Targeting Silence DENN-SV Gene And Its Reltionship On Human Breast Cancer Cell Line MCF-7Apoptotic Role With The NF-kappa B

Objective1.To build shRNA Eukaryotic Expression Vector Targeting DENN-SVGene，and screening a best target.2. Research the possible mechanisms ofanti-apoptotic effects of DENN-SV gene in human breast cancer cell lines inMCF-7.Methods1.This research designed four target sequences (DS-1, DS-2, DS-3, DS-4)based on DENN-SV sequence and shRNA design principles, and connectedthem to pRNAi-U6.1/Neo empty vector using recombinant DNA technology afterannealing, and transformed them into competent E.coli. Amplify strains, extractplasmidsand conduct digestion and DNA sequencing were obtained. Aftertransfecting recombinant eukaryotic expression vector into human breastcancer MCF-7cells, we used RT-PCR and Western blot inhibition to restrain theeffi ciency of DENN-SV mRNA expression.2.The strain containing the besttarget sequence has screened was amplified and extracted plasmid.Experimentwas divided into blank group, empty vector transfected group and the besttarget group.Proliferation of cell detected by MTT assay, got the growth curvesof MCF-7. The experiment was divided into two groups,each group is dividedinto a blank group, empty vector transfected group and a best target group, and48h after transfection given TRAIL25ng/ml for4hours, and the other group was not give TRAIL treatment,and collected cells.Cell apoptosis were detected byflow cytometry; Elisa assay content of NF-κB in cell lysates; using EMSA assaynucleoprotein samples in NF-κB activity.Results1.Sequencing results conformed that the designed sequence had beensuccessfully transfected into human breast cancer MCF-7cells. The expressionof GFP (green fl uorescent protein) was visible.RT-PCR results showedthat:there was no statistical significance between blank and negativegroups;four experimental groups (DS-1, DS-2, DS-3and DS-4) with the controlgroup and negative control group inhibition rate were statistically significant (P<0.05), and DS-1group and the remaining three group also has a significant (P<0.05). Western Blot analysis: there was not statistically significant betweenblank and negative groups; four experimental groups (DS-1, DS-2, DS-3andDS-4) with blank control group and negative control group inhibition rate wasstatistically significant (P <0.05), and DS-1group and the remaining three groupalso statistically significant (P <0.05). Both were the same, indicating that at thelevel of gene transcription and protein levels can proved that DS-1group hasthe best inhibition efficiency.2.Results of growth curves by MTT assay showedthat after transfection, the cell prolife ratio of experimental group reducedcantly (P<0.05). Flow cytometry TRAIL-induced apoptosis effect enhanced (P<0.05), there is statistically significant compared with the blank group and thenegative control group; Elisa detection of NF-κB results are shown at theexperimental group, the amount of NF-κB expression was significantlydecreased (P<0.05), EMSA detection of NF-κB results shown in experimentalgroup, NF-κB activity decreased (P <0.05). Conclusion1. In this study, we successfully constructed a recombinant vector of shorthairpin RNA gene targeting DENN-SV by RNAi technology,.we filter out the besttarget, and laid the foundation for further study of the anti-apoptotic mechanismof this gene and gene therapy of breast cancer.2. In this study, after interference of the expression of DENN-SV gene wegiven TRAIL treatment can lead to the increased rate of apoptosis of MCF-7cells. The mechanism of this phenomenon may be related to the activity ofNF-κB, and the phenomenon is not directly linked with the role of NF-κBrelations with TRAIL.