Eukaryotic Vector Of The Bmi-1 Gene ShRNA Construction And Its Effect On LoVo Cells

Background & Objective:Bmi-1 is a proto-oncogene belonging to the Polycomb group. It is involved in the regulation of growth and proliferation of cells. The bmi-1 gene overexpression was confirmed to relate to the occurrence,development, prognosis of the variety of human tumors. RNA interference is present research focus, small interference RNA (siRNA) can knockdown the expression of specific gene effectively and have no influence on normal gene function.Applications field of RNA interference technology have already expanded step by step from genomics research to medical fields,and RNA interference technology is used as methods of gene therapy.Using RNA interference not only provide an economic,rapid,high-efficient technical method of suppressing gene expression but also is possible to open up new idea for determination of gene function and gene therapy.In this study, colon cancer cells were transfected with vector pEGFP-N1 under the mediation of Lipofectamine so as to observe the transfection efficiency and the most optimal colon cancer cells.The bmi-1Retrovirus-mediated shRNA Expression Vector was constructed and then transfered into LoVo cells,puromycin were used to obtain stable transfected cloning cells and amplify culture.Its effect on mRNA and protein of bmi-1,as well as proliferation and apoptosis were investigated for exploring the possibility of bmi-1 gene as gene therapy target.This article aims at exploration specific blocking on bmi-1 expression in cancers having bmi-1 over-expression,and blocking bmi-1 may provide a new target for gene therapy and theoretical basis on clinical gene therapy for cancer.Methods:1.Bmi-1 gene mRNA in colon cancer tissue and adjacent tissues were detected by RT-PCR,and semi-quantification analysis were performed with gray scale scanning software.2.The complementary oligonucleotides encoding hairpin RNA which specifically targets bmi-1 gene were designed, and the nonsense oligonucleotides were designed also according to siRNA principle of design by molecular cloning technique.3.The recombinant vector pSIREN-VEGF-C were digested by BamHⅠ+ EcoRⅠdouble digestion, after electrophoresis identification ,gel recovery were used to get linear vector.4.Single strand were annealed to complementary oligonucleotides,then complementary oligonucleotides were connected with linear vector to construct pSIREN-bmi-1-2 and nonsense control vector pSIREN-bmi-1-C. Positive colonies were picked and identified by using EcoRⅠ+ BglⅡdouble digestion,then correct colonies were sended to biological company to sequence.5.Colon cancer cells were cultured according to conventional culture conditions of adherent tumor cells, and the optimal concentration of puromycin was obtained through gradient screening so as to establish stable LoVo cell line.6.Four different colon cancer cells were transfected with vector pEGFP-N1 under the mediation of Lipofectamine so as to observe the transfection efficiency and the most optimal colon cancer cells.7.The vectors pSIREN-bmi-1-1 ( it was stored in the laboratory ) , pSIREN-bmi-1-2 and nonsense control vector pSIREN-bmi-1-C were transfected into LoVo cells under the mediation of Lipofectamine,puromycin were used to obtain stable transfected cloning cells and amplify culture. 8.Bmi-1 mRNA expression were detected in stable transfected LoVo cells by fluorescence quantitative RT-PCR techniques, and semi-quantification analysis were performed with gray scale scanning software.9.Bmi-1 protein expression were detected in stable transfected LoVo cells by Western Blot techniques.10.Proliferation in pSIREN-bmi-1-1,pSIREN-bmi-1-2,pSIREN-bmi-1-C,blank control groups were detected by MTT Colorimetric Assay,morphological observation were used to distinguish pSIREN-bmi-1-1,pSIREN-bmi-1-2, pSIREN-bmi-1-C,blank control groups.11.Apoptosis in pSIREN-bmi-1-1,pSIREN-bmi-1-2,pSIREN-bmi-1-C,blank control groups were observed by Annexin V/PI double staining.Results:1.The expression of bmi-1mRNA in human colon cancer is higher than corresponding adjacent tissues by RT-PCR, the mean expression rates of on bmi-1 mRNA are 0.76±0.23, 0.35±0.19(P<0.05).2.The pSIREN-bmi-1-2 and pSIREN-bmi-1-C vectors were confirmed by restriction endonuclease digestion and DNA sequencing demonstrated that the length and orientation of inserted sequence was correct.3.The optimal transfection efficiency is 50% after transfecting vector pEGFP-N1 under the mediation of Lipofectamine in the middle of four different colon cancer cells,and this is the LoVo cell.4.The optimum concentration of obtaining stable transfected cloning cells was 5ug/mL.5.Bmi-1 mRNA expression level of pSIREN-bmi-1-2 group were showed remarkably lower than that of pSIREN-bmi-1-C and blank control group by fluorescence quantitative RT-PCR method, the rate of suppression on bmi-1 mRNA is 80%(p< 0.05);but there had no difference in pSIREN-bmi-1-1,pSIREN-bmi-1-C, blank control group(P>0.05).6.Bmi-1 protein expression level of pSIREN-bmi-1-2 group were showed remarkably lower than that of pSIREN-bmi-1-C and blank control group by Western Blot method, the rate of suppression on bmi-1 protein is 82%(p< 0.05);but there had no difference in pSIREN-bmi-1-1,pSIREN-bmi-1-C, blank control group( P>0.05 ) .We can infer that pSIREN-bmi-1-2 can inhibit bmi-1 protein expression,but pSIREN-bmi-1-1 had no such effect.7.MTT Colorimetric Assay demonstrated that pSIREN-bmi-1-2 group grow slower than other groups,but pSIREN-bmi-1-1 group, pSIREN-bmi-1-C group had no influence on cell growth.8.Detection of morphology were showed that each vectors and blank control group had no morphological difference.9.Vectors didn't induce cell apoptosis by detection of apoptosis staining when they were compared with blank control group.Conclusion:1.Bmi-1 mRNA expression in human colon cancer is higher than corresponding adjacent tissues by semi-quantitative RT-PCRr.2.Linear vector pSIREN-RetroQ was obtained by enzyme digestion, electrophoresis identification and gel recovery and the vectors expressing shRNA targeting bmi-1 were successfully constructed.3.LoVo cells were easily transfected into exogenous plasmids under the mediation of Lipofectamine in the middle of four different colon cancer cells.4.Recombinant vector pSIREN-bmi-1-2 could inhibit bmi-1 gene expression and proliferation in colorectal cancer LoVo cell, but didn't cause morphology changes and apoptosis.