学通路/机制的不同。磷酸酶张力蛋白同源物(Phosphatase and tensin homologue, PTEN)在肿瘤的形成过程中扮演着重要角色,同时PTEN表达降低与肿瘤淋巴结转移、肿瘤分级、肿瘤的TNM分期及微血管密度密切相关。一些研究报道显示PTEN参与调节肿瘤的放疗敏感性及化疗敏感性。更重要的是,最近有研究显示PTEN在肝癌细胞株及一些肿瘤组织中可能是MiR-21的一种靶基因。但在喉鳞状细胞癌(Laryngeal Squamous Cell Carcinoma, LSCC)和喉咽鳞状细胞癌(Hypopharyngeal Squamous Cell Carcinoma, HSCC)中MiR-21的表达与PTEN表达之间的关系尚未报道。在本研究研究中,我们首次研究了在LSCC和HSCC中MiR-21和PTEN的表达以及他们之间关系,从而使我们更进一步了解MiR-21在LSCC和HSCC形成及发展过程中的作用。
研究证实HPV感染是HNSCC一个重要的发病因子,尤其是在口-口咽鳞状细胞癌(Oral Squamous Cell Carcinoma, OSCC)中。更重要的是,在对感染的固定免疫应答及适应性免疫应答过程中,MicroRNAs参与调节免疫/炎症应答系统及凋亡通路。值得注意的是,免疫/炎症应答系统及凋亡通路在HPV的清除及免疫逃逸过程中伴有重要角色。因此,microRNAs的某种基因改变可能影响HPV状态和对OSCC的易感性。
在microRNAs分子内部或者他们的链接位点上当单核甘酸多态性(Single Nucleotide Polymorphism, SNP)可能对pri-microRNA模板的转录过程,microRNA前体加工成熟microRNA的过程或microRNA与其靶点基因的相互作用过程产生影响。据我们所知,在microRNA分子内部及与其相结合的靶点基因上的一些microRNA单核苷酸多态性对microRNA的功能和他们的靶点基因的表达产生重要的影响,从而导致肿瘤易感性。最近的研究已经研究了包括miR146rs2910164,miR149rs2292832,miR196rs11614913,and miR499rs3746444在内的一些microRNA SNPs同HNSCC发病风险之间的关系。考虑到HPV在OSCC发生过程中的作用,我们研究血清HPV16状态和这四种常见microRNA SNPs的共同作用对OSCC发病风险的影响。
第一部分MiR-21和PTEN在喉及喉咽鳞状细胞癌中的表达及临床意义
试验目的：
研究MiR-21和PTEN在喉及喉咽鳞状细胞癌中的表达,并分析它们的表达情况同患者临床特征之间的关系以及它们的表达情况两者之间的关系。
试验方法：
1.收集60例原发性HNSCC肿瘤组织(30例LSCC和30例HSCC),并收集距离肿瘤切缘2cm以上的形态学正常的临近正常粘膜作为对照。
2. TRIzol法分别提取肿瘤组织和正常粘膜组织中的富含小RNA的总RNA。
3.用Taqman microRNA试剂盒及ABI7900HTSequence Detection PCR仪器通过Real-time RT-PCR方法检测MiR-21在肿瘤组织和临近正常粘膜组织中的表达情况。
4.免疫组织化学染色被用于检测PTEN在60例肿瘤组织和60例临近正常粘膜组织中的表达情况。
5.统计分析用SPSS13.0统计学软件。MiR-21的相对表达用均数(mean)±标准差(SD)表示。在肿瘤组织中和临近正常粘膜之间的MiR-21和PTEN的不同表达分别用配对t检验和Wilcoxon符号秩检验。两组样本间的比较分别采用独立样本t检验或Mann-Whitney U检验。多组间的比较采用单向方差分析或Kruskall-Wallis检验。所有统计分析均为双侧,当P<0.05时,认为有统计学意义。
实验结果：
1.在LSCC和HSCC肿瘤组织中MiR-21的表达水平明显高于非肿瘤组织(P<0.05)。上调表达的MiR-21同临床分期(P=0.001),肿瘤大小(P=0.007),病理特征(P=0.025)及淋巴结转移(P=0.002)相关。
2.在LSCC和HSCC肿瘤组织中PTEN的免疫组化染色明显弱于其临近的正常粘膜组织(P<0.05),其下调表达的PTEN同肿瘤分期(P=0.025),肿瘤大小(P=0.017),淋巴结转移(P=0.040)相关。
3.在LSCC和HSCC肿瘤组织中的MiR-21的表达与PTEN的表达成负性相关。
实验结论：
1.MiR-21可能在LSCC和HSCC的发生、发展过程中扮演重要的角色。
2. PTEN在LSCC和HSCC中表达下调。
3.在LSCC和HSCC肿瘤组织中的MiR-21的表达与PTEN的表达成负性相关。
第二部分MicroRNA单核苷酸多态性增加HPV16相关的口咽鳞状细胞癌的发病风险
实验目的：
证实microRNA单核甘酸多态性可能改变血清HPV16状态同OSCC发病风险之间的关系的假说。
实验方法：
1.按照频率配对原则,325例原发OSCC的非西班牙裔白人患者及335例非癌症正常对照被招募。所有研究参与候选人签订知情同意书并完成一份关于人口资料及包括吸烟饮酒状态在内的肿瘤危险因子的流行病问卷调查表。
2.通过标准的ELISA检测研究参与者的血清HPV16的状态。
3.血液DNA提取试剂盒从3m1全血中提取基因组DNA。
4.应用限制性片段长度多态性聚合酶链反应(Polymerase chain reaction-restriction fragment-length polymorphism, PCR-RFLP)扩增包括miR146rs2910164,miRl49rs2292832,miR196rs11614913,and miR499rs3746444.的多态性在内的片段。
5.用卡方检验对选定的人口变量、吸烟、饮酒、microRNA基因频率及血清HPV16不同状态在病例组合对照组之间的分布差异进行评估。用单因素和多因素logistic回归分析计算其比值比(Odds ratios, ORs)和95%的置信区间(Confidence interval, CIs)来评估血清HPV16状态和microRNA基因型对OSCC发病风险的影响。我们也评估血清HPV16状态和microRNA的基因型对OSCC发病风险的联合作用,同时根据烟酒状态及肿瘤位点对这种作用进行分层分析。应用SAS9.1进行所有的统计分析。所有统计分析均为双侧,当P<0.05时,认为有统计学意义。
实验结果：
1.在OSCC患者中的血清HPV16阳性比对照组中常见。
2.总体上,没有发现这四种常见microRNA多态性同OSCC发病风险之间存在有统计意义的关系。
3.同携带miR499rs3746444TT基因型同时血清HPV16阴性的个体相比,我们发现携带miR499rs3746444CT或CC基因型同时血清HPV16阴性的个体的OSCC发病风险增高(OR=I.4,95%CI=1.0-2.0),我们发现携带miR499rs3746444TT基因型同时血清HPV16阳性的个体的OSCC发病风险增高(OR=3.4,95%CI=2.0-5.6),携带miR499rs3746444CT或CC基因型同时血清HPV16阳性的个体明显增加OSCC发病风险(OR=4.1,95%CI=2.1-8.0)。类似的结果对于miR146rs2910164,miR149rs2292832和miR196rs11614913的多态性也存在。
4.按照吸烟饮酒状态进行进一步的分层分析,我们发现我们研究的每种microRNA多态性改变HPV16感染相关的OSCC的发病风险,同时这种改变作用在从不吸烟人群中尤其明细；同口腔鳞状细胞癌(Oral Cavity Squamous Cell Carcinoma, OCSCC)相比,口咽部鳞状细胞癌(Oropharynx Squamous CellCarcinoma, OPSCC)的这种作用更为明显。
实验结论：
与血清HPV16阳性相关的口咽鳞状细胞癌的发病风险受microRNA多态性影响而发生改变。需要更多大样本量的研究去进一步证实我们的发现。
Head and neck squamous cell carcinoma (HNSCC) is a type of tumor which derives from the epithelium mucosa cell of oral cavity, oropharynx, laryngopharynx and larynx. And HNSCC is the fifth common malignant tumor worldwide. Tobacco use and alcohol consumption are considered as the most important risk factors of HNSCC. In addition, human papillomavirus (HPV) infection might be another important etiologic factor. Although comprehensive therapies including surgery, radiotherapy and chemotherapy was applied to increase the disease-free survival rate in recent years, patients with locally advanced HNSCC have a5-year overall survival rate hovering approximately30%. Therefore, there is a need to acquire deeper understanding of HNSCC biology and to identify predictive molecular markers, which might improve early diagnosis and patient selection for appropriate treatment and guide the development and evaluation of new therapies. In the past decades, researchers have mainly focused on the protein-coding genes including oncogenes and tumor suppressor genes, however, in recent years microRNAs as non-coding RNA gene products, have drawn much more attentions from researchers.
MicroRNAs ware evolutionarily highly conserved, singled-stranded, short non-coding RNAs of about22nucleotides that mediate gene expression through completely or partially base pairing with target mRNA at the3'-untranslated region, leading to mRNA cleavage or translational repression. MicroRNAs, as a new family of gene regulators, play central roles in a broad range of physiologic and pathologic processes including cell proliferation, differentiation, apoptosis, immune/inflammation response system, stress resistance and metabolism. Although their precise biological mechanisms largely remain unclear, a great many studies have demonstrated that microRNAs might be involved in human tumorigenesis as tumor suppressors or oncogenes by affecting cell growth and development and altered expressions of microRNAs are associated with the etiology, diagnosis, progress and prognosis of numerous cancers including HNSCC.
MiR-21is one of the most studied microRNAs in cancers and is upregulated in many solid tumors, including breast cancer, lung cancer, stomach cancer, glioblastoma, cholangiocarcinoma and pancreatic endocrine tumor. In head and neck cancer, high expression of MiR-21is also observed in tumor tissues. As microRNA expression is tissue-specific, the expression signatures could potentially suggest biological pathways/mechanisms that differ between the HNSCC subtypes. Phosphatase and tensin homologue (PTEN) plays an important role in tumorigenesis and decreased PTEN expression is associated with lymph node metastasis, tumor grade, tumor-node-metastasis (TNM) stage, and microvessel density. And several studies indicated that PTEN participated in mediating radiosensitivity and chemosensitivity in head and neck cancers. Most importantly, PTEN might be considered as one of target genes of MiR-21in HCC cell lines and some tumors. However, the association between MiR-21expression and PTEN expression has not been reported in Laryngeal squamous cell carcinoma (LSCC) and Hypopharyngeal squamous cell carcinoma (HSCC). Hence, in the present study we first investigated the expression of both MiR-21and PTEN and evaluated the association of MiR-21expression with PTEN expression in LSCC and HSCC to help us understand both function of MiR-21in tumorigenesis and its clinical significance.
It has been demonstrated that HPV infection is an important etiologic factor of HNSCC, especially oral squamous cell carcinoma (OSCC). Most importantly, in response to infection during both the innate and acquired immune response, microRNAs may be involved in the regulation of immune/inflammation response system and apoptosis pathways. It is worth noting that both immune/inflammation response system and apoptosis pathways play key roles in the HPV clearance and escape of immune surveillance. Therefore, certain genetic alterations of microRNAs might have influence on the HPV status and the susceptibility to OSCC.
A single nucleotide polymorphism (SNP) in microRNAs or their binding sites might affect the transcript of pri-microRNA transcripants, the processing of microRNA precursors to mature microRNAs, or microRNA-target interactions. To our knowledge, several SNPs in microRNAs and within their targets may have an effect on microRNA functions and their target gene expressions and thus may contribute to cancer susceptibility. Recent studies have investigated the association between HNSCC risk and several mircoRNA SNPs including miR146rs2910164, miR149rs2292832, miR196rs11614913, and miR499rs3746444polymorphisms. However, these studies show conflicting results. Considering the important roles of HPV in the development of OSCC, we evaluated the joint effects of HPV16serology status and these four common microRNA SNPs on risk of OSCC.
Part1Altered expressions of MiR-21and PTEN in human laryngeal and hypopharyngeal squamous cell carcinomas
Objective:
To examine the expressions of MiR-21and PTEN in laryngeal squamous cell carcinoma (LSCC) and hypopharyngeal squamous cell carcinoma (HSCC), and assess the associations of their expressions with clinical characteristics of patients as well as the association between the expressions of MiR-21and PTEN.
Methods:
1. We collected60cases of primary HNSCC tumor tissue including30LSCC and HSCC, and morphologically normal mucosa epithelium tissue adjacent to carcinomas (at least2cm from tumor margin) as control.
2. Total RNA enriched with small RNA was isolated from fresh frozen tumor tissue and adjacent normal mucosal epithelium tissue.
3. Real-time RT-PCR for MiR-21expression was analyzed by using Taqman microRNA assays and ABI7900HT Sequence Detection.
4. Immunohistochemistry Staining was carried out to study altered PTEN protein expression in60tumor tissues and60matched adjacent normal mucosa epithelium tissues.
5. Statistical analysis was performed using SPSS software (version13.0, SPSS Inc., Chicago, IL, USA). The relative expression of MiR-21was presented as mean±standard deviation (SD). The differences of MiR-21expressions between tumor and matched adjacent normal tissue were assessed by the Paired-Student's t test and the differences of PTEN expressions were analyzed by Wilcoxon Singed Ranks Test. Differences between two groups were examined by the Independent Samples t Test or Mann-Whitney U test. Multiple group comparisons were analyzed using One-way ANOVA or Kruskall-Wallis test. The criterion for statistical significance was set at P<0.05.
Results:
1. MiR-21was up-regulated in LSCC and HSCC compared to adjacent non-tumor tissues (P<0.05), and the up-regulated expression of MiR-21was associated with clinical stage (P=0.001), T classification (P=0.007), pathologic differentiation (P=0.025), and lymph node positivity (P=0.002).
2. PTEN IHC staining was notably weaker in tumor tissues than that in matched non-tumor tissues (P<0.05), and the down-regulated expression of PTEN was correlated with tumor staging (P=0.025), the extent of tumor (P=0.017), and lymph node positivity (P=0.040).
3. The level of MiR-21was reversely correlated with PTEN expression (P=0.006).
Conclusion:
1. MiR-21might play a central role in the progression of LSCC and HSCC.
2. PTEN was down-regulated in LSCC and HSCC.
3. The expression of MiR-21was negatively associated with the expression of PTEN in LSCC and HSCC.
Part2MicroRNA Variants Increase the Risk of Squamous Cell Carcinoma of Oropharynx Associated with HPV16
Objectives:
To test the hypothesis that microRNA polymorphisms might modify the association of HPV16seropositivity with OSCC risk.
Methods:
1.325non-Hispanic white patients with primary squamous cell carcinoma of oral and335cancer-free control individuals were selected by frequency matching. And all study participants signed an informed consent forms and completed an epidemiological questionnaire regarding demographic and risk factors including smoking and drinking status.
2. HPV16serologic detection was tested in the plasma of study participants by using a standard ELISA.
3. Genomic DNA was extracted from3ml of whole blood with the DNA Blood Mini Kit.
4. A polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) assay was applied to amplify the fragments that contain polymorphisms of miR146rs2910164, miR149rs2292832, miR196rs11614913, and miR499rs3746444.
5. The differences of the distributions of selected demographic variables, tobacco smoking, alcohol consumption, and microRNA genotypes frequencies between HPV16seropositivity and HPV16seronegative cases were evaluated using the chi-square test of association. To evaluate the association of serologic HPV16status and microRNA genotypes with OSCC risk, the odds ratios (ORs) and95%confidence interval (CIs) were calculated using both univariate and multivariate logistic regression analyses. We also assessed the joint effects of HPV16serology and microRNA genotypes on OSCC risk, and the joint effects were further stratified by smoking status and tumor site. Statistical Analysis System software (Version9.1; SAS Institute, Cary, NC) was used for all the statistical analyses. All tests were two-sided, and a p<0.05was considered the cutoff for statistical significance.
Results:
1. HPV16seropositivity was significantly more common in patients than that in controls.
2. Overall, no significant association of these four microRNA polymorphisms with the risk of OSCC was observed
3. Compared with individuals with miR499rs3746444TT genotype and HPV16seronegativity, an increased risk of OSCC was found among those with TT or CC genotypes and HPV16seronegativity (OR=1.4,95%CI=1.0-2.0), TT genotype and HPV16seropositivity (OR=3.4,95%CI=2.0-5.6) and CT or CC genotypes and HPV16seropositivity (OR=4.1,95%CI=2.1-8.0), respectively. Similar results were found for miR146rs2910164, miR149rs2292832and miR196rs11614913polymorphisms.
4. With further analysis stratified by smoking status and tumor site, we observed that each microRNA polymorphism altered risk of HPV16-associated OSCC, and such effect modification was particularly prominent in the never smokers, and for oropharyngeal cancer as opposed to oral cavity cancer.
Conclusion:
The risk of oropharynx squamous cell carcinoma (OPSCC) associated with HPV16seropositivity could be modified by microRNA polymorphisms. Larger studies are needed to verify our findings.
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71. Cai, Y., et al., A brief review on the mechanisms of miRNA regulation. Genomics Proteomics Bioinformatics,2009.7(4):p.147-54.
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73. Hu, Z., et al., Genetic variants of miRNA sequences and non-small cell lung cancer survival. J Clin Invest,2008.118(7):p.2600-8.
74. Chin, L.J., et al., A SNP in a let-7 microRNA complementary site in the KRAS 3' untranslated region increases non-small cell lung cancer risk. Cancer Res, 2008.68(20):p.8535-40.
75. Yang, H., et al., Evaluation of genetic variants in microRNA-related genes and risk of bladder cancer. Cancer Res,2008.68(7):p.2530-7.
76. Jazdzewski, K., et al., Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma. Proc Natl Acad Sci U S A,2008.105(20):p.7269-74.
77. Horikawa, Y., et al., Single nucleotide polymorphisms of microRNA machinery genes modify the risk of renal cell carcinoma. Clin Cancer Res,2008.14(23): p.7956-62.
78. Landi, D., et al., Polymorphisms within micro-RNA-binding sites and risk of sporadic colorectal cancer. Carcinogenesis,2008.29(3):p.579-84.
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81. Liu, Z., et al., Genetic variants in selected pre-microRNA genes and the risk of squamous cell carcinoma of the head and neck. Cancer,2010.116(20):p. 4753-60.
82. Kirnbauer, R., et al., A virus-like particle enzyme-linked immunosorbent assay detects serum antibodies in a majority of women infected with human papillomavirus type 16. Journal of the National Cancer Institute,1994.86(7): p.494-9.
83. Dahlstrom, K.R., et al., Human papillomavirus type 16 infection and squamous cell carcinoma of the head and neck in never-smokers:a matched pair analysis. Clin Cancer Res,2003.9(7):p.2620-6.
84. Chen, X., et al., Human papillomavirus seropositivity synergizes with MDM2 variants to increase the risk of oral squamous cell carcinoma. Cancer Res, 2010.70(18):p.7199-208.
85. Sonkoly, E., M. Stahle, and A. Pivarcsi, MicroRNAs and immunity:novel players in the regulation of normal immune function and inflammation. Semin Cancer Biol,2008.18(2):p.131-40.
86. Hebert, C., et al., High mobility group A2 is a target for miRNA-98 in head and neck squamous cell carcinoma. Mol Cancer,2007.6:p.5.
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88. Sonkoly, E., et al., MicroRNAs:novel regulators involved in the pathogenesis of psoriasis? PLoS One,2007.2(7):p. e610.
89. Lynam-Lennon, N., S.G. Maher, and J.V. Reynolds, The roles of microRNA in cancer and apoptosis. Biol Rev Camb Philos Soc,2009.84(1):p.55-71.
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54. Bentwich, I., et al., Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet,2005.37(7):p.766-70.
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56. Johnson, S.M., et al., RAS is regulated by the let-7 microRNA family. Cell, 2005.120(5):p.635-47.
57. Kloosterman, W.P. and R.H. Plasterk, The diverse functions of microRNAs in animal development and disease. Dev Cell,2006.11(4):p.441-50.
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60. Osada, H. and T. Takahashi, MicroRNAs in biological processes and carcinogenesis. Carcinogenesis,2007.28(1):p.2-12.
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62. Yanaihara, N., et al., Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell,2006.9(3):p.189-98.
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65. Chang, S.S., et al., MicroRNA alterations in head and neck squamous cell carcinoma. Int J Cancer,2008.123(12):p.2791-7.
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79. Hu, Z., et al., Common genetic variants in pre-microRNAs were associated with increased risk of breast cancer in Chinese women. Hum Mutat,2009. 30(1):p.79-84.
80. Christensen, B.C., et al., Mature microRNA sequence polymorphism in MIR196A2 is associated with risk and prognosis of head and neck cancer. Clin Cancer Res,2010.16(14):p.3713-20.
81. Liu, Z., et al., Genetic variants in selected pre-microRNA genes and the risk of squamous cell carcinoma of the head and neck. Cancer,2010.116(20):p. 4753-60.
82. Kirnbauer, R., et al., A virus-like particle enzyme-linked immunosorbent assay detects serum antibodies in a majority of women infected with human papillomavirus type 16. Journal of the National Cancer Institute,1994.86(7): p.494-9.
83. Dahlstrom, K.R., et al., Human papillomavirus type 16 infection and squamous cell carcinoma of the head and neck in never-smokers:a matched pair analysis. Clin Cancer Res,2003.9(7):p.2620-6.
84. Chen, X., et al., Human papillomavirus seropositivity synergizes with MDM2 variants to increase the risk of oral squamous cell carcinoma. Cancer Res, 2010.70(18):p.7199-208.
85. Sonkoly, E., M. Stahle, and A. Pivarcsi, MicroRNAs and immunity:novel players in the regulation of normal immune function and inflammation. Semin Cancer Biol,2008.18(2):p.131-40.
86. Hebert, C., et al., High mobility group A2 is a target for miRNA-98 in head and neck squamous cell carcinoma. Mol Cancer,2007.6:p.5.
87. Henson, B.J., et al., Decreased expression of miR-125b and miR-100 in oral cancer cells contributes to malignancy. Genes Chromosomes Cancer,2009. 48(7):p.569-82.
88. Sonkoly, E., et al., MicroRNAs:novel regulators involved in the pathogenesis of psoriasis? PLoS One,2007.2(7):p. e610.
89. Lynam-Lennon, N., S.G. Maher, and J.V. Reynolds, The roles of microRNA in cancer and apoptosis. Biol Rev Camb Philos Soc,2009.84(1):p.55-71.