MicroRNA-29a Exerts An Anticancer Effect By Modulating Sp1 In Glioma Cells

Background and Objective:Glioma is the common intracranial malignant tumor,accounting for 27% of the central nervous system tumors,and the trend of incidence is increasing year by year,the annual growth rate of 1%-2%,of which about 75% of the tumors were found in the middle-late stage.Although through comprehensive treatment such as surgery,radiation and chemotherapy,the 5-year survival rate is still very low.The pathogenesis of glioma is still not completely clear,Extensive date shows that the comprehensive factors such as physical,chemical,virus,and genetic are closely related with the occurrence of glioma.Our study found that the expression of transcription factor Sp1 was elevated in glioma cells,which was consistent with previous studies that Sp1 was able to promote tumorigenesis and progression,but the concrete mechanism of action was unclear.The regulation of gene expression by microRNAs is achieved by complete or incomplete complementation of the 3'UTR seed sequence of a particular target gene,and studies have shown that changes in microRNAs expression are detected in the progression of tumors.It is predicted that multiple microRNAs can target the 3'UTR region of Sp1 and we find that microRNA-29a(miR-29a)is low in gliomas,so we speculate that there exists a connection between the transcription factor Sp1 and miR-29 a.This study aimed to explore the microRNA regulatory mechanism of Sp1 expression in gliomas,and to provide the exact experimental theoretical basis for the diagnosis and treatment of glioma.Methods:(1)The expression level of Sp1 mRNA and miR-29 a were detected by Real-Time PCR in human normal glial cells and 4 glioma cells respectively.To detect the expression of Sp1 protein using Western blot.(2)The expression of Sp1 mRNA and protein was detected after miR-29 a overexpression by miR-NC/29 a mimcs and si-NC/Sp1,RealTime PCR and Western Blot respectively after transient transfection with Lipofectamine 2000.(3)Bioinformatics analysis software TargetSpy and online database Targetscan,miRBase prediction shows that miR-29 a can target Sp1 3'UTR region,with dual fluorescence experiment testing whether miR-29 a directly binds with the Specific sequence of Sp1 3'UTR region,used to confirm its direct targeting effect.(4)Transwell assay was used to detect the metastatic and invasive ability of U87 and A172 cells.After transient transfection of miR-29 a in U87 and A172 cells,Real-Time PCR and Western Blot were used to detect the interference effect of Sp1,next,Transwell was used to detect the metastatic and Invasive changes of U87 and A172 cells following Sp1 interference;and Transwell assay was used to detect the changes of cell metastasis and invasion ability after Simulator miR-29 a was transiently transfected into U87 and A172 cells.Results:(1)The expression of Sp1 mRNA and protein in glioma cell lines U87?A172?U251 and SHG44 was increased compared with OLN93,the expression of transcription factor Sp1 in gliomas is highly expressed.(2)The expression of miR-29 a was down-regulated in glioma cell lines U87?A172?U251 and SHG44 compared with OLN93,the expression level of miR-29 a was found to be decreased in gliomas.(3)MiR-29 a inhibited the expression of Sp1 by direct targeting of Sp1 3'UTR region in U87 and A172 cells: the expression of Sp1 mRNA and protein was down-regulated after miR-29 a overexpressed in U87 and A172 cells.To confirm the direct targeting effect of miR-29 a and Sp1,The results of the double fluorescence experiments showed that When U87 and A172 cells were transfected with wild-type vector,the expression was down regulated,whereas the co-transfected mutant vector did not change,Confirming that miR-29 a directly targets the Sp1 3 ' UTR region.(4)Phenotypic study of miR-29 a overexpressed in U87 and A172 cells: After overexpression of miR-29 a in U87 and A172 cells,The metastasis and invasion ability of cells were all significantly inhibited After interference with Sp1,the metastasis and invasion ability of cells were curbed universally.Conclusion:In glioma cells,the expression of miR-29 a is low and Transcription factor Sp1 is highly expressed,and miR-29 a is capable of direct targeting of 3'UTR Specific sequences of Sp1.Of miR-29 a and Sp1 functional analysis showed that miR-29 a can inhibit tumor metastasis and invasion,while Sp1 can enhance the ability of tumor metastasis and invasion.After the overexpression of miR-29 a,the expression of Sp1 mRNA and protein were down-regulated.Therefore,miR-29 a may be a tumor suppressor gene,During the progression of gliomas,by inhibiting the expression of Sp1 to inhibit the development of tumors.By studying the inhibitory effect of miR-29 a on Sp1 gene,it may be helpful to improve our understanding of the pathogenesis of gliomas and provide a reliable theoretical basis for the diagnosis and treatment of gliomas.