Inhibitory Effect Of MiRNA Expression Vector Targeting Survivin On Proliferation Of Human Colorectal Carcinoma HT-29 Cells

Objective:To investigate the effects of microRNA target survivin on transfected human colorectal carcinoma (HT-29) cell apoptosis, proliferation and survivin expression.Methods:(1)Four pairs of micromRNA targeting to the different sites of survivin were designed. micromRNA targeting survivin was synthesized and transfected Ht-29 cells by lipofectin.(2) Ht-29 cells were cultured in the 6 orifices. Four groups of genome and Empty plamid were transfected into cells.After 48 hours, we will observe the the group of recombinant plasmid by the fourescent inverted microscope and detecte trasfection efficiency by the flow cytometry instrument.In finally,we will choose the highst rate of trasfection group for the purpose of genome.(3)We will recovery the purpose genome group and the empty plasmid group strains and cuitivate in LB medium.The alkali denaturation method is used to extract obtained corresponding plasmid DNA which will be measured of contration and purity.(4) The cultured cells were divided into control, liposome, negative control and positive control groups Transient transfected cells were collected and the proliferation index and apoptosis of HT-29 cells were detected by flow cytometry and electron microscope. The inhibitory effects were determined by PT-PCR and Western blot.Result:(1) The plasmid pcDNATM6.2-GW/Em GFP-miR was successfully constructed by sequencing.(2)The proliferation index and apoptosis rate of the positive control group was also significantly higher compared with other groups (t=20.05, P<0.01; t=18.75, P<0.01; t=18.59, P<0.01; t=16.40, P<0.01; t=15.84, P<0.01; t=15.92, P<0.01). Survivin mRNA and protein expression level were specifically suppressed in transfected HT-29 cells (t=0.68, P<0.01; t=0.58, P<0.01; t=0.61, P<0.01; t=0.64, P<0.01:t=0.62, P<0.01; t=0.67, P<0.01).Conclusion:(1) PcDNATM6.2-GW/Em GFP-miR that tartet survivin was successfully constructed.(2)Survivin microRNA can effectively decrease the expression of survivin mRNA and protein, induce colorectal carcinoma HT-29 cell apoptosis and inhibit cell proliferation.