MiR-148a-3p Regulates Proliferation,Migration And Invasion Of Glioma Cells By Targeting CUL5

Purpose:Glioma is the most common malignant tumor of the central nervous system.The traditional treatment has a poor prognosis and a short median survival time,especially in patients with high-grade infiltrating glioblastoma,which causes serious damage to the nervous system.MicroRNAs(miRNAs)are a class of non-coding,single-stranded,small-molecule RNAs.They regulate cell biological processes by regulating messenger RNAs which involved in cellular inflammation,growth,stress response,differentiation,and apoptosis.Recent studies have shown that abnormal miRNA expression levels are closely related to the development of gliomas.They are involved in the molecular pathogenesis of gliomas and are potential tumor molecular markers and therapeutic targets.Previous studies have shown that miR-148a-3p promotes the development of glioma and is negatively correlated with patient survival.This study is based on the human glioma cell line,aiming to study the molecular mechanism of miR-148a-3p promoting the progression of glioma,and laying the foundation for the clinical application as a new diagnosis and treatment of glioma of miR-148a-3p.Methods: 1.We utilized Bioinformatics software to predict the target gene of miR-148a-3p.2.We constructed miR-148a-3p overexpression and anti-expression and its negative control lentivirus in glioma cells and screened for stable expressing miR-148a-3p glioma cells by puromycin,using qRT-PCR to detect the expression efficiency of miR-148a-3p.3.We utilized real-time PCR and Western blot to test the expression levels of target genes in glioma cells which overexpressing and inhibiting expression of miR-148a-3p.4.We utilized dual luciferase reporter gene assay to verify the targeting relationship between CUL5 and miR-148a-3p.5.We utilized real-time PCR and Western blot to detect the mRNA and protein expression level of CUL5 in glioma cell lines and normal glial cells.6.We constructed lentivirus expressing the CUL5 and co-expressing miR-148a-3p and CUL5 and its negative control lentivirus in glioma cells and screened for stable expressing glioma cells by transfecting puromycin.7.We utilized CCK-8 and plate cloning experiments to detect the effect of CUL5 on the proliferation of glioma.8.We utilized scratch and Transwell experiments to detect the effect of CUL5 on the migration ability of gliomas.9.We utilized transwell assay to test the effect of CUL5 on the invasion ability of glioma.10.Finally,the function of CUL5 gene was rescued,We utilized cck-8 and plate cloning,scratches and Transwell assay to detect the effect of miR-148a-3p expression on the proliferation,migration and invasion of CUL5 inhibiting glioma cells.Results: 1.Bioinformatics software initially predicted that CUL5 was one of the target genes of miR-148a-3p.2.Successfully constructed a stable cell line that overexpresses and suppresses the expression of miR-148a-3p in U87,U251,BT325 and T98 G.3.Overexpression of miR-148a-3p can inhibit CUL5 mRNA and protein levels,and supression of miRNA-148a-3p can promote CUL5 expression.4.Dual luciferase reporter gene experiments demonstrated that CUL5 was a direct target gene of miR-148a-3p.5.CUL5 was expressed in both glioma cell lines and normal glial cells.Comparing with normal glial cells HA1800,the mRNA and protein expression levels of CUL5 up-regulated in glioma cell lines U87,U251,T98 G,BT325(The difference was statistically significant(P < 0.05)).6.CCK-8 and plate cloning experiments show that CUL5 can inhibit the proliferation of glioma cell lines;scratch and Transwell experiments show that CUL5 can inhibit the migration of glioma cell lines;Transwell experiments show that overexpression of CUL5 can inhibit Invasion of glioma cell lines.7.Overexpression of miR-148a-3p inhibited the inhibition of proliferation,migration and invasion of glioma induced by overexpression of CUL5.Conclusions: 1.CUL5 is one of the direct target genes of miR-148a-3p.2.Successfully constructed a glioma stable cell line that overexpresses and inhibits the expression of miR-148a-3p and overexpressing CUL5 and simultaneously overexpressing miR-148a-3p and CUL5.3.CUL5 inhibits the proliferation,migration and invasion in glioma cells.4.MiR-148a-3p promotes the proliferation,migration and invasion in glioma cells by targeting CUL5.