[Experimental research of targeting hTERT gene inhibited in hepatocellular carcinoma therapy by RNA interference]

RNA interference (RNAi) is a new gene blocking technology that silences target gene at post-transcription level induced by the small interference RNA (siRNA). RNAi has been demonstrated great prospect in gene functional research and gene therapy areas. Nowadays, RNAi has been reported to be used to inhibit the expression of endogenous genes including cyclophilin, GAPDH, p53, and c-myc; and there were some progresses in the therapy of the diseases caused by AIDS and hepatitis viruses with RNAi. However, hTERT gene, which was highly expressed in hepatocellular carcinoma and other malignant neoplasm, has not been researched by RNAi. In present research, we utilized RNAi to inhibit hTERT gene expression in vitro and in vivo, investigated the feasibility and specificity of gene therapy for hepatocellular carcinoma.

Small interference RNAs homologous to hTERT gene were designed,pTZU6+1-shRNA-hTERT vector was constructed and transfected into hepatocellular carcinoma SMMC-7721 cells and transplanted SMMC-7721 tumor in nude mice to induce RNAi. The changes of hTERT gene expression and tumor cell proliferation in both siRNA treatment groups and control group were determined by flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), immunochemistry in vitro and in vivo.

The expression of hTERT had been obviously inhibited by RNAi in vitro. The inhibition rate of cell growth was 37.5% after pTZU6+1-shRNA-hTERT vector was transfected to hepatocellular carcinoma SMMC-7721 cells; the phase of cell cycle indicated the reduction of S phase, while G(1)/G(0) phase increased. The mRNA level of hTERT decreased from 99.4% to 53.1%, its protein expression reduced from 86.3% to 46.6%. The tumor size reduced after treated with pTZU6+1-shRNA-hTERT vector in vivo; hTERT mRNA level decreased from 99.1% to 76.2%, and its protein expression decreased from 87.2% to 61.8% in siRNA treatment group. In contrast, there were no changes in control groups in vitro and in vivo.

RNAi inhibits the hTERT gene expression and proliferation of hepatocellular carcinoma SMMC-7721 cells with specificity, and is a possible new approach for neoplasm gene therapy.