Stable inhibition of hTERT gene by siRNA in hepatocarcinoma cells

AIM: To elucidate the time-efficiency relation of stable inhibition of hepatocarcinoma cell proliferation by RNA interference in vitro. METHODS: The stable screening-inhibition technique of RNAi was adopted to suppress the expression of hTERT stably. After small hairpin RNA (shRNA) targeting hTERT gene was designed, recombinant vector pGenesil-shRNA-hTERT was constructed and transfected into hepatocarcinoma SMMC-7721 cells, which were stably selected by G418 to establish hepatocarcinoma cell lines stablely expressing pGenesil-shRNA-hTERT. Real-time RT-PCR, MTT and PCR-TRAP 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 SMMC-7721 cells expressing stably pGenesil-shRNA-hTERT. In the cell lines expressing stably pGenesil-shRNA-hTERT, hTERT mRNA was obviously suppressed, and the telomerase activities were significantly decreased. Hepatocarcinoma SMMC-7721 cell proliferation significantly inhibited in pGenesil-shRNA-hTERT 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 anti-tumor gene therapy.