The Experimental Study In RNA Interference Targeting Human Telomerase Reverse Transcriptase Gene In Colorectal Cancer Gene Therapy

Part I:Constuction of shRNA recombinant eukaryotic expression vector and the effect on apoptosis of colorectal cancer SW480 cellsObjective To construct the recombinant eukaryotic plasmid containing small hairpin RNA (shRNA) targeting hTERT and investigate the effect of RNA-mediated interference hTERT (human telomerase reverse transcriptase,hTERT) expression on the biological behaviour of colorectal cancer sw480 cells.Methods Three small interfering RNA (siRNA) targeting hTERT gene were synthesized chemically, one pair of siRNA fragments which had the most silencing effect was synthesized into oligodeoxynucleotide fragment,and constructed into eukaryotic vector pGPU6/GFP/Neo.The recombinant eukaryotic vector pGPU6/GFP/Neo was identified by restriction enzyme digestion and sequencing.The constructed recombinant eukaryotic vector pGPU6/GFP/Neo-hTERT-shRNA was transfected into human colorectal cancer SW480 cells by lipofectamine.Cells carrying efficiency and morphology changes was observed by fluorescence microscope.The expression level of hTERT mRNA in SW480 cells in different times was detected by RT-PCR analysis. The telomerase activity of SW480 cells after transfected 48h was examined by TRAP-PCR-ELISA analysis.The hTERT protein expression of SW480 cells was detected by immunohistochemical method.The distribution of cell cycle was analyzed by FCCS.The apoptosis changes of SW480 cells were monitored by TUNEL assay.The changes of mitochondrial membrane potential (MMP) were detected by laser confocal microscope. The ultrastructure changes of SW480 cells after transfected was examined by TEM (The transmission electron microscope).Results The shRNA fragment was successfully inserted into the eukaryotic plasmid pGPU6/GFP/Neo to constitute recombinant eukaryotic plasmid pGPU6/GFP/Neo-hTERT-shRNA.The transfection efficiency of SW480 cells was higher when the ratio of plasmid and Lipofectamine at 1:2.5 and transfected 48h, and the transfected percentage of cell is 59%. RT-PCR assay showed that the hTERTmRNA expression level of hTERT-shRNA group reduced remarkably, compared with the blank group, liposome group, NC-shRNA group, its inhibition rate was respectively 39.2%,33.28%, 27.95%. TRAP-PCR-ELISA showed that the telomerase activity in the hTERT-shRNA group after transfection decreased for 18.7% significantly,compared with another three groups,the difference was statistic significance(P<0.05).Immunohistochemistry assay showed that the number of stained positive SW480 cell in hTERT-shRNA group was less markedly than that of in other three groups. Through pathological image software assay, the difference between hTERT-shRNA group and blank group had clear statistic significance (P<0.05), as well as there were statistic significance compared with Lipofectamine group and NC-shRNA group (P<0.01).FACS method showed that the SW480 cells number at phase G0/G1 increased obviously in hTERT-shRNA group. This pointed out that the silence S W480 cells increased after transfection plasmid hTERT-shRNA, compared with NC-shRNA group, the proliferation index(PI) went down about 14.2%,there was obvious statistics significance (P<0.05). TUNEL assay showed that the number of apoptotic cells for hTERT-shRNA group increased remarkably and the apoptosis index(AI) was evidently higher than that of other groups, it had obvious statistics significance (P<0.01). Transmission Electron Microscope assay showed that nucleolus fissions seen in some cells, MMP went down markedly and fluorescent intensity of Rh 123 enhanced for hTERT-shRNA group, compared with other groups, the difference had statistic significance(P<0.01). Transmission Electron Microscope:the volume of SW480 cells got smaller significantly, the minovillus and protrusions reduce, some disappeared even, chromatin gathered unevenly along the nuclear membrane. Lunularosome could be seen and more vacuolizations increased in part of cells.Conclusions Recombinant eukaryotic expression vector pGPU6/GFP/ Neo-hTERT-shRNA targeting hTERT gene was successfully construct.It c ould silence hTERT gene effectively, be able to inhibit the proliferation andgrowth of human colorectal cancer SW480 cells, reduce the telomer ase activity and promote apoptosis of tumor cells finally. Objective To investigate the treatment effect of recombinant plasmid pGPU6/GFP/Neo-hTERT-shRNA targeting hTERT gene on human colorectal cancer SW480 cells in transplanted nude mice.Methods Human colorectal cancer SW480 cells were subcutaneously implanted under the skin of the right armpit to establish nude mice models of colorectal cancer, after the tumors grew to a definite size, the mice were randomly divided into three groups:normal saline (NS group), NC-shRNA group and hTERT-shRNA group. Each group was respectively treated for 6 consecutive times, the growth status of the tumor was observed, tumor volume was measured, tumor growth curve was drawn, tumor tissue morphology was observed with HE staining, the expression of hTERT protein in the tumor was detected by immunohistochemistry, cell apoptosis was inspected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling(TUNEL), the expression of hTERT mRNA was checked by RT-PCR and the telomerase activity was detected by PCR-TRAP-ELISA.Results All nude mouce after implanted SW480 cells subcutaneously at the 14th day had formed tumors and the diameters of tumor nodules were up to 5-7mm.The transplanted nude mice models had been constructed successfully. The rate is of 100% into tumors.After nude mouce starting treatment, the growth of tumor volume in hTERT-shRNA group became slower than the NS group and the NC-ShRNA group, and significantly slower at the beginning of the 18th days. HE assay showed that partial tumor tissue in hTERT-shRNA group presented necrosis and tumor cells morphology changed obviously. Immunohistochemistry detection showed that the expression levels of hTERT protein in tumor tissue of hTERT-shRNA group decreased, and a small amount of hTERT protein positive cells had been seen. TUNEL assay showed that the number of apoptotic cells in hTERT-shRNA group increased significantly and distributed densely. Compared with the NS group and the NC-shRNA group, the apoptosis index in hTERT-shRNA group increased by 29.4% and 31.1% separately,the difference was statistically significant (P<0.01). RT-PCR analysis showed that the expression levels of hTERT mRNA in hTERT-shRNA group compared with NS group and NC-shRNA group decreased by 52.1% and 48.3% respectively,and the differences were significant(P<0.01).PCR-TRAP-ELISA analysis showed that the telomerase activity of hTERT-shRNA group was observably lower than NS group and NC-shRNA group, the inhibition rates were 48.5% and 53.0% respectively, the differences were statistically significant (P<0.05).Conclusions Recombinant plasmid pGPU6/GFP/Neo-hTERT-shRNA inhibits the growth and promotes apoptosis of implanted human colorectal cancer by down-regulating the expression of hTERT mRNAå’ŒhTERT protein in tumor tissues.