miR-543对胶质瘤SHG-44细胞侵袭、迁移的影响及机制研究  

Research on the Effect and Mechanism of miR-543 on Invasion and Migration of SHG-44 Glioma Cells

作　　者：赵云东[1] 宋玉琴[2] 刘娜[3] 孙冬弢[1] ZHAO Yundong;SONG Yuqin;LIU Na;SUN Dongtao(Second Affiliated Hospital of Shenyang Medical College,Shenyang 110035;Sixth People＇s Hospital of Shenyang,Shenyang 110035;Seventh People＇s Hospital of Shenyang,Shenyang 110035)

机构地区：[1]沈阳医学院附属第二医院,沈阳110035 [2]沈阳市第六人民医院,沈阳110035 [3]沈阳市第七人民医院,沈阳110035

出　　处：《宁夏医科大学学报》2018年第7期770-775,共6页Journal of Ningxia Medical University

基　　金：辽宁省科学技术计划项目(20170540873)

摘　　要：目的探讨miR-543在胶质瘤SHG-44细胞侵袭、迁移中的作用、分子靶点及可能的作用机制。方法采用miR-543 mimic、inhibitor及各自NC序列转染胶质瘤SHG-44细胞,获得5组细胞,分别分为Control组(无转染)、mimic NC组(转染mimic NC片段)、inhibitor NC组(转染inhibitor NC片段)、miR-543 mimic组(转染miR-543 mimic)、miR-543 inhibitor组(转染miR-543 inhibitor);Real-time PCR检测各组转染后miR-543表达水平;划痕实验检测细胞迁移能力;Transwell小室实验检测细胞侵袭能力;明胶酶谱实验检测细胞上清基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)活性;Real-time PCR及Western blot检测细胞中微小RNA 543(miR-543)潜在作用靶点局部黏着斑激酶(FAK)、上皮间质转化调控蛋白(TWIST1)、多囊肾病蛋白2(PKD2)mRNA及蛋白水平的表达;Western blot检测细胞中蛋白激酶B(AKT)通路活化情况。结果与Control组相比,mimic NC组及inhibitor NC组细胞的侵袭及迁移能力、FAK、TWIST1、PKD2、MMP-2、MMP-9表达及AKT的磷酸化水平差异均无统计学意义(P均>0.05);与mimic NC组相比,miR-543 mimic组能够有效抑制SHG-44细胞的迁移及侵袭能力,抑制MMP-2、MMP-9活性,抑制FAK、TWIST1、PKD2分子和蛋白水平的表达,抑制AKT的磷酸化(P均<0.01);与inhibitor NC组相比,miR-543 inhibitor组细胞迁移能力增强,侵袭能力升高,MMP-2、MMP-9活性增强,FAK、TWIST1、PKD2表达上调,AKT通路磷酸化水平升高(P均<0.05)。结论 miR-543可能通过对下游靶基因的调节影响AKT通路活化,进而影响胶质瘤SHG-44细胞的迁移及侵袭。Objective To investigate the effect,molecular targets and possible mechanisms of miR-543 on invasion and migration of SHG-44 glioma cells. Methods miR-543 mimic,inhibitor and corresponding NC sequences were transfected into SHG-44 glioma cells. Cells were divided into five groups,which were Control group,miR-543 mimic group,mimic NC group,miR-543 inhibitor group and inhibitor NC group. Real-time PCR was used to detect the expression of miR-543. Wound healing assay was used to detect cell migration ability. Transwell assay was used to detect cell invasion ability. Gelatin zymography was used to detect the activity of MMP-2 and MMP-9. Real-time PCR and Western blot were used to detect the m RNA and protein expression levels of FAK,TWIST1 and PKD2. The activation of AKT pathway was detected by Western blot.Results Compared with the Control group,the mimic NC group and the inhibitor NC group had no significant difference of the ability in cell invasion and migration（P〉0.05）. In addition,the expression of FAK,TWIST1,PKD2,MMP-2,MMP-9 and p-AKT also had no distinct difference（P〉0.05）. But compared with the mimic NC group,miR-543 mimic transfection effectively inhibited the migration and invasion of SHG-44 cells,degraded the activity of MMP-2,MMP-9 and the expression of FAK,TWIST1,PKD2（P〈0.01）. The phosphorylation of AKT was also significantly higher（P〈0.01）. However,compared with the inhibitor NC group,miR-543 inhibitor group increased the ability of cell migration and invasion,and the activity of MMP-2 and MMP-9 upregulated the expression of FAK,TWIST1,PKD2 and the phosphorylation of AKT（P〈0.01）. Conclusion miR-543 may affect the activation of AKT pathway through the regulation of downstream target genes,thereby affecting the migration and invasion of SHG-44 glioma cells.

关 键 词：胶质瘤 微小RNA-543 蛋白激酶B 

分 类 号：R739.41[医药卫生—肿瘤]