miRNA-451调控人脑胶质瘤细胞上皮-间质转化发生的机制研究

miRNA-451调控人脑胶质瘤细胞上皮-间质转化发生的机

制研究

张亚辉;郭红宝;南阳;甄英伟;王乐;管强;俞凯;黄强;钟跃

【期刊名称】《中华神经外科杂志》 【年(卷),期】2017(033)004

【摘要】目的 研究miRNA-451过表达后通过靶标钙结合蛋白39(CAB39)调控人脑胶质瘤细胞上皮-间质转化发生的机制,以及miRNA-451过表达对胶质瘤细胞侵袭和迁移能力的影响.方法 培养U251胶质瘤细胞,将其分为空白对照组、无义序列组以及miRNA-451 mimics处理组(简称miRNA-451组).采用实时定量PCR检测转染后U251细胞miRNA-451的表达;以Western blot法检测CAB39、E-钙黏素、N-钙黏素、波形蛋白、TWIST、人基质金属蛋白酶2(MMP-2)、人基质金属蛋白酶9(MMP-9)、Snail、蛋白激酶B(AKT)、磷酸化蛋白激酶B(P-AKT)的表达水平;通过细胞免疫荧光实验进一步验证N-钙黏素、波形蛋白的表达水平;采用划痕实验和Transwell实验检测细胞侵袭和迁移能力的变化.结果 与空白对照组及无义序列组相比,miRNA-451组的miRNA-451相对表达水平明显升高(均P ＜0.05).Western blot结果显示,与空白对照组及无义序列组相比,miRNA-451组CAB39、N-钙黏素、波形蛋白、TWIST、MMP-2、MMP-9、Snail、P-AKT的表达水平降低(均P＜0.05),而E-钙黏素的表达水平升高(均P ＜0.05).细胞免疫荧光实验结果显示,miRNA-451组的N-钙黏素、波形蛋白的表达水平相对于空白对照组明显降低(均P ＜0.05).划痕操作后24 h、48 h miRNA-451组的划痕修复率与空白对照组及无义序列组相比均明显降低(均P＜0.05).转染48 h后miRNA-451组穿过滤膜的细胞数目与

空白对照组及无义序列组相比显著减少(均P ＜0.05).结论 miRNA-451可能通过降低靶标CAB39的表达调控PI3K/AKT通路,抑制人脑胶质瘤细胞中上皮-间质转化现象的发生,从而抑制人脑胶质瘤细胞的侵袭和迁移能力.%Objective To investigate the mechanism of miRNA-451 overexpression in the regulation of epithelial-mesenchymal transition(EMT) in human glioma cells through target calcium binding protein 39 (CAB39) and to explore the effects of miRNA-451 overexpression on invasion and migration of glioma cells.Methods The U251 glioma cells were cultured and divided into 3 groups including control group,nonsense sequence group and miRNA-451 mimics treatment group (briefly miRNA-451 group).The expression of miRNA-451 was detected by RT-PCR in glioma cells after transfection.The

expression

proteins

of

CAB39,E-cadherin,N-

cadherin,Vimentin,TWIST,MMP-2,MMP-9,Snail,AKT,P-AKT were detected by Western blot.Cell immunofluorescence assay was used to further validate the expression levels of N-cadherin and Vimentin.The wound healing and transwell experiments were performed to detect changes in cell invasion and migration abilities.Results The miRNA-451 expression level in the miRNA-451 group was significantly higher compared with those in the control and nonsense sequence groups (both P ＜ 0.05).The results of Western blot revealed that the expression levels of CAB39,N-cadherin,Vimentin,TWIST,MMP-2,MMP-9,Snail,P-AKT

in

miRNA-451

group were decreased (all P ＜ 0.05) and the expression level of E-

cadherin was increased as compared with those in the control and nonsense sequence groups (all P ＜ 0.05).Cell immunofluorescence assay showed that the relative expressions of N-cadherin and Vimentin in miRNA-451 group were significantly lower than those in control group (both P ＜ 0.05).The results of the wound healing showed that the scar repair rate of miRNA-451 group was significantly lower than those in the control and nonsense groups at 24 hrs or 48 hrs post would healing (all P ＜ 0.05).The number of cells in the miRNA-451 group was significantly decreased as compared with the other two groups at 48 hrs post transfection (both P ＜ 0.05).Conclusion The regulation of PI3K/AKT pathway by miRNA-451 might be through the decrease of CAB39 expression,which could cause the inhibition of EMT in human glioma cells and their invasion and migration. 【总页数】6页(392-397)

【关键词】微RNAs;神经胶质瘤;上皮-间质转化;肿瘤侵润;微RNA-451 【作者】张亚辉;郭红宝;南阳;甄英伟;王乐;管强;俞凯;黄强;钟跃

【作者单位】300052 天津医科大学总医院神经外科,天津市神经病学研究所神经肿瘤实验室;300052 天津医科大学总医院神经外科,天津市神经病学研究所神经肿瘤实验室;300052 天津医科大学总医院神经外科,天津市神经病学研究所神经肿瘤实验室;300052 天津医科大学总医院神经外科,天津市神经病学研究所神经肿瘤实验室;300052 天津医科大学总医院神经外科,天津市神经病学研究所神经肿瘤实验室;300052 天津医科大学总医院神经外科,天津市神经病学研究所神