MiR-218Inhibits Glioma Cells Proliferation、High Invasiveen Ess And Migratory Throug Downregulation Of The Slit2-robo1Pathway

Part I The expression and meaning of miR-218and Robo1in humanglioma tissueObjective：To explore the expression and meaning of miR-218and Robo1in humanglioma tissue.Method:The expression levels of miR-218in40cases of different grades of gliomastissues and10control brain tissues were detected by means of qRT-PCR.The expressionlevels of Slit2and Robo1were evaluated using Western blot and immunocytochemistry.Whilst the relationships between miR-218and Slit2-Robo1pathway were analyzed.Results:Quantitive real-time RT-PCR analysis revealed that miR-218exression werereduced in Ⅰ, Ⅱ-grade glioma tissues(P<0.05)and markedly reduced in Ⅲ, Ⅳ-gradeglioma tissues(P<0.01) versus normal brain tissuees,that Slit2is distinctly expressed bynormal cerebral neurons,but at very low levels in all-grade glioma tissues, moreover,withthe progression of malignant glioma,the expression of Slit2is gradually decreased or evensilenced.However,Robo1is not only expressed in normal brain neurons,but alsoover-expressed in different grades of gliomas(P<0.05).Conclusion:MiR-218exression were markedly reduced in all-grade glioma tissues,meanwhile the expression of Slit2in all-grade glioma tissues were decreased;there was apositive correlation between the expression of Slit2and miR-218; the expression of Robo1in all-grade glioma tissues were markedly increased, there was a negative correla tionbetween the expression of Robo1and miR-218. PartⅡ MiR-218inhibits glioma cells proliferation、high invasiveenessand migratory through downregulation the Slit2-Robo1pathwayObjective：To explore the effect on Slit2-Robo1pathway through miR-218over-expression in human U251glioma cellsMethod:Human U251glioma cells were transfected with miR-218mimics and mimics NC,the expression levels of mRNA and protein expression of Robo1and Slit2aftermiR-218transfection were detected by qRT-PCR and westemb1otting analysis.Thechanges of cell growth and proliferation were determined by MTT method and the changesof cell were determined by cycle flow cytometry assay.The migration and invasion ofglioma cells were detected by Transwell assay.The change of tumor growth in vivo wasevaluated by animal experiment. A computational search revealed a conserved target site ofmiR-218within the3＇-untranslated region of Robo1.Luciferase reporter assay wasperformed to examine the effects of miR-218on expression of potential target geneRobo1.Results:MiR-218was up-regulated after the U251cells transfection with miR-218mimics.over-expression of miR-218decreased mRNA and protein levels of Robo1,andincreased mRNA and protein levels of Slit2.The proliferation was significantly decreasedby the MTT assay and the cell cycle was arrested in G0/G1phase by flow cytometry assayafter transfection with miR-218mimics. Nude mouse tumorigenicity assay suggested thatmiR-218may play a crucial role in the proliferation of glioma cells in vitro. Transwellassay demonstrated that the migration and invasion capability of tumor cells decreasedafter transfection with miR-218mimics.Luciferase reporter assay suggested that miR-218might inhibit Robo1expression through interaction between miR-218and3′UTR ofRobo1.Conclusion: MiR-218may inhibit glioma cells proliferation、high invasiveness andmigratory through downregulation the Slit2-Robo1pathway;and the biological effect ofmiR-218in glioma cells is probably have relation to regulating its potential target geneRobo1. PartⅢ MiR-218inhibits glioma cell proliferation、high invasiveeness andmigratory through negatively regulating the expression of Robo1geneObjective：To explore the mechanism on miR-218inhibits glioma cells proliferation、high invasiveeness and migratory through negatively regulating the expression of Robo1gene.Method:Downregulating the expression of Robo1in U251cells by siRobo1,the expe riment were divided into:①control group;②m iR-218mimics group;③Robo1siContgroup;④Robo1siRNA group.The expression levels of protein expression of Robo1aftertransfection were detected by westemb1otting analysis.The changes of cell growth andproliferation were determined by MTT method and the changes of cell cycle weredetermined by cycle flow cytometry assay.The migration and invasion of glioma cells weredetected by Transwell assay.Results:Westemb1otting analysis revealed that Robo1exression were markedlyreduced in miR-218mimics group and Robo1siRNA group versus control group andRobo1siCont group（P＜0.01）.The Robo1exression levels in Robo1siRNA group wereequal to in miR-218mimics group（P＞0.05）.MTT assays showed that downregulation ofRobo1significantly inhibited the proliferation of U251cells and the cell cycle was arrestedin G0/G1phase by flow cytometry assay compared with control group.Furthermore,transwell assay demonstrated that the migration and invasion capability of tumor cellsdecreased after transfection with Robo1siRNA. Importantly,downregulation of Robo1inU251cells could inhibit proliferation and block G0/G1transition,which was consistentwith the effect of miR-218overexpression in U251cells.Conclusion:MiR-218inhibits glioma cells proliferation、high invasiveeness andmigratory through negatively regulating the expression of Robo1gene.