miR-548x and miR-4698 controlled cell proliferation by affecting the PI3K/AKT signaling pathway in Glioblastoma cell lines

Abstract

AbstractGlioblastoma multiforme (GBM) is the most aggressive and prevalent form of brain tumor cancers that originate from glial cells. This study proposed to investigate the effect of miR-548x and miR-4698 on the proliferation and the PI3K/AKT signaling pathway in glioblastoma cell lines. The molecular features of glioblastoma were studied using KEGG and TCGA sites. Next, by using miRwalk 2.0 and TargetScan version 7.1, the microRNAs that target critical genes in the PI3k/AKT pathway were selected according to score. The pre-miR-548x and pre-miR-4698 were cloned in a pCDH plasmid to produced lentiviral vector. The expression levels of miR-548x, miR-4698 and target genes were detected by qRT-PCR. The MTT, cell cycle, annexin and colony formation assay was used to detect the cell proliferation. MiR-548x and miR-4698 predicted target genes (Rheb, AKT1, mTOR, PDK1) were also evaluated by luciferase assay. The expression of AKT was detected by western blotting. Our results described that overexpression of miR-548x and miR-4698 could inhibit proliferation of A-172 and U251 cells. Also, miR-548x promoted the cell cycle arrest of GBM cell lines. The luciferase reporter assay results showed the 3′ UTR of PDK1, RHEB, and mTOR are direct targets of the miR-548x and miR-4698. Too, the western blot analysis revealed that miR-548x and miR-4698 could downregulate the AKT1 protein expression. Overall, our findings suggest that miR-548x and miR-4698 could function as tumor suppressor genes in glioblastoma by controlling the PI3K/AKT signaling pathway and may act as gene therapy for clinical treatment of glioblastoma multiforme.