Study On The Role And Mechanism Of MiR-195/205 In Human Brain Glioma And TGF-β1 Signal Pathway

Part 1:Expression of miR-195.miR-205 in human brain glioma tissue and TGF-β1 in the glioma patients peripheral blood and the study of their relationObjective:To investigate the expression of miR-195 and miR-205 in human brain glioma tissues and TGF-β1 in the glioma patients peripheral blood and analyze the correlation of their expression.Methods:Quantitative real-time PCR(q RT-PCR)was used to detect miR-205 and miR-195 evels in human glioma tissue samples and U87 cells treated with different concentrations of TGF-β1.Enzyme-linked immunosorbent assay(ELISA)was performed to determine TGF-β1 in the same 31 cases glioma patients peripheral blood.At the same time,12 cases normal brain tissues samples and 31 cases Non-cancer volunteers peripheral blood samples as control group.Results:(1)qRT-PCR results showed that compared to control group,the expression of miR-205 levels in human glioma tissue samples were significantly reduced,the expression of miR-195 levels in human glioma tissue samples were significantly increased.(P<0.01).(2)ELISA results showed that compared to control group,the expression of TGF-β1 in glioma patients peripheral blood were significantly increased.(P<0.01).(3)In U87 cells treated with different concentrations of TGF-β1,the expression of miR-205 levels were significantly reduced,the expression of miR-195 levels were significantly increased.TGF-β1 concentration was negatively correlated with miR-205 mRNA level,but positively correlated with miR-195 mRNA.(P<0.01).(4)The analysis of correlation showed that expression level of TGF-β1 in glioma patients peripheral blood was negatively correlated with miR-205 mRNA levels in human glioma tissue samples(R2=0.769),but positively correlated with miR-195 mRNA in human glioma tissue samples(R2=0.794).Conclusion:It was shown that miR-205 was decreased in glioma tissue,but miR-195 and TGF-β1 was increased.In addition,TGF-β1 concentration was negatively correlated with miR-205 mRNA level,but positively correlated with miR-195 mRNA.In addition,miR-205 was down-regulated and miR-195 was up-regulated by TGF-β1 in a dose-dependent manner.Part 2:Effects of miR-195、miR-205 and TGF-β1 on proliferation and invasion of U87 cellsObjective:To observe the effects of up-regulation of miR-195、miR-205 and TGF-β1 on proliferation,apoptosis,invasion and migration of U87 cells.Methods:First,miR-195 mimics were transfected into human glioma cell lines U87.Second,cell proliferation was detected by colony formation assay and MTT.Cell invasion was gauged by Transwell Matrigel invasion assay.To further detect the effect on TGF-β1 on U87 cell proliferation and invasion,TGF-β1 combined with or without its inhibitor LY364947 was used to treat U87 cells,the cell prolif-eration,invasion were determined according to the previous protocol.Results:Clone formation experiment results showed that compared with the NC group,over-expressing miR-195 or TGF-β1 increased the number of colony,but over-expression of miR-205 reduced the number of colony(P<0.05).MTT test showed that the proliferation ability of U87 cells was significantly increased after up-regulating miR-195 or TGF-β1 expression,but the proliferation ability was significantly decreased after up-regulating miR-205 expression,(P<0.05).Transwell invasion assay results showed that,in U87,over-expression of miR-195 or TGF-β1 can increase the number of cells through the Matrigel membrane(P<0.05),but over-expression of miR-205 can reduce the number of cells through the Matrigel membrane(P<0.05).Conclusion:it was shown that miR-205 over-expression inhibited U87 proliferation and invasion efficiently,miR-195 and TGF-β1 over-expression promoted U87 proliferation and invasion efficiently.Part 3:Study on interaction mechanism about miR-205,miR-195 and SMADs in glioma cellsObjective:To explore the interaction mechanism about miR-205、miR-195 and SMADs(signal molecule of TGF-β1)in glioma cells.Methods:U87 cells were transfected with mimics or inhibitors of miR-205 and miR-195.SMAD proteins were assayed by western blotting.Luciferase assay and co-immunoprecipitation(Co-IP)were used to determine the relationships between miR-205 and SMAD2,miR-195 and SMAD7.Results:It was demonstrated that miR-205 prohibited SMAD2 and phosphorylation of SMAD2(p-SMAD2)expression,but did not affect levels of SMAD3,p-SMAD3,SMAD4 and SMAD7.miR-205 inhibitor enhanced expression of SMAD2 and P-SMAD2 in U87 cells.Otherwise,miR-195 inhibited the expression of SMAD7,there was no influence on the expression of t-SMAD2,t-SMAD3 and SMAD4.In addition,miR-195 inhibitor enhanced expression of SMAD7 in U87 cells.Luciferase activity assay demonstrated that miR-205 enormously decreased the luciferase activity of SMAD2 3’UTR in U87 cells.There was no effect on luciferase activity of 3’UTR of SMAD3,SMAD4 and SMAD7.miR-195 inhibited extremely the luciferase activity of SMAD7 3’UTR in U87 cells.It was also shown that there was no function of miR-195 on SMAD2,SMAD3 and SMAD4.Moreover,high expression of miR-205 significantly inhibited the heteromer formation of SMAD2 and SMAD4.High expression of miR-195 increased heteromer formation.The luciferase activity assay showed that miR-205 and miR-195 inhibitors enhanced luciferase activity of SMAD2-3’UTR and SMAD7-3’UTR,respectively,in U87 cells.miR-205 inhibitor but not miR-195 inhibitor significantly increased the heteromer formation of SMAD2 and SMAD4.Conclusion:1.miR-205 over-expression inhibited the expression of SMAD2 efficiently,miR-195over-expression inhibited the expression of SMAD7 efficiently.2.High expression of miR-205 significantly inhibited the heteromer formation of SMAD2 and SMAD4,high expression of miR-195 increased heteromer formation of SMAD2 and SMAD4.