MicroRNA-148a Promotes Human Glioma Cell Migration And Invasion By Targeting GADD45

Objective:This study aims to investigate the effect of mir-148a on the migration and invasion of glioma cells by searching for the target gene of mir-148a and verifying the interaction between mir-148a and target genes.Methods1.We used the bioinformatics prediction software TargetScan,miRanda,PicTar,PITA to screen target genes of mir-148a;2.We used RNA pull down to screen mir-148a target genes;3.We used luciferase reporter gene to verify the target gene of mir-148a;4.We used qRT-PCR and Western Blot methods to detect the expression of gene RNA and protein;5.We used transwell migration and scratch experiments to evaluate cell migration and invasion of gliomas.Result:1.We used the bioinformatics prediction software TargetScan,miRanda,PicTar,and PITA to predict target genes for mir-148a,and 7402,434,and 1847 mir-148a targets were obtained respectively,and total of 312 genes are the intersection of three prediction software prediction results.GADD45A-2 is one of potential target gene of mir-148a.2.We performed RNA pull down using biotin assay and found GADD45A-2 was the target gene of mir-148a.3.We used fluorescence reporter gene activity assays to confirm that mir-148a binds to the GADD45 3’UTR region and inhibits the fluorescence intensity of its reporter gene.This result indicates that miR-148a regulates gene expression through this site on the 3’ UTR of GADD45A gene,and GADD45 is a target gene.of mir-148a.We also found GADD45 expression was significantly downregulated in glioma cell line compared with normal glial cell(P<0.05).4.We constructed a GADD45 overexpression lentiviral vector(pLent-GADD45)and infected glioma U251 and U87 cell lines to establish a GADD45 overexpression stable cell line.5.We found that GADD45 was down-regulated in gene and protein levels when mir-148a was over-expressed,and the opposite was found when mir-148a was inhibited.Combined with the above results,it was demonstrated that mir-148a regulates GADD45.6.We found that mir-148 regulated U87,U251 migration and invasion ability mediated by GADD45 by using transwell migration and scratch assay experiments.Conclusion:1.GADD45 is the target gene of mir-148a.2.Mir-148a promotes human glioma cell migration and invasion by targeting GADD45.