The Effection Of Down Regulation Of Htert By Rnai On The Expression Of H2ax And Dnmt3b

Objectives: To find a new and effective method of gene therapy, and to further investigate the new biological functions of hTERT, RNAi technicque was applied to inhibite hTERT mRNA in SMMC-7721 cells. Cell growth rate were observed and mRNA expression levels of H2AX and hDNMT3b were evaluated by RT-PCR.Methods: U6 promoter was amplified by PCR using the vector pRS as template. The amplified gene fragment was subsequently cloned into pGFP-sh vector to obtain RNAi expression vector pGFP-U. The hTERT cDNA sequences (AGA CCA AGC ACT TCC TCT ACT) that directly targeting coding region of hTERT (AF015950: 980-1000bp) were designed by full length gene targeting technique. The target sequences and its corresponding antisense sequences followed by the terminator of RNA polymerase III (TTTTTT) were linked by 9 bp loop sequences and cloned to the downstream of the U6 promoter in pGFP-U vector, resulting in the RNAi vector pGFP-shTERT to express a hTERT-specific short hairpin RNA (shRNA) in vivo. The RNAi vector pGFP-shTERT and the vancant vector pGFP-U were transferred into SMMC-7721 cells respectively by Liposome-mediated transfection. At 48 hours post transfection, the transfected cells were observed under the fluorescence microscope and selected by applying G418 in H-DMEM medium. MTT assay was applied to evaluate the effects of shRNA on cell growth. The total RNA was isolated by TRIzol, The semi-quantitative two-step RT-PCR which took the total RNA as template was used to determine the interference efficiency. The H2AX and DNMT3b gene expression level were evaluated also by RT-PCR.Results: pGFP-U and pGFP-shTERT vectors were confirmed by sequencing identification and digestion with restriction enzymes. The transfecting cells gave high levels of GFP expression. Compared with pGFP-U vector, pGFP-shTERT vector cause efficient down regulation of hTERT gene expression significantly, inhibited cell growth, increased H2AX expression level, and on the other hand, no obvious effect on DNMT3b expression.Conclusions: 1. The RNAi expression vector pGFP-U, the recombinant plasmid pGFP-shTERT aimed at hTERT, and the SMMC-7721 generating shRNA targeting hTERT are constructed successfully. 2. It shows that down regulating of hTERT gene expression by RNAi can inhibit the proliferation of liver cancer SMMC-7721. The results also indicate that hTERT interacts with H2AX and exerts important roles in the regulation of H2AX expression, while there is little evidence in the interaction of hTERT and DNMT3b.