XIAP在甲状腺乳头状癌中的表达及基因沉默对细胞生物学行为的影响
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摘要
近年来,甲状腺癌的发病率呈上升趋势,以乳头状癌的升高最为明显。甲状腺乳头状癌是最常见的甲状腺恶性肿瘤,约占所有甲状腺恶性肿瘤的60-80%,占全身恶性肿瘤的1.0-1.5%。甲状腺乳头状癌存在增殖失控、凋亡受阻等生物学特性,且侵袭能力较强,淋巴结转移率高,术后复发率较高,二次手术效果不佳,目前尚缺乏内科及生物学治疗方法。
X-连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)是凋亡抑制蛋白(inhibitor of apoptosis protein,IAP)家族最重要的成员之一,它被认为是家族中最具有特征性及功能最强的内源性Caspase抑制因子,在生理学上是细胞死亡的关键性调节因子。研究显示,XIAP在正常的组织中呈低表达或不表达状态,而在多种恶性肿瘤中高表达,如在肺癌、胸腺癌、胰腺癌、宫颈癌,白血病中高表达,在卵巢癌也有许多相关报告。体外试验及动物实验均研究表明,XIAP通过抑制细胞凋亡及促进细胞增殖、生存、迁移、侵袭及肿瘤血管形成等在肿瘤发生发展过程中发挥瘤基因的重要作用。由于XIAP在恶性肿瘤中过表达,而在正常的组织中低表达甚至不表达,且最新研究显示敲除正常大鼠XIAP基因后大鼠未发现明显病理变化,提示敲除XIAP可能对正常细胞无毒性。基于以上特性,XIAP基因被认为是具有潜力的肿瘤治疗靶点,已成为肿瘤治疗和药物开发的热门靶基因。
RNAi是近几年发展起来的新技术,是外源性和内源性双链RNA在生物体内诱导同源靶基因的mRNA特异性降解,导致转录后基因沉默的现象。RNAi具有高度序列特异性、抑制基因表达的高效性、稳定性及可传播性。目前常用的RNAi实验方法由直接转染双链siRNA及载体(质粒或病毒)表达发卡RNA两种,由于慢病毒载体能转染分裂期与非分裂期细胞,具有转染率高,基因抑制表达作用强,持续时间长等特性,且病毒的遗传物质能整合到宿主的基因组。通过慢病毒载体介导的RNAi,使长效抑制目的基因表达成为可能。
尽管目前已经证明XIAP与某些恶性肿瘤发生发展存在密切相关,而且也展现出作为肿瘤治疗靶点的潜力,但目前有关甲状腺乳头状癌与XIAP之间的文献较少,为此我们研究XIAP在甲状腺乳头状癌中的表达情况及用RNAi对其细胞生物行为的影响,以期对甲状腺乳头状癌提出新的生物学治疗策略。
第一部分XIAP在甲状腺乳头状癌中的表达及意义
1.目的
(1)检测XIAP在甲状腺乳头状癌、甲状腺腺瘤及正常甲状腺组织中的表达
(2)研究XIAP的表达与甲状腺乳头状癌临床病理参数的关系
2.材料与方法
(1)收集病例资料:收集本院接受手术并病理证实甲状腺乳头状癌、甲状腺腺瘤及甲状腺正常组织标本,详细记录病人资料。
(2)研究XIAP在甲状腺乳头状癌、甲状腺腺瘤及甲状腺正常组织的表达:免疫组织化学染色法。
3.结果
(1)XIAP在甲状腺乳头状癌组织中是高表达的,81.6%(40/49)标本为阳性表达。
(2)对49例甲状腺乳头状癌患者的临床资料进行卡方检验及t检验,研究XIAP与临床病理参数之间的关系。男性患者中XIAP阳性率为76.9%(10/13),女性患者中XIAP阳性率为80.6%(29/36),两组之间无显著性差异(P=0.780)。XIAP阳性表达患者年龄为46.18±15.24岁, XIAP阴性表达患者年龄为43.22±11.35岁,两组之间无显著性差异(P=0.520)。术前发生淋巴结转移XIAP阳性表达率为86.7%(13/15),术前未发生淋巴结转移XIAP阳性表达率为79.4% (27/34),两组之间不存在显著性差异(P=0.546)。
XIAP阳性表达患者肿瘤直径为1.98±0.59厘米, XIAP阴性表达患者肿瘤直径为1.53±0.52厘米,两组之间有显著性差异(P=0.041)。术前发生甲状腺外浸润XIAP阳性表达率为95.4%(21/22),术前未发生甲状腺外浸润XIAP阳性表达率为70.4%(19/27),两组之间存在显著性差异(P=0.024)。处于疾病早期(Early stage,I+II期)的甲状腺乳头状癌患者中XIAP阳性率为69.6%(16/23),进展期(Advanced stage,III+IV期)甲状腺乳头状癌患者中XIAP阳性率为92.3% (24/26),两组相比有显著性差异(P=0.040),进展期XIAP表达水平高于早期。经典型的甲状腺乳头状癌患者中XIAP阳性率为63.2%(12/19),滤泡型的甲状腺乳头状癌患者中XIAP阳性率为92.8%(13/14),其它型的甲状腺乳头状癌患者中XIAP阳性率为93.7%(15/16),其中经典型与滤泡型、其它型相比有显著性差异(P分别为0.045、0.031),经典型XIAP阳性率低于滤泡型及其它型;而滤泡型、其它型患者XIAP阳性率无显著性差异(P=0.922)。(3) Spearman相关性分析:XIAP与肿瘤直径(r=0.328,P=0.031)、甲状腺外浸润(r=0.317,P=0.037)、临床分期(r=0.289,P=0.046)呈正相关关系。
4.结论
(1) XIAP在甲状腺乳头状癌组织中是高表达的,81.6%(40/49)标本为阳性表达。
(2) XIAP的表达在不同肿瘤大小、是否浸润、临床分期、病理类型的患者之间有显著性差异,而在不同的性别、不同年龄的患者、是否转移无显著性差异。
(3) Spearman相关性分析,XIAP与肿瘤大小、浸润、临床分期呈正相关。
第二部分慢病毒介导的甲状腺乳头状癌细胞XIAP基因沉默
1.目的
(1)构建针对XIAP慢病毒介导的干扰载体,并生产高滴度慢病毒颗粒。
(2)观察慢病毒转染甲状腺乳头状癌细胞株后XIAP的表达情况,从而筛选出最有效的shRNA。
2.材料与方法
(1)针对XIAP慢病毒介导的干扰载体的构建:采用吉凯基因公司的慢病毒载体系统,构建针对目的基因的RNA干扰慢病毒载体。
(2) RNA干扰慢病毒的生产及滴度测定:三种质粒共转染293T细胞生产病毒,使用梯度稀释法测定滴度。
(3)使用Real-time PCR和Western Blot筛选高效干扰序列。
3.结果:
(1)成功构建针对XIAP慢病毒介导的干扰载体。
(2)包装生产了浓度为2×108TU/ml慢病毒颗粒,K1及CGTH W3的MOI值分别为10和20。
(3)成功筛选出高效干扰序列,在K1细胞系中,XIAP mRNA水平降低79%,蛋白水平降低了87%;在CGTH W3细胞系中,XIAP mRNA水平降低了72%,蛋白水平降低了76%。
4.结论
(1)成功构建XIAP特异的RNAi慢病毒表达质粒载体。
(2)包装并生产高滴度的慢病毒颗粒。
(3)利用Real-Time PCR和Western Blot筛选高效干扰序列,经慢病毒转染后XIAP基因mRNA和蛋白水平显著被抑制。
第三部分XIAP基因沉默对甲状腺乳头状癌生物学行为影响
1.目的
(1)观察XIAP基因沉默对细胞增殖的影响
(2)分析XIAP基因沉默对细胞周期的影响
(3)探讨XIAP基因沉默对细胞凋亡的影响
(4)研究XIAP基因沉默对细胞侵袭的影响
2.材料与方法
(1)细胞增殖实验:采用MTT法评价XIAP基因沉默后对细胞增殖的影响。
(2)细胞周期分析:利用流式细胞术测定细胞DNA含量,Modtif软件分析细胞周期。
(3)细胞凋亡实验:采用Annexin-IV和PI双染流式细胞仪分析XIAP基因沉默后对细胞凋亡的影响,并用Western blot检测细胞凋亡相关蛋白及Real-time PCR检测细胞凋亡相关基因的表达情况。
(4)细胞侵袭能力实验:利用Transwell实验评价XIAP基因沉默后细胞的侵袭能力。
3.结果
(1) XIAP基因沉默细胞增殖分别降低51%、43%,明显低于未干预组;阴性对照慢病毒感染组的细胞增殖未受明显影响。
(2) XIAP基因沉默细胞G2/M期分别增加18.4%、14.1%,与未干预组及阴性对照慢病毒感染组相比有显著性差异。
(3) XIAP基因沉默细胞凋亡率分别增加14.2%、8.4%,与未干预组及阴性对照慢病毒感染组相比有显著性差异。
(4) XIAP基因沉默细胞穿膜能力分别降低50.7%、44.9%,明显低于未干预组及阴性对照慢病毒感染组。
4.结论
(1)敲除XIAP基因可抑制甲状腺乳头状癌细胞增殖,使其滞留在G2/M期。
(2)沉默XIAP基因可促进甲状腺乳头状癌细胞凋亡。
(3)静默XIAP基因可抑制甲状腺乳头状癌细胞侵袭。
The incidence of thyroid carcinoma has risen especially Papillary thyroid carcinoma in recent years. Papillary thyroid carcinoma is the most common type of thyroid carcinoma, it accounts for 60-80% of thyroid malignancies and 1-2% of all human malignant tumor. There are biological characters present in papillary thyroid carcinoma such as outcontrol of proliferation and apoptosis arrest, strong capability of invasion, high incidence of lymph nodes metastasis, high recurrence rate after operation, unfavorable result after second surgery, and lack of internal and biological therapy approaches generally.
XIAP(X-linked inhibitor of apoptosis protein) is one of the most important members in IAP family, and is well known as hallmark of the family, it also has been recognized as the strongest endogenous caspase suppress factor, key regulate factor of cell death in physiology. The Studies have shown that low or not XIAP expression in normal tissue, but in many malignant neoplasms, such as lung cancer, thymus cancer, pancreatic cancer, cervical cancer, leukemia and oophoroma, have over expression. The experiments in vitro and animal have pointed out XIAP play a tumor gene role in tumor development and progress through suppression of cell apoptosis and promotions of cell proliferation, survival, migration, invasion and tumor vessel formation. Due to the over expression in malignancies and low even not expression in normal human tissue, and the latest research demonstrates mice XIAP-deficient mice do not have any obvious pathology defects that prompt maybe knockout of XIAP has no shortcoming to do with normal cells. On account of all these characteristics above, XIAP gene is considered as a potential oncotherapy target point and has become a popular target gene in tumor therapy and pharmaprojects.
RNA interference (RNAi) is a new technique which has been developed in recent years, it can be defined as a phenomenon in which ectogenesis and endogenous double strand RNA (dsRNA) degrade the homologous gene mRNA, then lead to gene silence after transcription. There are many characters in RNA interference, such as sequence specificity, high performance of gene express suppression, stability and transmissibility. There are two RNA interference experiment methods used commonly: direct transfection double strand siRNA and vector (plasmid and virus) express hairpin RNA. Lentivirus can be used in transfecting dividing cells or non-dividing cells, and has the characters such as high transfection efficiency, strong suppression of gene expression, long time duration, it also can cassette hereditary substance into genome of host cells.It becomes possible of prolonged action suppresses target gene expression through lentivirus vector mediated RNA interference.
In spite of the proof of the intimate relationship between XIAP and some malignant tumors’development and progression, also XIAP presents potential as a tumor therapy target, but there are few documents about the relationship between XIAP and papillary thyroid carcinoma right now. With regards to all reasons above, we study the expression of XIAP in papillary thyroid carcinoma and the cell biological effect mediated by RNA interference, and expecting to bring up a new biology therapy strategy for papillary thyroid carcinoma.
Chapter 1 Overexpression of X-linked inhibitor of apoptosis protein in papillary thyroid carcinoma
1. Aim
(1) To detect the expression of XIAP in papillary tyroid carcinoma, thyroid adenoma and normal thyroid tissue
(2) To analysis the relationship between XIAP and clinical parameters
2. Materials and Methods
(1) Case data collection: this study was conducted on a total 49 paraffin embeded papillary thyroid carcinoma samples, 33 thyroid adenoma and 25 normal thyroid tissue samples, which were diagnosed by pathology department at our hospital.
(2) Immunohistochemistry for XIAP expression using a rabbit monoclonal anti- XIAP antibody.
3. Results
(1) XIAP expression was strongly positive in 40 of 49 papillary thyroid carcinoma samples(81.6%).
(2) We analysed the clinical data of papillary thyroid carcinoma patients by Chi-Square test and t test. The XIAP expression of male was 76.9%(10/13),and among female patients was 80.6%(29/36), Compared to the group with positive XIAP expression, XIAP expression was not associated with gender(p=0.780). The ages of positive XIAP expression was 46.18±15.2 years, and the ages of negative XIAP expression was 43.22±11.35 years, compared to the group with positive XIAP expression, XIAP expression was not associated with age(p=0.520). XIAP expression of metastatic samples was 86.7%, and the expression of non metastatic samples was 79.4%, there was not significant difference between two groups(p=0.546).The diameters of positive XIAP expression was 1.98±0.59cm, and the diameters of negative XIAP expression was 1.53±0.52cm, compared to the group with positive XIAP expression, XIAP expression was significantly associated with tumor diameter(p=0.041). XIAP expression of extrathyroid invasion samples was 95.4%(21/22), and the expression with non invasion samples was 70.4%(19/27), there is significant difference between two groups(p=0.024). XIAP expression was 69.6%(16/23) among early stage patients, and 92.3%(24/26) among advanced stage patients, there was significant difference between two groups(p=0.040). XIAP expression was 63.2%(12/19) among classical histology, 92.8%(13/14) among follicular variant, and 93.7%(15/16) among others histology classification, there was significant difference among group classic variant and group follicular and others(p=0.045, 0.031).
(3) Positive expression of XIAP was associated with tumor diameters(r=0.328, P=0.031), invasion(r=0.317, P=0.037), clinical stage(r=0.289, P=0.046), while not age, gender, metastasis and histology classification by spearman correlation analysis.
4. Conclusions
(1) XIAP expression was strongly positive in 40 of 49 papillary thyroid carcinoma samples(81.6%).
(2) XIAP expression was significantly associated with the tumor diameters invasion, clinical stage and histology classification. In contrast,no correlation was found between gender or age at the expression level of XIAP protein of papillary thyroid cacinoma patients.
(3) Positive expression of XIAP was associated with tumor diameters, invasion, clinical stage, while not with gender, age, metastasis and and histology classification by spearman correlation analysis.
Chapter 2 Silencing of XIAP in papillary thyroid carcinoma mediated by lentivirus vector
1. Aim
(1) To construct XIAP RNAi lentivirus vector, and produced high titer lentivirus particals.
(2) Select the most effective shRNA through XIAP expression after transfection of papillary thyroid carcinoma cell line.
2. Materials and Methods
(1) XIAP RNAi lentiviral vectors were constructed by Genechem lentiviral RNAi Expression System
(2) XIAP RNAi lentiviral vectors were packaged by co-transfecting 293FT with 3 plasmids. The titer of lentivirus was measured by hole dilution system
(3) XIAP mRNA level was analyzed by real time RT-PCR, XIAP protein level was determined by Western Blot.
3. Result
(1) The lentivirus vector targeting XIAP was successfully constructed,.
(2) The concentration of lentivirus were 2×108TU/ml after packaged and produced, the MOI of K1 and CGTH W3 were 10 and 20,respectively.
(3) High performance interference sequence was selected, and after transfection, the mRNA level decreased 79%, the protein level decreased 87% in K1 cell line; and the mRNA level decreased 72%, the protein level decreased 76% in CGTH W3 cell line.
4. Conclusion
(1) The construction of lentivirus vectorand targeting XIAP was successful.
(2) The package and production of high titer lentivirus particle were successful.
(3) The selected siRNA sequence demonstrated significant silencing effect of XIAP at both mRNA and protein levels.
Chapter 3 Effects of XIAP gene silence on biological behavior of papillary thyroid carcinoma cells
1. Aim
(1) To measure the function of XIAP in proliferation of papillary thyroid carcinoma cell lines
(2) To determine the function of XIAP in cell cycle of papillary thyroid carcinoma cell lines
(3) To analyse the function of XIAP in apoptosis of papillary thyroid carcinoma cell lines
(4) To elucidate the function of XIAP in invasion of papillary thyroid carcinoma cell lines
2. Materials and Methods
(1) Proliferation of papillary thyroid carcinoma was measured by MTT
(2) Cell cycle analysis is performed by flow cytometry.
(3)Apoptosis of cells were determined by flow cytometry with Annexin-V and PI Staining, and we also Western blotting and Real time PCR to test the protein and RNA level which is relevant to apoptosis
(4) Invasion of cells were tested by Transwell methods,
3. Results
(1) The knockdown of XIAP has reduced the proliferation level, 51% and 43%, respectively.
(2) After XIAP knockdown, about increased 18.4% and 14.1% of cells were at G2/M phase.
(3) XIAP silencing could increase the level of apoptosis, 14.2% and 8.4%, respectively.
(4) Knockdown of XIAP decreased the number of invaded cells about 50.7% and 44.9%, respectively.
4. Conclusion
(1) The knockdown of XIAP could restrain the proliferation of papillary thyroid carcinoma cells, and made it detained at G2/M phase.
(2) The silence of XIAP could promote the apoptosis of papillary thyroid carcinoma.
(3) The silence of XIAP could suppress the invasion of papillary thyroid carcinoma.
X-连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)是凋亡抑制蛋白(inhibitor of apoptosis protein,IAP)家族最重要的成员之一,它被认为是家族中最具有特征性及功能最强的内源性Caspase抑制因子,在生理学上是细胞死亡的关键性调节因子。研究显示,XIAP在正常的组织中呈低表达或不表达状态,而在多种恶性肿瘤中高表达,如在肺癌、胸腺癌、胰腺癌、宫颈癌,白血病中高表达,在卵巢癌也有许多相关报告。体外试验及动物实验均研究表明,XIAP通过抑制细胞凋亡及促进细胞增殖、生存、迁移、侵袭及肿瘤血管形成等在肿瘤发生发展过程中发挥瘤基因的重要作用。由于XIAP在恶性肿瘤中过表达,而在正常的组织中低表达甚至不表达,且最新研究显示敲除正常大鼠XIAP基因后大鼠未发现明显病理变化,提示敲除XIAP可能对正常细胞无毒性。基于以上特性,XIAP基因被认为是具有潜力的肿瘤治疗靶点,已成为肿瘤治疗和药物开发的热门靶基因。
RNAi是近几年发展起来的新技术,是外源性和内源性双链RNA在生物体内诱导同源靶基因的mRNA特异性降解,导致转录后基因沉默的现象。RNAi具有高度序列特异性、抑制基因表达的高效性、稳定性及可传播性。目前常用的RNAi实验方法由直接转染双链siRNA及载体(质粒或病毒)表达发卡RNA两种,由于慢病毒载体能转染分裂期与非分裂期细胞,具有转染率高,基因抑制表达作用强,持续时间长等特性,且病毒的遗传物质能整合到宿主的基因组。通过慢病毒载体介导的RNAi,使长效抑制目的基因表达成为可能。
尽管目前已经证明XIAP与某些恶性肿瘤发生发展存在密切相关,而且也展现出作为肿瘤治疗靶点的潜力,但目前有关甲状腺乳头状癌与XIAP之间的文献较少,为此我们研究XIAP在甲状腺乳头状癌中的表达情况及用RNAi对其细胞生物行为的影响,以期对甲状腺乳头状癌提出新的生物学治疗策略。
第一部分XIAP在甲状腺乳头状癌中的表达及意义
1.目的
(1)检测XIAP在甲状腺乳头状癌、甲状腺腺瘤及正常甲状腺组织中的表达
(2)研究XIAP的表达与甲状腺乳头状癌临床病理参数的关系
2.材料与方法
(1)收集病例资料:收集本院接受手术并病理证实甲状腺乳头状癌、甲状腺腺瘤及甲状腺正常组织标本,详细记录病人资料。
(2)研究XIAP在甲状腺乳头状癌、甲状腺腺瘤及甲状腺正常组织的表达:免疫组织化学染色法。
3.结果
(1)XIAP在甲状腺乳头状癌组织中是高表达的,81.6%(40/49)标本为阳性表达。
(2)对49例甲状腺乳头状癌患者的临床资料进行卡方检验及t检验,研究XIAP与临床病理参数之间的关系。男性患者中XIAP阳性率为76.9%(10/13),女性患者中XIAP阳性率为80.6%(29/36),两组之间无显著性差异(P=0.780)。XIAP阳性表达患者年龄为46.18±15.24岁, XIAP阴性表达患者年龄为43.22±11.35岁,两组之间无显著性差异(P=0.520)。术前发生淋巴结转移XIAP阳性表达率为86.7%(13/15),术前未发生淋巴结转移XIAP阳性表达率为79.4% (27/34),两组之间不存在显著性差异(P=0.546)。
XIAP阳性表达患者肿瘤直径为1.98±0.59厘米, XIAP阴性表达患者肿瘤直径为1.53±0.52厘米,两组之间有显著性差异(P=0.041)。术前发生甲状腺外浸润XIAP阳性表达率为95.4%(21/22),术前未发生甲状腺外浸润XIAP阳性表达率为70.4%(19/27),两组之间存在显著性差异(P=0.024)。处于疾病早期(Early stage,I+II期)的甲状腺乳头状癌患者中XIAP阳性率为69.6%(16/23),进展期(Advanced stage,III+IV期)甲状腺乳头状癌患者中XIAP阳性率为92.3% (24/26),两组相比有显著性差异(P=0.040),进展期XIAP表达水平高于早期。经典型的甲状腺乳头状癌患者中XIAP阳性率为63.2%(12/19),滤泡型的甲状腺乳头状癌患者中XIAP阳性率为92.8%(13/14),其它型的甲状腺乳头状癌患者中XIAP阳性率为93.7%(15/16),其中经典型与滤泡型、其它型相比有显著性差异(P分别为0.045、0.031),经典型XIAP阳性率低于滤泡型及其它型;而滤泡型、其它型患者XIAP阳性率无显著性差异(P=0.922)。(3) Spearman相关性分析:XIAP与肿瘤直径(r=0.328,P=0.031)、甲状腺外浸润(r=0.317,P=0.037)、临床分期(r=0.289,P=0.046)呈正相关关系。
4.结论
(1) XIAP在甲状腺乳头状癌组织中是高表达的,81.6%(40/49)标本为阳性表达。
(2) XIAP的表达在不同肿瘤大小、是否浸润、临床分期、病理类型的患者之间有显著性差异,而在不同的性别、不同年龄的患者、是否转移无显著性差异。
(3) Spearman相关性分析,XIAP与肿瘤大小、浸润、临床分期呈正相关。
第二部分慢病毒介导的甲状腺乳头状癌细胞XIAP基因沉默
1.目的
(1)构建针对XIAP慢病毒介导的干扰载体,并生产高滴度慢病毒颗粒。
(2)观察慢病毒转染甲状腺乳头状癌细胞株后XIAP的表达情况,从而筛选出最有效的shRNA。
2.材料与方法
(1)针对XIAP慢病毒介导的干扰载体的构建:采用吉凯基因公司的慢病毒载体系统,构建针对目的基因的RNA干扰慢病毒载体。
(2) RNA干扰慢病毒的生产及滴度测定:三种质粒共转染293T细胞生产病毒,使用梯度稀释法测定滴度。
(3)使用Real-time PCR和Western Blot筛选高效干扰序列。
3.结果:
(1)成功构建针对XIAP慢病毒介导的干扰载体。
(2)包装生产了浓度为2×108TU/ml慢病毒颗粒,K1及CGTH W3的MOI值分别为10和20。
(3)成功筛选出高效干扰序列,在K1细胞系中,XIAP mRNA水平降低79%,蛋白水平降低了87%;在CGTH W3细胞系中,XIAP mRNA水平降低了72%,蛋白水平降低了76%。
4.结论
(1)成功构建XIAP特异的RNAi慢病毒表达质粒载体。
(2)包装并生产高滴度的慢病毒颗粒。
(3)利用Real-Time PCR和Western Blot筛选高效干扰序列,经慢病毒转染后XIAP基因mRNA和蛋白水平显著被抑制。
第三部分XIAP基因沉默对甲状腺乳头状癌生物学行为影响
1.目的
(1)观察XIAP基因沉默对细胞增殖的影响
(2)分析XIAP基因沉默对细胞周期的影响
(3)探讨XIAP基因沉默对细胞凋亡的影响
(4)研究XIAP基因沉默对细胞侵袭的影响
2.材料与方法
(1)细胞增殖实验:采用MTT法评价XIAP基因沉默后对细胞增殖的影响。
(2)细胞周期分析:利用流式细胞术测定细胞DNA含量,Modtif软件分析细胞周期。
(3)细胞凋亡实验:采用Annexin-IV和PI双染流式细胞仪分析XIAP基因沉默后对细胞凋亡的影响,并用Western blot检测细胞凋亡相关蛋白及Real-time PCR检测细胞凋亡相关基因的表达情况。
(4)细胞侵袭能力实验:利用Transwell实验评价XIAP基因沉默后细胞的侵袭能力。
3.结果
(1) XIAP基因沉默细胞增殖分别降低51%、43%,明显低于未干预组;阴性对照慢病毒感染组的细胞增殖未受明显影响。
(2) XIAP基因沉默细胞G2/M期分别增加18.4%、14.1%,与未干预组及阴性对照慢病毒感染组相比有显著性差异。
(3) XIAP基因沉默细胞凋亡率分别增加14.2%、8.4%,与未干预组及阴性对照慢病毒感染组相比有显著性差异。
(4) XIAP基因沉默细胞穿膜能力分别降低50.7%、44.9%,明显低于未干预组及阴性对照慢病毒感染组。
4.结论
(1)敲除XIAP基因可抑制甲状腺乳头状癌细胞增殖,使其滞留在G2/M期。
(2)沉默XIAP基因可促进甲状腺乳头状癌细胞凋亡。
(3)静默XIAP基因可抑制甲状腺乳头状癌细胞侵袭。
The incidence of thyroid carcinoma has risen especially Papillary thyroid carcinoma in recent years. Papillary thyroid carcinoma is the most common type of thyroid carcinoma, it accounts for 60-80% of thyroid malignancies and 1-2% of all human malignant tumor. There are biological characters present in papillary thyroid carcinoma such as outcontrol of proliferation and apoptosis arrest, strong capability of invasion, high incidence of lymph nodes metastasis, high recurrence rate after operation, unfavorable result after second surgery, and lack of internal and biological therapy approaches generally.
XIAP(X-linked inhibitor of apoptosis protein) is one of the most important members in IAP family, and is well known as hallmark of the family, it also has been recognized as the strongest endogenous caspase suppress factor, key regulate factor of cell death in physiology. The Studies have shown that low or not XIAP expression in normal tissue, but in many malignant neoplasms, such as lung cancer, thymus cancer, pancreatic cancer, cervical cancer, leukemia and oophoroma, have over expression. The experiments in vitro and animal have pointed out XIAP play a tumor gene role in tumor development and progress through suppression of cell apoptosis and promotions of cell proliferation, survival, migration, invasion and tumor vessel formation. Due to the over expression in malignancies and low even not expression in normal human tissue, and the latest research demonstrates mice XIAP-deficient mice do not have any obvious pathology defects that prompt maybe knockout of XIAP has no shortcoming to do with normal cells. On account of all these characteristics above, XIAP gene is considered as a potential oncotherapy target point and has become a popular target gene in tumor therapy and pharmaprojects.
RNA interference (RNAi) is a new technique which has been developed in recent years, it can be defined as a phenomenon in which ectogenesis and endogenous double strand RNA (dsRNA) degrade the homologous gene mRNA, then lead to gene silence after transcription. There are many characters in RNA interference, such as sequence specificity, high performance of gene express suppression, stability and transmissibility. There are two RNA interference experiment methods used commonly: direct transfection double strand siRNA and vector (plasmid and virus) express hairpin RNA. Lentivirus can be used in transfecting dividing cells or non-dividing cells, and has the characters such as high transfection efficiency, strong suppression of gene expression, long time duration, it also can cassette hereditary substance into genome of host cells.It becomes possible of prolonged action suppresses target gene expression through lentivirus vector mediated RNA interference.
In spite of the proof of the intimate relationship between XIAP and some malignant tumors’development and progression, also XIAP presents potential as a tumor therapy target, but there are few documents about the relationship between XIAP and papillary thyroid carcinoma right now. With regards to all reasons above, we study the expression of XIAP in papillary thyroid carcinoma and the cell biological effect mediated by RNA interference, and expecting to bring up a new biology therapy strategy for papillary thyroid carcinoma.
Chapter 1 Overexpression of X-linked inhibitor of apoptosis protein in papillary thyroid carcinoma
1. Aim
(1) To detect the expression of XIAP in papillary tyroid carcinoma, thyroid adenoma and normal thyroid tissue
(2) To analysis the relationship between XIAP and clinical parameters
2. Materials and Methods
(1) Case data collection: this study was conducted on a total 49 paraffin embeded papillary thyroid carcinoma samples, 33 thyroid adenoma and 25 normal thyroid tissue samples, which were diagnosed by pathology department at our hospital.
(2) Immunohistochemistry for XIAP expression using a rabbit monoclonal anti- XIAP antibody.
3. Results
(1) XIAP expression was strongly positive in 40 of 49 papillary thyroid carcinoma samples(81.6%).
(2) We analysed the clinical data of papillary thyroid carcinoma patients by Chi-Square test and t test. The XIAP expression of male was 76.9%(10/13),and among female patients was 80.6%(29/36), Compared to the group with positive XIAP expression, XIAP expression was not associated with gender(p=0.780). The ages of positive XIAP expression was 46.18±15.2 years, and the ages of negative XIAP expression was 43.22±11.35 years, compared to the group with positive XIAP expression, XIAP expression was not associated with age(p=0.520). XIAP expression of metastatic samples was 86.7%, and the expression of non metastatic samples was 79.4%, there was not significant difference between two groups(p=0.546).The diameters of positive XIAP expression was 1.98±0.59cm, and the diameters of negative XIAP expression was 1.53±0.52cm, compared to the group with positive XIAP expression, XIAP expression was significantly associated with tumor diameter(p=0.041). XIAP expression of extrathyroid invasion samples was 95.4%(21/22), and the expression with non invasion samples was 70.4%(19/27), there is significant difference between two groups(p=0.024). XIAP expression was 69.6%(16/23) among early stage patients, and 92.3%(24/26) among advanced stage patients, there was significant difference between two groups(p=0.040). XIAP expression was 63.2%(12/19) among classical histology, 92.8%(13/14) among follicular variant, and 93.7%(15/16) among others histology classification, there was significant difference among group classic variant and group follicular and others(p=0.045, 0.031).
(3) Positive expression of XIAP was associated with tumor diameters(r=0.328, P=0.031), invasion(r=0.317, P=0.037), clinical stage(r=0.289, P=0.046), while not age, gender, metastasis and histology classification by spearman correlation analysis.
4. Conclusions
(1) XIAP expression was strongly positive in 40 of 49 papillary thyroid carcinoma samples(81.6%).
(2) XIAP expression was significantly associated with the tumor diameters invasion, clinical stage and histology classification. In contrast,no correlation was found between gender or age at the expression level of XIAP protein of papillary thyroid cacinoma patients.
(3) Positive expression of XIAP was associated with tumor diameters, invasion, clinical stage, while not with gender, age, metastasis and and histology classification by spearman correlation analysis.
Chapter 2 Silencing of XIAP in papillary thyroid carcinoma mediated by lentivirus vector
1. Aim
(1) To construct XIAP RNAi lentivirus vector, and produced high titer lentivirus particals.
(2) Select the most effective shRNA through XIAP expression after transfection of papillary thyroid carcinoma cell line.
2. Materials and Methods
(1) XIAP RNAi lentiviral vectors were constructed by Genechem lentiviral RNAi Expression System
(2) XIAP RNAi lentiviral vectors were packaged by co-transfecting 293FT with 3 plasmids. The titer of lentivirus was measured by hole dilution system
(3) XIAP mRNA level was analyzed by real time RT-PCR, XIAP protein level was determined by Western Blot.
3. Result
(1) The lentivirus vector targeting XIAP was successfully constructed,.
(2) The concentration of lentivirus were 2×108TU/ml after packaged and produced, the MOI of K1 and CGTH W3 were 10 and 20,respectively.
(3) High performance interference sequence was selected, and after transfection, the mRNA level decreased 79%, the protein level decreased 87% in K1 cell line; and the mRNA level decreased 72%, the protein level decreased 76% in CGTH W3 cell line.
4. Conclusion
(1) The construction of lentivirus vectorand targeting XIAP was successful.
(2) The package and production of high titer lentivirus particle were successful.
(3) The selected siRNA sequence demonstrated significant silencing effect of XIAP at both mRNA and protein levels.
Chapter 3 Effects of XIAP gene silence on biological behavior of papillary thyroid carcinoma cells
1. Aim
(1) To measure the function of XIAP in proliferation of papillary thyroid carcinoma cell lines
(2) To determine the function of XIAP in cell cycle of papillary thyroid carcinoma cell lines
(3) To analyse the function of XIAP in apoptosis of papillary thyroid carcinoma cell lines
(4) To elucidate the function of XIAP in invasion of papillary thyroid carcinoma cell lines
2. Materials and Methods
(1) Proliferation of papillary thyroid carcinoma was measured by MTT
(2) Cell cycle analysis is performed by flow cytometry.
(3)Apoptosis of cells were determined by flow cytometry with Annexin-V and PI Staining, and we also Western blotting and Real time PCR to test the protein and RNA level which is relevant to apoptosis
(4) Invasion of cells were tested by Transwell methods,
3. Results
(1) The knockdown of XIAP has reduced the proliferation level, 51% and 43%, respectively.
(2) After XIAP knockdown, about increased 18.4% and 14.1% of cells were at G2/M phase.
(3) XIAP silencing could increase the level of apoptosis, 14.2% and 8.4%, respectively.
(4) Knockdown of XIAP decreased the number of invaded cells about 50.7% and 44.9%, respectively.
4. Conclusion
(1) The knockdown of XIAP could restrain the proliferation of papillary thyroid carcinoma cells, and made it detained at G2/M phase.
(2) The silence of XIAP could promote the apoptosis of papillary thyroid carcinoma.
(3) The silence of XIAP could suppress the invasion of papillary thyroid carcinoma.
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