SiRNA沉默 HBx对 HepG2.2.15细胞人端粒酶逆转录酶基因表达的影响

目的探讨应用RNA干扰技术沉默HBx对HepG2．2．15细胞中hTERT基因表达的影响。方法（1）将pSIHBV／X质粒转染HepG2．2．15细胞，RT-PCR法评估沉默效率。（2）MTT法检测转染24h、48h、72h后细胞增殖情况。（3）Real-timePCR和Westernblot检测hTERT表达情况。结果测序结果显示pSIHBV／X质粒构建正确；RT-PCR检测HBVX基因的沉默效率为53．6％；MTT检测结果显示转染24h、48h、72h后H印G2．2．15细胞的增殖受抑制，与对照组比较差异有统计学意义（P〈0．05）；Real-timePCR和Western blot检测结果均显示，转染pSIHBV／X质粒后HepG2．2．15细胞中hTERT基因的mRNA水平和蛋白水平表达分别有不同程度的下调，与对照组比较差异有统计学意义（P〈0．05）。结论siRNA沉默HBx基因可抑制HepG2．2．15细胞中癌症标志基因hTERT的表达。

Effect of HB x siRNA on hTERT gene expression of human hepatocellular carcinoma cell HepG 2.2.15

Objective To observe the effect of silencing HBx by RNA interference on the expres- sion of hTERT in HepG2. 2. 15 cells. Methods The pSIHBV/X plasmid was transfected into HepG 2.2.15 cells, RT-PCR was used to evaluate the efficaey of transfection. MTT was conducted to check the cell proliferation afer transfection 24 h, 48 h,72 h. The expression of hTERT assayed by Real- time PCR and Western blot. Results The results of sequencing indicated that the recombinant plasmid pSIHBV/X sequence was correct. Efficient RNA interference of HBx gene was 53.6%. The cell pro- liferation was inhibited after 24 h, 48 h, and 72 h of the transfection, and there were significantly differences between pSIHBV/X group and control （P〈0.05）. The results of real time PCR and western blot analysis showed that the mRNA and protein expression of hTERT was decreased after transfection. Compared with control groups, hTERT mRNA and protein in test groups was significantly lower （P〈0.05）. Conclusion Silencing HBx gene by siRNA can inhibit the expression of cancer marker gene hTERT in HepG2.2.15 cell.