过表达miR-1182抑制脑胶质瘤细胞恶性表型的机制研究

Mechanism of micro RNA-1182 overexpression in inhibiting malignant phenotype of glioma cells

目的探讨微小RNA(miR)-1182在脑胶质瘤中的表达情况及其过表达对脑胶质瘤细胞恶性表型的调控作用及机制。方法(1)从中国脑胶质瘤基因组图谱计划(CGGA)官方网站中下载198例脑胶质瘤标本的miR-1182表达水平数据及患者的生存期信息,比较不同病理类型、不同WHO分级脑胶质瘤组织间miR-1182表达水平的差异,并采用Kaplan-Meier生存曲线分析miR-1182的表达水平与患者生存期的关系。(2)体外常规培养人脑胶质瘤细胞系A172、LN229、T98G、U87、U251及人正常星形胶质细胞系NHA,采用实时定量PCR(qPCR)法检测各组细胞中miR-1182的表达水平。(3)将U87、U251细胞分别分为miR-1182转染组与阴性序列转染组,分别转染miR-1182模拟物和miR-1182阴性序列,转染48 h后采用5-乙炔基-2'-去氧尿苷(EdU)染色检测2组细胞的增殖能力,流式细胞仪检测2组细胞的凋亡水平,Transwell实验检测2组细胞的迁移能力,Western blotting实验检测2组细胞中周期蛋白(c-Myc、c-JUN、CCND1、P21)及磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)、上皮-间充质转化(EMT)通路相关蛋白[PI3K、磷酸化(p)-PI3K、Akt、p-Akt、N-钙黏蛋白、β-连环蛋白、波形蛋白]的表达水平。结果(1)WHOⅢ、Ⅳ级脑胶质瘤组织中miR-1182的表达水平均明显低于WHOⅡ级,差异均有统计学意义(P<0.05)。miR-1182低表达脑胶质瘤患者的中位生存期[(701.00±11.14)d]明显短于miR-1182高表达患者[(1812.00±23.21)d],差异有统计学意义(P<0.05)。(2)与NHA细胞比较,A172、LN229、T98G、U87、U251细胞中miR-1182的表达水平均明显降低,差异均有统计学意义(P<0.05),其中U87、U251细胞降低最明显。(3)与阴性序列转染组比较,miR-1182转染组U87、U251细胞的增殖能力明显减弱,细胞凋亡率明显升高,穿膜细胞数明显减少,c-Myc、c-JUN、CCND1蛋白的表达水平明显降低,P21蛋白的表达水平明显升高,PI3K、p-PI3K、Akt、p-Akt蛋白的表达水平明显降低,N-钙黏蛋白、β-连环蛋白、波形蛋白的表达水平明显降低,差异均有统计学意义(P<0.05)。结论miR-1182低表达脑胶质瘤患者的预后较差。miR-1182在脑胶质瘤细胞中呈低表达。过表达miR-1182可通过调控细胞周期蛋白及PI3K/Akt、EMT通路相关蛋白的表达来抑制脑胶质瘤细胞的恶性表型。

Objective To investigate the micro RNA(miR)-1182 expression in glioma,and explore the regulation role and mechanism of miR-1182 overexpression in malignant phenotype of glioma cells.Methods(1)The data of miR-1182 expressions of 198 glioma samples and survival of these glioma patients were downloaded from the official website of Chinese Glioma Genome Atlas(CGGA),and the differences of miR-1182 expression levels among glioma tissues of different pathologic types and different WHO grades were compared.Kaplan-Meier survival curve was used to analyze the relation between miR-1182 expression level and patient survival.(2)Human glioma cell lines A172,LN229,T98G,U87,and U251,and human normal astrocyte cell line NHA were routinely cultured in vitro,and the miR-1182 expression levels in each group were detected by real-time quantitative PCR(qPCR).(3)U87 and U251 cells were divided into miR-1182 transfection group and negative control group;the miR-1182 mimics and miR-1182 negative control sequence were transfected,respectively.After 48 h of transfection,5-ethynyl-2'-deoxyuridine(EdU)staining was used to detect the cell proliferation ability,flow cytometry was used to detect the cell apoptosis,Transwell assay was used to detect the cell migration ability,and Western blotting was used to detect the expression levels of cyclin(C-myC,C-Jun,CCND1,and P21),phosphatidylinositol 3 kinase/protein kinase B(PI3K/Akt),and epithelial-mesenchymal transformation(EMT)pathway related proteins(N-cadherin,β-catenin,and vimentin).Results(1)The miR-1182 expressions in glioma tissues of WHO grading III and IV were significantly lower as compared with those in glioma tissues of WHO grading II(P<0.05).The median survival time in patients from the low miR-1182 expression group([701.00±11.14]d)was significantly shorter than that in the high miR-1182 expression group([1812.00±23.21]d,P<0.05).(2)As compared with that in NHA cell group,the miR-1182 expression levels in A172,LN229,T98G,U87 and U251 cell groups were significantly decreased(P<0.05),and the decrease was most significant in U87 and U251 cell groups.(3)As compared with the negative control group,the U87 and U251 cells in miR-1182 transfection group had significantly weaker proliferation ability,significantly higher apoptosis rate,significantly decreased number of transmembrane cells,significantly decreased protein expression levels of C-MyC,C-Jun and CCND1,significantly increased P21 protein expression level,significantly decreased expression levels of PI3K,phosphorylated(p)-PI3K,Akt and p-Akt,and significantly decreased expression levels of N-cadherin,β-catenin and vimentin(P<0.05).Conclusions Glioma patients with low miR-1182 expression have poor prognosis.Low miR-1182 expression is noted in glioma cells.Overexpression of miR-1182 can inhibit the malignant phenotype of glioma cells,which may be related to cell cycle-related proteins,PI3K/Akt,and EMT pathway ralated proteins.