论文部分内容阅读
目的构建人端粒酶逆转录酶(Human telomerase reverse transcriptase,hTERT)基因小干扰RNA(Small interferingRNA,siRNA)质粒,并探讨其对不同p53状态卵巢癌细胞SKOV3(缺p53)和OVCAR3(含p53)生长和凋亡的影响。方法设计并合成针对hTERT基因的siRNA,与表达的质粒pGenesil-1连接,构建重组表达质粒pG1、pG2和pGCR(阴性对照质粒),分别转染卵巢癌细胞SKOV3和OVCAR3,并设未转染细胞对照组和转染空载体对照组。转染后48 h,荧光显微镜观察并计算转染效率;实时荧光定量PCR法检测转染细胞中hTERT基因mRNA的表达;Western blot检测转染细胞中hTERT、p53和p21蛋白的表达;端粒重复序列扩增(Telomeric repeat amplification portocol,TRAP)法检测转染细胞端粒酶的活性;CCK-8法检测细胞生长情况;流式细胞仪和AnnexinⅤ-PE/7-AAD双染法分别检测细胞周期和细胞凋亡的情况。结果已成功构建了靶向hTERT siRNA重组质粒pG1、pG2和阴性对照质粒pGCR,具有较高的转染效率,与pGCR转染组相比,pG1和pG2转染组hTERT基因和蛋白表达(P<0.05)及端粒酶活性均明显降低,pG1和pG2转染组均可抑制两种细胞的生长,且对OVCAR3细胞的抑制作用强于SKOV3细胞;pG1和pG2转染组G0-G1期细胞比例明显下降,S期细胞比例明显增多(P<0.05);在OVCAR3细胞中,hTERT表达受抑后均出现了明显的凋亡,同时伴随p53和p21蛋白表达的增加,在缺乏p53基因的SKOV3细胞中并未导致明显的凋亡,其p21蛋白表达明显低于pGCR转染组(P<0.05)。结论构建的靶向hTERT siRNA重组表达质粒在体外能有效和特异地沉默卵巢癌细胞hTERT基因的表达,降低端粒酶活性,并引起肿瘤细胞的生长抑制和凋亡,其机制可能与抑癌基因p53及p21上调有关。
Objective To construct a small interfering RNA (siRNA) plasmid targeting human telomerase reverse transcriptase (hTERT) and to investigate its effect on the expression of SKOV3 (lacking p53) and OVCAR3 (including p53) Effects of growth and apoptosis. Methods siRNA targeting hTERT gene was designed and synthesized. The recombinant plasmid pGenesil-1 was designed and synthesized. The recombinant plasmids pG1, pG2 and pGCR (negative control plasmid) were constructed and transfected into SKOV3 and OVCAR3 ovarian cancer cells respectively. The untransfected cells Control group and empty vector control group. 48 h after transfection, the transfection efficiency was observed by fluorescence microscope and the transfection efficiency was calculated. The expression of hTERT mRNA was detected by real-time fluorescence quantitative PCR. The expression of hTERT, p53 and p21 proteins in transfected cells was detected by Western blot. The telomerase activity in transfected cells was detected by Telomeric repeat amplification protocol (TRAP). The cell growth was detected by CCK-8 assay. The cell cycle was detected by flow cytometry and AnnexinⅤ-PE / 7-AAD double staining And the situation of apoptosis. Results The recombinant plasmids pG1 and pG2 targeting hTERT siRNA and pGCR negative control plasmid were successfully constructed. Compared with pGCR transfection group, the expression of hTERT gene and protein in pG1 and pG2 transfection group was significantly higher than that in pGCR transfection group (P < 0.05) and telomerase activity were significantly decreased, both pG1 and pG2 transfected cells inhibited the growth of both cells, and the inhibition of OVCAR3 cells stronger than SKOV3 cells; pG1 and pG2 transfected G0-G1 phase cell ratio (P <0.05). In OVCAR3 cells, the expression of hTERT was significantly inhibited, accompanied by the increase of p53 and p21 protein expression, in the absence of p53 gene in SKOV3 cells Did not result in obvious apoptosis, the p21 protein expression was significantly lower than pGCR transfection group (P <0.05). CONCLUSION: The recombinant plasmid targeting hTERT siRNA can effectively and specifically silence the expression of hTERT gene in vitro, reduce the telomerase activity, and induce the growth inhibition and apoptosis of tumor cells. The mechanism may be related to tumor suppressor gene p53 and p21 upregulation.