摘要
目的 寻找微小RNA（miR）-7在表皮生长因子受体（EGFR）通路下游的作用靶点,探讨其抑制胶质瘤生长的内在机制.方法 瞬时转染人CHG5胶质瘤细胞miR-7寡核苷酸序列,实时定量逆转录聚合酶链反应（RT-qPCR）检测miR-7水平；噻唑蓝（MTT）比色法绘制细胞生长曲线,Transwell小室实验检测瘤细胞迁移能力,软琼脂克隆形成实验检测瘤细胞致瘤潜能；Western blot方法检测磷酸肌醇3激酶（PI3K）、Raf-1、细胞周期素（Cyclin） D1、磷酸化苏氨酸激酶（p-AKT）和磷酸化丝裂原细胞外激酶1/2（p-MEK1/2）的蛋白表达；利用TargetScan和Saner软件共同分析预测miR-7的潜在靶点；荧光素酶实验验证miR-7与PI3K和Raf-1间的靶关系.结果 与脂质体组和无义序列组比较,miR-7组瘤细胞增殖速度减慢,瘤细胞迁移力降低,肿瘤克隆能力减弱（P＜0.05）；Western blot结果显示EGFR下游通路成员表达均有不同程度下降；荧光素酶实验证实PI3K和Raf-1均是miR-7的直接作用靶点.结论 miR-7通过同时调控EGFR下游PI3K/ATK和Raf/MEK/ERK两条通路发挥肿瘤生长抑制作用.
Objective To search for the targets of microRNA （miR）-7 in the epidermal growth factor receptor （EGFR） downstream pathway,and study the internal mechanism of glioma growth inhibition by miR-7.Methods After CHG5 glioma cells were transiently transfected with miR-7 sequence,reverse transcriptase quantitative PCR （RT-qPCR） method was used to detect the gene transfection.Methyl thiazol tetrazolium （MTT） assay was used to draw cell growth curves,Transwell assay to detect cell migration,and the soft agar colony formation assay to detect tumorigenicity.Western blotting was used to detect the expression of phosphatidylinositol 3 kinase （PI3K）,Raf-1,Cyclin D1,p-protein kinase B （AKT） and pmitogen extracellular kinase 1/2 （p-MEK1/2）.The TargetScan and Sanger softwares were used to co-predict the potential targets of miR-7.Luciferase experiments were used to test the relationship between miR-7 and PI3K or Raf-1.Results As compared with control and scramble groups,the ability of proliferation and migration,and clone of cells in miR-7 group were obviously declined （P 〈 0.05）.Western blotting results showed that the expression of EGFR downstream members was decreased.Luciferase experiments confirmed that both PI3K and Raf-1 were the direct targets of miR-7.Conclusion miR-7 can inhibit glioma growth by simultaneously regulating PI3K/ATK and Raf/MEK/ERK pathways both in EGFR downstream pathway.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2014年第1期98-101,共4页
Chinese Journal of Experimental Surgery
基金
国家973计划子项目（2010CB529405）
国家自然科学基金资助项目（81000901）
天津市科技创新体系及条件平台建设计划重大项目（10SYSYJC28800）
天津市滨海新区医药卫生科研基金资助项目（2012BWKZ001）
