Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.
Molecular Virology lab, Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran.
Sci Rep. 2020 Jan 31;10(1):1558. doi: 10.1038/s41598-020-57588-5.
Glioblastoma 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.
胶质母细胞瘤（GBM）是源自神经胶质细胞的最具侵袭性和最常见的脑肿瘤癌。本研究旨在探讨 miR-548x 和 miR-4698 对胶质母细胞瘤细胞系增殖和 PI3K/AKT 信号通路的影响。使用 KEGG 和 TCGA 网站研究胶质母细胞瘤的分子特征。接下来，通过使用 miRwalk 2.0 和 TargetScan 版本 7.1，根据分数选择靶向 PI3k/AKT 通路关键基因的 microRNAs。将 pre-miR-548x 和 pre-miR-4698 克隆到 pCDH 质粒中，以产生慢病毒载体。通过 qRT-PCR 检测 miR-548x、miR-4698 和靶基因的表达水平。通过 MTT、细胞周期、 Annexin 和集落形成测定法检测细胞增殖。还通过荧光素酶测定评估 miR-548x 和 miR-4698 预测的靶基因（Rheb、AKT1、mTOR、PDK1）。通过 Western blot 检测 AKT 的表达。我们的结果表明，miR-548x 和 miR-4698 的过表达可抑制 A-172 和 U251 细胞的增殖。此外，miR-548x 促进 GBM 细胞系的细胞周期停滞。荧光素酶报告基因测定结果表明，PDK1、RHEB 和 mTOR 的 3'UTR 是 miR-548x 和 miR-4698 的直接靶标。此外，Western blot 分析表明，miR-548x 和 miR-4698 可以下调 AKT1 蛋白表达。总之，我们的研究结果表明，miR-548x 和 miR-4698 可通过控制 PI3K/AKT 信号通路作为胶质母细胞瘤的肿瘤抑制基因发挥作用，并且可能作为胶质母细胞瘤多形性的临床治疗的基因治疗。
