【作者】 王建；

【导师】 任常山；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2008， 博士

【摘要】 目的端粒酶逆转录酶（hTERT）是端粒酶的催化亚基,能够以端粒酶RNA为模板逆转录合成端粒DNA。它的表达与端粒酶活性呈正相关,限于生殖细胞、干细胞和90%以上的恶性肿瘤细胞,而在大多数正常人体细胞没有表达。由于hTERT与肿瘤的发生、发展关系密切,近年来成为研究的热点。随着研究的深入,发现hTERT的功能不仅仅是合成端粒,在抵抗细胞凋亡、促进细胞增殖等方面也发挥重要作用。为了进一步研究hTERT新的生物学功能,探讨其与肿瘤相关的作用机制,本文应用RNA干扰技术进行如下研究:第一部分,化学合成siRNA-hTERT,观察其在HeLa细胞中抑制hTERT的表达情况,筛选出有效的干扰序列;第二部分,检测hTERT基因沉默后HeLa细胞中癌症相关基因表达的变化;第三部分,研究hTERT基因沉默诱导HeLa细胞凋亡的途径。方法第一部分,实验分8组:A组为空白对照组,细胞不加处理因素;B组为转染试剂对照组,只加转染试剂;C组为阴性对照组,转染非特异siRNA;S_1～5组为siRNA-hTERT组,转染特异siRNA。根据hTERT基因序列及其二级结构,依据Tuschl等siRNA设计原则,设计合成5条针对hTERT基因的siRNA;利用专用脂质体转染试剂RNAi-Mate将siRNA转染至HeLa细胞中,采用半定量RT-PCR法和Western blot筛选有效的干扰序列;MTT法检测siRNA对细胞生长增殖的抑制效应。第二部分,按Trizol法分别抽提S₃组、S₄组和空白组细胞总RNA,并进一步纯化总RNA,应用琼脂糖凝胶电泳判断评价总RNA的质量,分离mRNA;参照Schena等的方法逆转录cDNA探针并标记mRNA,分别用Cy5-dUTP和Cy3-dUTP标记不同组的mRNA;将含混合探针的杂交液与芯片变性后,将杂交液滴于芯片点样区,用盖玻片覆盖,置于杂交盒中,杂交过夜;使用激光共聚焦荧光扫描仪扫描芯片,用ImaGene软件分析数据;采用Western blot法验证差异表达基因。第三部分,实验分5组:空白对照组,转染试剂对照组,非特异阴性对照组,S₃、S₄特异干扰组。转染后48小时,流式细胞仪检测各组细胞凋亡率;转染后48小时比色法测定各组Caspase-3和Caspase-8的活性;转染后48小时,提取各组细胞胞浆蛋白,Western blot法检测各组细胞胞浆Cyt c含量。结果第一部分,RNAi-Mate能有效的将siRNA转染至细胞内,各特异性siRNA转染组hTERT mRNA表达水平有不同程度的下降,以S₃、S₄转染组最为显著,而在各对照组内hTERT mRNA水平无明显变化;Western blot显示的蛋白表达变化与mRNA变化趋势相一致;MTT结果显示,转染24后,两个特异干扰组S₃、S₄与阴性对照组比较,细胞增殖抑制率均显著增高（P＜0.01）,两个特异干扰组之间比较无显著性差异（P＞0.05）。第二部分,芯片分析结果显示,在干扰组与对照组之间的差异表达基因中,EGFR、VEGF、bcl-2三个基因表达下调,TRAIL基因表达上调;Western blot结果显示,各基因的蛋白水平变化与芯片筛选结果一致。第三部分,运用流式细胞术进行细胞凋亡率分析显示,各对照组之间的细胞凋亡率差异无统计学意义（P＞0.05）,而两个干扰组与对照组比较,凋亡率均显著增高（P＜0.01）,两个干扰组之间比较无显著性差异（P＞0.05）;转染后48小时,各组的caspase-8活性比无显著性差异（P＞0.05）,而两个干扰组的caspase-3活性比与对照组比较差异有显著性（P＜0.01）,均表现为活性比增高;Western blot结果显示,两个干扰组的胞浆Cyt c蛋白含量较对照组也有显著的增高。结论1、化学合成的靶向hTERT的siRNA能有效抑制HeLa细胞中hTERT的表达,并能抑制HeLa细胞增殖。2、靶向hTERT的siRNA能下调EGFR、VEGF、bcl-2基因的表达,上调TRAIL基因表达。3、靶向hTERT的siRNA通过激活线粒体途径诱导HeLa细胞凋亡。更多还原

【Abstract】 ObjectiveThe human telomerase reverse transcriptase（hTERT） is the catalytically active component of the telomerase complex.hTERT catalyzes the telomere elongation by reverse transcription,using RNA component of telomerase as template.Its expression correlates with telomerase activity and is restricted to germ cells,stem cells and to more than 90%of human cancers,whereas most normal human somatic cells have no hTERT expression.As close relations with the development and progression of many malignant tumors,telomerase and hTERT were in the spotlight of research.With research going deeply,some new phenomenas and results were revealed and indicated that telomerase has important functions in anti-apoptosis and cell proliferation.To further study the new biological functions of hTERT,The main contents of this research are as following:The first part:To investigate the efficency of construct the small interferening RNA（siRNA）of suppress the expression of hTERT in HeLa cell line after knocked down hTERT gene by siRNA synthesized in chemosynthesis;The second part:To study the effects of silencing of hTERT on transcriptional profile of cancer associated genes in HeLa cells;The third part:To investigate the molecular mechanism of induction of apoptosis by siRNA targeting hTERT in HeLa Cells.MethodsThe first part:Five double-stranded siRNAs targeting coding of hTERT gene were designed by full length gene targeting technique or selected randomly.RNAi-Mate mediated transient transfection was conducted to transmit the siRNA into HeLa cells.Five pairs of specific targeted sequence were selected in the coding region of hTERT gene.mRNA and protein levels of hTERT were detected by reverse transcription-polymerase chain reaction（RT-PCR） and Western blot.Proliferation inhibition rate of HeLa cells was analyzed by MTT assay.The second part:Total RNA were extraeted from untreated control group,S₃ group and S₄ group,respectively,with Trizol one-step method,RNAs were further purified with QIANGEN Rneasy Kit.Agarose gel electrophoresis was used to analyze the intensity ratio between 28s and 18s to evaluate the quality of total RNA.cDNA probes were reversely transeribed from mRNA according to the methods bySehena et al.Cy5-dUTP and Cy3-dUTP were used to label mRNA probes from differeni tissues.After the degeneration of hybridization soluteion of mixed probes and microarray,hybridization soluteion was dropped onto the sampling area of microarray,covered with a cover sliP,placed into the hybridization cabin,sealed with parafilm,and then hybridized in 42℃water bath for 16h.The microarrays were scanned with confocal fluorescence scanner and the data was analyzed by ImaGene software to plot the value of Cy3 and Cy5.The third part:To detect apoptosis rate by flow cytometer after transfection for 48h;Measurement of relative Caspase-3 and Caspase-8 activity by colorimetric assay;Cytoplasm Cyt c were detected by Western blotting.ResultsThe first part:Sequence specific siRNAs targeting hTERT down-regulated mRNA expression significantly,protein expression level was decreased after transfection of siRNA-hTERT by means of western blot.After treatment of siRNA,proliferation inhibition rate of HeLa cells were increased（P＜0.01）.The second part:The hybridized microarray was scanned.The analysis for the date revealed that there were 3 down-regulated gene（EGFR,VEGF and bcl-2） and 1 up-regulated gene（TRAIL）. Wester blot analysis demonstrated that the expression change of bcl-2,EGFR,VEGF and TRAIL was in accord with chip analytic result.The third part:After transfection for 48h,annexin-V and PI double-staining FCM analysis showed that the cell apoptosis rate in two RNAi-hTERT groups was noticeably higher than in three control groups（P＜0.01）,and there was no significant difference on the cell apoptosis rate between three control groups（P＞0.05）.After transfection for 48h,the difference of Caspase-8 enzymatic activity ratio between each groups was of no significance（P＞0.05）,but the Caspase-3 enzymatic activity ratio in two RNAi-hTERT groups was noticeably higher than in three control groups（P＜0.01）.Wester blot analysis demonstrated that the level of cytosolic Cyt c was significantly increased after RNAi-hTERT.Conclusion1.Specific siRNA targeting hTERT could effectively inhibit hTERT expression and HeLa cell proliferation.2.Specific siRNA targeting hTERT could up-regulate the expression of TRAIL,and down-regulate the expression of EGFR,VEGF and bcl-2.3.Specific siRNA targeting hTERT induces apoptosis of HeLa cells via activating mitochondrial signal transduction pathway.更多还原