학술논문
靶向YB-1的microRNA表达载体的构建及其对MB-MDA-231细胞增殖的影响 / MicroRNA137 suppressed human breast cancer cell MB-MDA231 proliferation by directly targeting YB-1 gene
Document Type
Academic Journal
Source
激光杂志 / Laser Journal. 32(4):67-69
Subject
Language
Chinese
ISSN
0253-2743
Abstract
目的：构建靶向YB-1基因的microRNA表达载体,研究其对乳腺癌细胞MB-MDA231增殖的影响。方法：采用microRNA靶基因预测软件预测可能作用于YB-1基因的micro RNA,构建其过表达载体,利用Lipofectamine2000转染细胞MB-MDA231,荧光显微镜下评估转染效果,real-time PCR检测microRNA137表达,real-time PCR、Western blot分别检测YB-1mRNA、蛋白水平的表达,双荧光酶素分析microRNA137作用位点,MTT检测MB-MDA231细胞转染后增殖情况;结果：酶切、测序结果表明pGensill-pre-mir137载体构建成功,real-time PCR结果表明pGensill-pre-mir137实验组细胞较对照组mir137表达明显增高（P〈0.05）;RT-PCR、Westernblot显示实验组转染后YB-1mRNA和蛋白水平表达较对照组均降低（P〈0.05）;双荧光素酶分析显示共转染microRNA137表达载体与荧光报告载体后,荧光素酶活性较对照组降低30%;MTT法检测显示转染实验组较对照组细胞增值能力降低（P〈0.05）。结论：靶向YB-1基因的microRNAl37表达载体构建成功,microRNA137可通过靶向YB-1的3’非编码区,抑制MB-MDA231 YB-1基因的表达而影响其增殖。
AIM：To investigate the inhibition effects of microRNA targeting Y-box binding protein 1（YB-1）gene on the proliferation of human breast cancer cell MB-MDA231.Methods：A series of bioinformatics analysis were performed to predict possible microRNA targeting the 3 UTR of YB-1 gene.According to the results from bioinformatics analysis,the microRNA overexpression pGensill-pre-mir137 plasmid was constructed.The vector was transfected into MB-MDA231 cells by lipofectamine,and the expression of microRNA was detected by real-time PCR.Then the expressions of YB-1 mRNA and protein were analyzed by real-time PCR and Western blot analysis respectively.To evaluate the relationship of microRNA with 3＇UTR of YB-1 gene,the dual luciferase assays were performed,and MTT assays were used to observe the inhibitory effect of microRNA on cell proliferation in vitro.Results：pGensil 1-pre-mir 137 plasmid was successfully constructed and identified by enzyme restriction digestion and sequence analysis.Compared with control groups,the expression of microRNA137 was increased remarkably（P0.05）, the expression of YB-1 mRNA and protein level were significantly reduced（P 0.05）.In MB-MDA231 cotransfected by pGensill-pre-mir137 and pMIR-Luc-YB1 plasmids,luciferase reporter activities reduced by 30% compared with that in MB-MDA231 cotransfected by pGensill and pMIR-Luc-YB 1 plasmids.The growth ability of cells in experimental group were lower than that of control groups（P0.05）.Conclusions The microRNA137 overexpression plasmid is successfully constructed,and microRNA 137 can suppress human breast cancer cell proliferation by directly targeting 3＇ UTR of YB-1 gene.
AIM：To investigate the inhibition effects of microRNA targeting Y-box binding protein 1（YB-1）gene on the proliferation of human breast cancer cell MB-MDA231.Methods：A series of bioinformatics analysis were performed to predict possible microRNA targeting the 3 UTR of YB-1 gene.According to the results from bioinformatics analysis,the microRNA overexpression pGensill-pre-mir137 plasmid was constructed.The vector was transfected into MB-MDA231 cells by lipofectamine,and the expression of microRNA was detected by real-time PCR.Then the expressions of YB-1 mRNA and protein were analyzed by real-time PCR and Western blot analysis respectively.To evaluate the relationship of microRNA with 3＇UTR of YB-1 gene,the dual luciferase assays were performed,and MTT assays were used to observe the inhibitory effect of microRNA on cell proliferation in vitro.Results：pGensil 1-pre-mir 137 plasmid was successfully constructed and identified by enzyme restriction digestion and sequence analysis.Compared with control groups,the expression of microRNA137 was increased remarkably（P0.05）, the expression of YB-1 mRNA and protein level were significantly reduced（P 0.05）.In MB-MDA231 cotransfected by pGensill-pre-mir137 and pMIR-Luc-YB1 plasmids,luciferase reporter activities reduced by 30% compared with that in MB-MDA231 cotransfected by pGensill and pMIR-Luc-YB 1 plasmids.The growth ability of cells in experimental group were lower than that of control groups（P0.05）.Conclusions The microRNA137 overexpression plasmid is successfully constructed,and microRNA 137 can suppress human breast cancer cell proliferation by directly targeting 3＇ UTR of YB-1 gene.