摘要
目的探讨miR-186靶向Smad6对人胶质瘤细胞增殖的影响。方法运用生物信息学软件预测miR-186的下游靶基因。双荧光素酶报告实验验证miR-186与Smad6的3′-非编码区(3′-UTR)的结合作用。人胶质瘤细胞系U87转染miR-186 mimics或miR-186 inhibitor后,qRT-PCR检测miR-186及Smad6 mRNA的表达;Western blot检测Smad6蛋白的表达水平。Smad6过表达质粒或对照质粒与miR-186 mimics或mimics NC共转染,Western blot检测Smad6蛋白的表达水平;CCK-8和克隆形成实验检测细胞的增殖。结果生物信息学软件预测出Smad6为miR-186的下游靶基因之一。双荧光素酶报告实验发现,相比各自对照组,转染miR-186 mimics或miR-inhibitor后,野生型Smad6 3′-UTR荧光强度相应地明显减弱或增强,而突变了的Smad6 3′-UTR荧光强度没有明显变化。相较于各自的对照组,人胶质瘤细胞系U87转染miR-186 mimics后,Western blot检测发现Smad6蛋白表达下调,而转染miR-186 inhibitor后,Smad6蛋白的表达升高。miR-186 mimics与Smad6过表达质粒共转染后,CCK-8和克隆形成实验检测细胞的增殖情况,发现Smad6过表达可以逆转miR-186 mimics引起的细胞增殖的抑制作用。结论miR-186可通过靶向调控Smad6的表达进而发挥其抑制人胶质瘤细胞增殖的作用。
Objective To investigate the effect of miR-186 targeting Smad6 on regulating proliferation of human glioma cells.Methods The target gene of miR-186 was predicted by bioinformatics tools.The double luciferase reporter assay was used to verify the binding effect of miR-186 on the 3′non-coding region(3′-UTR)of Smad6.The human glioma cell line U87 was transfected with miR-186 mimics or miR-186 inhibitor,and the expression levels of miR-186 and Smad6 were detected by qRT-PCR.The expression level of Smad6 protein was detected by Western blot.Smad6 overexpression plasmid or control plasmid was co-transfected with miR-186 mimics or mimics NC,and the expression level of Smad6 protein was detected by Western blot.CCK-8 and clonal formation assay were used to detect cell proliferation.Results Bioinformatics tools predicted that Smad6 was one of the target gene of miR-186.The dual luciferase reporter assay showed that the fluorescence intensity of the wild-type Smad6 3′-UTR region was significantly weakened or enhanced after the transfection of miR-186 mimics or miR-186 inhibitor when compared to the respective control groups,while the fluorescence intensity was not significantly changed between the mutant-type Smad6 3′-UTR groups and the respective control groups.After transfecting miR-186 mimics or miR-186 inhibitor in human glioma cell line U87,the protein expression level of Smad6 was down-regulated or up-regulated when compared with the respective control groups.After co-transfection of miR-186 mimics with Smad6 overexpression plasmid,CCK-8 and clonal formation experiments were performed to detect cell proliferation,it was found that Smad6 overexpression could reverse the inhibitory effect of miR-186 mimics on cell proliferation.Conclusion miR-186 inhibits the proliferation of human glioma cells by targeting Smad6.
作者
刘艳
邵阿末
曾金艳
胡亚玲
许路
LIU Yan;SHAO Amo;ZENG Jinyan;HU Yaling;XU Lu(Wuxi Higher Health Vocational and Technology School,Wuxi,Jiangsu 214028,China;Central Laboratory,People′s Hospital of Wuxi City,Wuxi,Jiangsu 214023,China)
出处
《重庆医学》
CAS
2019年第16期2718-2722,共5页
Chongqing medicine
基金
江苏省无锡市卫生和计划生育委员会科研面上项目(MS201765)
