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目的检测人端粒酶逆转录酶(Human telomerase reverse transcriptase,hTERT)基因沉默对卵巢癌细胞株A2780生长抑制和凋亡的影响,并探讨其与p53、p21基因表达激活的相关性。方法设计合成针对hTERT基因的shRNA干扰质粒,与可表达荧光蛋白的pGenesil-1.1质粒连接,构建重组质粒pG1、pG2和阴性质粒pGCR,转染A2780细胞,采用荧光定量RT-PCR检测转染细胞hTERT基因mRNA的表达,Western blot检测细胞hTERT、P53和P21蛋白的表达,TRAP法检测细胞的端粒酶活性,CCK-8法检测细胞的生长,流式细胞术分析细胞周期,AnnexinⅤ-PE/7AAD双染和TUNEL法检测细胞凋亡。结果酶切及测序结果证实重组质粒pG1、pG2和pGCR构建正确,质粒pG1和pG2转染可明显下调A2780细胞hTERT的表达水平和端粒酶活性,而上调细胞P53和P21蛋白的表达水平;沉默hTERT基因表达能够引起A2780细胞的生长抑制和G0-G1期细胞比例明显下降,并出现明显的细胞凋亡。结论针对hTERT基因的shRNA干扰质粒在体外能有效和特异地沉默卵巢癌A2780细胞hTERT基因的表达,降低端粒酶活性,并引起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 investigate the effect of human telomerase reverse transcriptase (hTERT) gene silencing on the growth and apoptosis of ovarian cancer cell line A2780 and to explore its relationship with p53 and p21 gene expression. Methods shRNA interference plasmids targeting hTERT gene were designed and synthesized and ligated with pGenesil-1.1 plasmid, which can express fluorescent protein. The recombinant plasmids pG1 and pG2 were constructed and transfected into A2780 cells. Fluorescent quantitative RT-PCR was used to detect hTERT The mRNA expression of hTERT, P53 and P21 were detected by Western blot. The telomerase activity was detected by TRAP, the cell growth was detected by CCK-8, the cell cycle was analyzed by flow cytometry, AnnexinⅤ-PE / 7AAD Double staining and TUNEL assay for apoptosis. Results Restriction endonuclease digestion and sequencing confirmed that recombinant plasmids pG1, pG2 and pGCR were constructed correctly. Plasmid pG1 and pG2 transfection could significantly down-regulate the expression of hTERT and telomerase in A2780 cells, but up-regulate the expression of P53 and P21 protein. hTERT gene expression can cause A2780 cell growth inhibition and G0-G1 phase cells decreased significantly, and significant apoptosis. Conclusion shRNA interference plasmid targeting hTERT gene can effectively and specifically silence the expression of hTERT gene in ovarian cancer A2780 cells in vitro and reduce the activity of telomerase and cause cell growth inhibition and apoptosis. The mechanism may be related to the expression of tumor suppressor gene p53 and p21 related to the increase.