Stable inhibition of hTERT gene by siRNA in hepatocarcinoma cells

    ZHOU Xi, ZHANG Pengª²Hui

    1Department of Ophthalmology, First Affiliated Hospital, 2Faculty of Laboratory Medicine, Chongqing University of Medical Sciences, Chongqing 400016, China

    ¡¾Abstract¡¿ AIM: To elucidate the timeª²efficiency relation of stable inhibition of hepatocarcinoma cell proliferation by RNA interference in vitro. METHODS: The stable screeningª²inhibition technique of RNAi was adopted to suppress the expression of hTERT stably. After small hairpin RNA (shRNA) targeting hTERT gene was designed, recombinant vector pGenesilª²shRNAª²hTERT was constructed and transfected into hepatocarcinoma SMMCª²7721 cells, which were stably selected by G418 to establish hepatocarcinoma cell lines stablely expressing pGenesilª²shRNAª²hTERT. Realª²time RTª²PCR, MTT and PCRª²TRAP were utilized to detect the alterations of hTERT mRNA expressions, telomerase activity and cell proliferation. RESULTS: The effectiveness of RNAi existed continually and stably in hepatocarcinoma SMMCª²7721 cells expressing stably pGenesilª²shRNAª²hTERT. In the cell lines expressing stably pGenesilª²shRNAª²hTERT, hTERT mRNA was obviously suppressed, and the telomerase activities were significantly decreased. Hepatocarcinoma SMMCª²7721 cell proliferation significantly inhibited in pGenesilª²shRNAª²hTERT group compared to that in negative control group. CONCLUSION: RNAi may continually and stably suppress hTERT mRNA expression and carcinoma cell proliferation, which is a potential new approach for antiª²tumor gene therapy. ......