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目的:探讨DNA载体途径的RNA干扰技术抑制人端粒酶逆转录酶(hTERT)基因表达及诱导喉癌细胞Hep-2凋亡的作用。方法:构建靶向hTERTmRNA的质粒pshRNA1、pshRNA2及对照质粒pshRNA3、pEGFP,并将其分别转染喉鳞癌Hep-2细胞。共聚焦荧光显微镜观察质粒转染及表达情况;RT-PCR测定hTERTmRNA表达;Western印迹测定hTERT蛋白表达;末端重复片断扩增-酶联免疫吸附(TRAP-ELISA)方法测定细胞端粒酶活性;MTT法研究细胞增殖活性变化;原位细胞凋亡(TUNEL)及透射电子显微镜研究细胞凋亡。结果:①共聚焦荧光显微镜下见大量的细胞呈现绿色荧光,与其他组比较,pshRNA1、2组呈现荧光的死亡细胞显著增加。②pshRNA1、2转染细胞后hTERTmRNA及hTERT蛋白表达显著降低;其端粒酶活性明显受到抑制:转染后2 d pshRNA1组活性为:0.159±0.039、pshRNA2组活性为0.163±0.028,与空白对照组1.523±0.076比较,差异有非常显著性(P<0.005)。③pshRNA1、2转染细胞后可显著抑制细胞增殖、诱导细胞凋亡。这两组细胞透射电镜可见典型细胞凋亡特征。结论:DNA载体途径的RNA干扰技术能有效抑制hTERT基因的表达及端粒酶活性并诱导癌细胞凋亡,此法可能成为抑制肿瘤细胞的新途径。
Objective: To investigate the effect of RNA interference of DNA vector pathway on human telomerase reverse transcriptase (hTERT) gene expression and apoptosis of Hep-2 cells in laryngeal carcinoma cells. Methods: Plasmids pshRNA1 and pshRNA2 targeting hTERT mRNA and pshRNA3 and pEGFP were constructed and transfected into Hep-2 cells. The expression of hTERT mRNA was detected by RT-PCR, the expression of hTERT protein was detected by Western blotting, the telomerase activity was detected by TRAP-ELISA and MTT Law to study changes in cell proliferation activity; apoptosis in situ (TUNEL) and transmission electron microscopy. Results: (1) A large number of cells showed green fluorescence under the confocal fluorescence microscope. Compared with other groups, the number of apoptotic cells in pshRNA1,2 group increased significantly. ② The expression of hTERT mRNA and hTERT protein was significantly decreased after transfected with pshRNA1,2, and the telomerase activity was significantly inhibited. The activity of pshRNA1 group was 0.159 ± 0.039 at 2 d after transfection, and 0.163 ± 0.028 at pshRNA2 group. Compared with the blank control group 1.523 ± 0.076, the difference was significant (P <0.005). ③pshRNA1,2 transfected cells can significantly inhibit cell proliferation and induce apoptosis. Transmission electron microscopy of these two groups showed typical apoptotic features. Conclusion: RNA interference of DNA vector pathway can effectively inhibit the expression of hTERT gene and the activity of telomerase and induce the apoptosis of cancer cells. This method may be a new way to suppress tumor cells.