Role Of MicroRNA-16 And Its Target Gene Wip1 On Invision And Proliferation In Glioblastoma Cells And Stem Cells

Background and purpose Glioblastoma is the most common fatal brain tumor,is a kind of polygenic disorders involving a large number of signaling pathways regulating and abnormal expression of multiple genes,including mi RNAs precise regulation of gene expression,but the detailed mechanism is not clear.In recent years found that Glioma stem cells(Glioma stem-like cells,GSCs)is considered to be the root cause of malignant Glioma invasion,radiation and chemotherapy resistance and relapse.Although comprehensive treatment and targeted therapy,the median survival in patients with is still less than 15 months.And glioblastoma cells and the molecular mechanism of stem cell growth and has important clinical value,can promote a more effective treatment or drug research and development,is expected to improve the patient's survival.Micro RNAs(mi RNAs)is a kind of by the length of the endogenous gene encoding is about 22 nucleotides noncoding single-stranded RNA molecules,according to its function can be divided into proto-oncogenes function of mi RNAs,tumor-suppressor genes function of mi RNAs and regulate stem cell function of mi RNAs,they in tumor occurrence or development through a variety of molecular mechanisms involved in classical genetic and epigenetic regulation.Glioma associated mi RNAs in the study,the proto-oncogenes function of mi R-21,mi R-221/222,mi R-10 b,mi R-17-92 gene cluster,a tumor suppressor gene function of mi R-128,mi R-34 a,mi R-7,mi R124 / mi R-137,etc.mi R-16 lower the expression was first discovered in chronic lymphocytic leukemia,may have the function of the tumor-suppressor genes.Has found a variety of regulating stem-mi Rs GSCs and neural stem cells,participate in the regulation of BMPs,SHH, NOTCH and so on multiple signaling pathways,which control the proliferation and differentiation of the GSCs mi R-124,mi R-125 and mi R-37 b,etc.Regulating the self-renewal of mi R-7,mi R-9,mi R-34 a,mi R-124,mi R-128,mi R-326 and mi R-326,etc.Regulating the chemotherapy drug resistance and radiation resistance of mi R-181-b,etc.Our preliminary experimental results confirmed that mi R-16 in glioma tissues and cell lines were significantly lower expression,and may be associated with the pathogenesis of glioma.Through micro RNA.org,Targetscan and mi R Walk database analysis,forecasting Wip1 is probably one of the target genes of mi R-16.Wip1(wild-type p53 induced phosphatase gene 1)is a proto-oncogene discovered in recent years,the study found that high expression in a variety of tumors,associated with the proliferation and prognosis of tumor,can by acting on the downstream target genes of p53 and Wip1-p53 pathway is involved in cell cycle and cell apoptosis process,which is not sensitive to tumor radiotherapy,chemotherapy drug resistance and poor prognosis.A study shows the p53 and glioma and its closely related to cancer stem cells,but its regulatory mechanism is not yet clear.Based on this,in this study,we discussed the over-expression of mi R-16 in U87 and U251 glioblastoma cells and its biological functions such as stem cell proliferation and invasion,and mi R-16 and Wip1 target gene p53 and its correlation,for further in-depth study of mi R-16 and the role of target genes in the human brain glioblastoma mechanism lay the foundation.Methods Glioblastoma U87 and U251 cells and their stem cells(GSC-U87 and GSC-U251)of training.Stem cells differentiation experiment and immunofluorescence staining method appraisal;Lipfectamine2000 liposome method in chemical synthesis of mi R-16 oligonucleotide random sequence(mi R-16 mimics and mi R-16 mimics negative control)transfection into human brain glioblastoma cell line U87 U251 and stem cell(GSC-U87 and GSC-U251),build mi R-16 of glioblastoma cells and stem cell model; q RT-PCR technique to detect after transfection cells mi R-16,Wip1 and p53 m RNA expression;Tablet cloning and Ed U lab testing mi R-16 effect on cell growth,proliferation,and flow cytometry instrument detection mi R-16 to the effect of cell apoptosis and cycle;Transwell invasion and scratch test mi R-16 effects on cell migration,invasion ability;Dual luciferase reporter gene experiment detect the relationship between mi R-16 and Wip1 3'-UTR;Western blot detecting mi R-16 Wip1 predicted target genes and its downstream gene p53 protein expression level.Results Glioma cell stem cell culture,the differentiated cells expressed GFAP,CD133 and Nestin expression,and GSC and GSC-87-251 cells express CD133 and Nestin,GFAP expression,indicates the cultivation experiment of GSC-87 and GSC-251 cells for glioma stem cells;After transfection mi R-16 mimics significantly increased the expression of mi R-16,Wip1 m RNA and protein expression decreased,p53 m RNA and protein expression increased;Over-expression of mi R-16 make U87 and U251 cell and stem cell growth,proliferation and invasive ability to drop,S phase cells decreased,the increase in the number of G1 phase cell cell early apoptosis rate increased.Conclusion mi R-16 suppression U87 and U251 cells and stem cells proliferation and invasion ability,promoting apoptosis;mi R-16 by inhibiting its target genes expression of Wip1 and make the high p53 expression,thus in the glioblastoma tumor suppressor role.