Cisplatin Inhibits The Proliferation Of Osteosarcoma Cell Line Saos-2via MiR-376c/TGFA Pathway

Objective:DDP is wide spectrum anti-cancer drug, for Platinum of metal complex collection property, role may wish to n-of agent, main role target points for DNA, is cell cycle non heterosexual drug, but due to big dose chemotherapy of HIV side effects, and tumor cell of mutations, and original made or following made of chemotherapy drug resistance drug, and early lung transfer, and local relapse, problem, led to near20years to flesh tumor patients of survival rate cannot continues to significantly improve. Looking for an efficient, practical, new side effects of osteosarcoma treatment methods, will, to a large extent change the current treatment of osteosarcoma patients. With the development of molecular biology and genetics, targeted therapy of osteosarcoma has become the focus of study. MicroRNA (miRNA) are found in viruses and in higher organisms, evolution of highly conserved on endogenous non-coding RNA. Broad participation of miRNA regulation consists of an individual’s growth, apoptosis, proliferation and differentiation of many vital processes, recently found that miRNA and cancer have a very close relationship. Now, in a variety of abnormal expression of tumor found in the miR-376family, reports miR-376c significantly lower expression in multiple tumor-inhibiting proliferation and metastasis of tumors. The research group reported that targeted inhibition of miR-376c in the early expression of TGFA. At the same time, reports that the TGFA people played an important role in the proliferation and metastasis of osteosarcoma and targeted regulation of miRNA was of the TGFA rarely reported, DDP via miR-376c/TGFA pathway inhibition activity of osteosarcoma are not to be reported. Cut raised miR-376c this issue to be DDP TGFA functional effect of inhibition of osteosarcoma cell proliferation, with a view to looking for prevention and treatment of bone Sarcoma targets provide a solid foundation.Method:1, building the TGFA shRNA vector, liposome transfection of cells Saos-2, QPCR and Western blot detection of TGFA silent efficiency;2TGFA-shRNA Transfection, and BrdU by MTT method for detection of Saos-2cell growth and DNA synthesis;3, DDP combined with TGFA ORF clone Saos-2, With BrdU and MTT method for detection of Saos-2cell growth and DNA synthesis;4, chemical synthesis of miR-376c mimics and build a miR-376c Sponge carriers using QPCR detection of expression of miR-376c;5, DDP combined with miR-376c and miR-376c Sponge handle, and BrdU by MTT method for detection of Saos-2cell growth and DNA synthesis;6, By luciferase reporter gene detection miR-376c target regulation of TGFA;7, DDP combined with miR-376c Sponge Saos-2, PI3K/AKT Western blot detection pathway activity;8, miR-376c Saos-2cells in conjunction with TGFA, Western blot detection of activity of PI3K/AKT pathways.Result:1, TGFA-shRNA transfected Saos-2cells, expression of silence around70%TGFA;2, TGFA shRNA transfection can significantly inhibit the growth of Saos-2cells and cellular DNA synthesis;3, DDP when concentration of2.5mg/1can significantly inhibit expression of TGFA, DDP combined with TGFA ORF clone when dealing with Saos-2cells may respond to inhibition of DDP on cell proliferation,4, MiR-376c mimics miR-376c overexpression by about50times times, cut miR-376c Sponge miR-376c express70%;5, compared to the DDP DDP combined with miR-376c cells alone, may be even more pronounced inhibition of Saos-2cell proliferation and DDP combined with miR-376c Sponge cells compared to DDP alone, Response inhibition of DDP on cell line Saos-2;6, miR-376c can be used with TGFA3’UTR direct interactions and inhibits the expression of TGFA;7, miR-376c Sponge and DDP combined with Saos-2cells compared to DDP alone, immutable PI3K and AKT protein expression, but you can reply to phosphorylation of DDP on PI3K and AKT phosphorylation inhibits;8, TGFA combined treatment Saos-2cell and miR-376c compared to miR-376c single, immutable PI3K and AKT protein expression, but you can reply to DDP PI3K and AKT phosphorylation inhibits the phosphorylation.Conclusion:1, DDP can be lowered through TGFA inhibit Saos-2cell proliferation of osteosarcoma;2, DDP Saos-2raised miR-376c through inhibition of osteosarcoma cell proliferation;3, Saos-2of osteosarcoma cells, targeted inhibition of miR-376c expression of TGFA;4, DDP can be lowered through TGFA inhibition activity of PI3K/AKT pathways;5, miR-376c through targeted inhibition of TGFA inhibiting PI3K/AKT pathway activity;6, DDP inhibits proliferation of osteosarcoma cells by miR-376c/TGFA means.41figures,20tables,116...