The Effects And The Mechanisms Of Notch1Gene Silenced By RNAi On Proliferation And Apoptosis In Human Osteosarcoma Cell MG63Line

Objective:In this study,we intend to construc the shRNA recombinant plasmid to block theNotch1signaling pathway, and to observe the cell proliferation,apoptosis, Bcl-2and Bax protein expression changes..Methods:(1) Design three pairs of Notch1gene loci shRNA fragments of eukaryotic expressionvector, liposome-mediated transfection of genetically modified MG63cells.Notch1protein and mRNA expression were detected by qRT-PCR and WesternBlotting after shRNA transfection and filter out the strongest interferenceshRNA.(2) the most Interference efficient shRNA stable transfected into MG63cells, andMG63cell proliferation and apoptosis change detected by MTT proliferationassay, Annexin V staining and flow cytometry flow.(3) using Western blotting method to detect Bcl-2and Bax protein changes in theMG63cells of after blocking Notch1signaling pathwayResults:(1) successfully constructed the recombinant plasmid containing Notch1shRNA fragments, andnamed as shRNAa, b, c, the Notch1gene mRNA and protein expression were loweredafter after transient MG63cells, shRNAa recombinant plasmid have strongest silencingeffect, inhibition rate was79.66%on RNA levels (p <0.05).(2) After the stably transfected MG63cells using shRNAa to blocking the Notch1signalingpathway, MTT proliferation assay, Annexin V method of staining and flow cytometry wereused to detect the cell proliferation and apoptosis status, compared with the control group,the cell proliferation rate less slow (p <0.05), increased apoptosis (p <0.05) (3) use shRNAa stable transfected MG63cells to blocking Notch1signaling pathway, Westernblotting detection found the Bcl-2protein expression was decreased, and Bax proteinexpression was increased inMG63-shRNAa cellsConclusions:(1) successfully constructed the shRNA recombinant plasmid targeting Notch1gene, and itcan effectively blocking Notch1signaling pathway.(2) Silencing Notch1gene by RNAi could significantly inhibit the proliferation ofMG63cells and induce the cell apoptosis．The mechanisms may be related withregulating the expression of Bcl-2gene and Bax gene.