The Function Study Of Let-7a In Glioma Cells And Glioma Stem Cells

Objective:Investigate the effect and mechanism of let-7a in glioma cells and glioma stem cells(GSCs).Methods:Transient transfection of Let-7a mimics to U251 and U87 cells to investigate the effects of let-7a on the proliferation,migration,invasion of glioma cells and self-renewal of GSCs.The serum-free culturing technology was used to enrich GSCs and qRT-PCR was performed to identify the expression level of let-7a.Proliferation of glioma cells and GSCs were determined by the CCK-8 assay.Wound healing assay and Transwell assay were used to detect the role of let-7a on cell migration and invasion.The self-renewal ability of GSCs was mesured by tumor sphere formation assay.The target genes of let-7a were identified using bioinformatics analysis and dual-luciferase activity assay.Western blot analysis was used to detect the protein expression levels of E2F2.SiRNA-E2F2 was used to knockdown E2F2 in glioma cells and GSCs.The effect of the E2F2 on cell proliferation and GSCs self-renewal was detected by CCK-8 assay and tumor sphere formation assay.RESULTS:1)48h posttransfection with Let-7a mimics,q-PCR detected the expression levels of let-7a in U251,U87 cells and the GSCs,indicating that let-7a was dramatically elevated compared with cells transfected with miR-NC.2)Overexpression of let-7a suppresses proliferation,migration and invasion of the glioma cells.3)Upregulation of let-7b inhibits the proliferation and self-renewal ability of glioma stem cells.4)Bioinformatics analysis predicts E2F2 and IMP2 are candidate targets of let-7a,qRT-PCR and western blot analysis showed that mRNA and protein expression levels of the E2F2 were decreased in U251,U87 cell and U251-GSC,U87-GSC after overexpressing let-7a.Expression of IMP2 mRNA levels and protein levels were suppressed in U251,U87 cells,but there was no significant difference in IMP2 mRNA and protein levels in U251-GSC,U87-GSC after overexpressing let-7a.5)Dual-luciferase assay indicated that the overexpression of let-7b reduced the activity of a luciferase reporter fused to the wild-type E2F2 3’UTR.E2F2 is a direct target of let-7a in glioma cells.6)Synthesized siRNA-E2F2 was transfected to suppress the protein expression levels of E2F2 in U251,U87 cells and GSCs.Downregulation of E2F2 inhibits the proliferation of glioma cells and the self-renewal of GSCs.Conclusion:Overexpression of let-7a reduced the proliferation,migration and invasion of U251 and U87 cells.Let-7a also inhibited the proliferation and self-renewal of GSCs.As a direct target gene of let-7a,E2F2 regulates the proliferation of glioma cells and self-renewal of GSCs.