摘要
目的探讨mi R-203对胞质多聚腺苷酸化元件结合蛋白4(cytoplasmic polyadenylation element binding protein4,CPEB4)基因的靶向作用及对人脑胶质瘤U87细胞增殖的影响。方法采用免疫组化法检测CPEB4在人脑胶质瘤U87细胞中的表达,生物信息学方法预测mi R-203与CPEB4基因的靶向配对关系,并应用Dual-Luciferase誖报告系统鉴定;将mi R-203 mimics及si RNA转染U87细胞,Real-time PCR法检测mi R-203和CPEB4 m RNA水平,Western blot法检测CPEB4蛋白的表达,平板克隆形成试验检测癌细胞的增殖情况。结果光镜下可见U87细胞均匀分布,但细胞形态多样性明显,胞质内有CPEB4蛋白高表达,呈棕褐色;mi R-203与CPEB4基因靶向配对良好,mi R-203能够抑制CPEB4 m RNA水平。过表达mi R-203能够降低CPEB4 m RNA和蛋白的表达,抑制U87细胞的增殖。结论mi R-203通过负性调控人脑胶质瘤U87细胞中CPEB4基因的表达,进而抑制癌细胞的增殖。
Objective To investigate the targeting of miR-203 to cytoplasmic polyadenylation element binding protein 4（CPEB4）as well as its effect on proliferation of human glioma U87 cells. Methods The expression of CPEB4 in U87 cells was determined by immunohistochemical assay, while the targeting of miR-203 to CPEB4 was analyzed by bioinformatic method and identified by Dual-Luciferase誖report system. U87 cells were transfected with miR-203 mimics and siRNA, in which the miR-203 and CPEB4 mRNA levels were determined by Real-time PCR, and the expression of CPEB4 protein by Western blot. The proliferation level of U87 cells was determined by plate colon formation test. Results U87 cells were distributed evenly under optical microscope, while showed obvious diversity in morphology. CPEB4 in brown color was highly expressed in cytoplasm. The miR-203 was well complementary with CPEB4 gene and inhibited the CPEB4 mRNA expressed. However, the over-expression of miR-203 decreased the expression of CPEB4 mRNA and protein and inhibited the proliferation of U87 cells. Conclusion The miR-203 may inhibit the proliferation of U87 cells through negatively regulating CPEB4 expression.
出处
《中国生物制品学杂志》
CAS
CSCD
2016年第8期819-822,828,共5页
Chinese Journal of Biologicals
