Construction Of Liver-specific RNAi Expression Vectors And The HTERT Inhibition In SMMC-7721 Cells

At present, cancer gene therapy has been limited by the lack of tissue and cancer specificity. To target cancer cells without damaging non-targeting tissue cells, we chose tissue-specific promoter to induce shRNA expression, which has the effect to inhibit targeting gene. Recent studies show that apoA-I promoter can induce gene expression in liver specifically, and it has been certificated in our previous work, we use apoA-I promoter here to induce the expression of shRNA and the shRNA inhibition effect, and the promoter trancfection effiency is improved by different enhancers which contain CMV, SV40 enhancers. Telomerase activity is mainly regulated by the human telomerase reverse transcriptase(hTERT) gene. Our objective was to investigate the effect of short hairpin RNA (shRNA) on hTERT expression in liver cancer cells, which induced by apoA-I promoter. Short hairpin RNA expression vectors targeting the messenger RNA of hTERT were constructed, named as pRS-shT1, pRS-shT2, pRS-shT3, pRS-shT4, induced by U6 promoter on the vector pRS. Cells were treated with shRNA expression vectors directed against 3 different hTERT sites, the control vectors that included mismatched shRNA, named as pRS-shT5. The Polâ…¡promoter expression of hTERT was determined by reverse-transcriptase polymerase chain reaction and then the best inhibition effect shRNA was constructed to be inserted into the vector PDA-EGFP which contained apoA-I promoter. Then three different enhancers were inserted into upstream of apoA-I promoter. In this system, the problem of off-target effect caused by apoA-I promoter, that is, RNA Pol II promoter was solved, by insert MAZ and Ribozyme box. We found that treatment of shRNA expression vectors induced a significant decrease in hTERT messenger RNA expression, the level of hTERT protein, telomerase activity, and cell viability. All of these effects were seen regardless of the target site, and the shRNA control showed none of these effects. Two enhancers improve the transfection efficiency of apoA-I promoter.In addition, studies have shown that EGFR (epidermal growth factor receptor), expression of EGFR signals in liver cancer cell is twice than in normal liver cells, EGFR over-expresses in 92% of liver cancer cell membrane, and EGFR itself has the effect of inhibition to the development of tumor cells. Moreover, upregulation of EGFP expression also upregulates hTERT gene expression, so based on the above-mentioned study, we constructed the RNA interference expression vector to target EGFR gene, which was induced by modified apoA-I promoter, and of hTERT and EGFR expression vectors were co-transfected to obtain a double-down effect of hTERT expression, and the effect were test by western-blot.Our research showed that pRS-shT2 vector had the best RNAi effect, which was ready for the use as target sequences for further study. CMV enhancer showed the advantages comparing with SV40 enhancer, test by GFP fluorescence and RT-PCR. The inhibition valuation of the complete RNAi expression vector was confirmed.