基于生物信息学分析研究miR-182-5p通过靶向调控EP300促进乳腺癌细胞的侵袭和转移

近期研究表明,miR-182-5p对多种癌症的侵袭和转移具有重要作用,但其在乳腺癌侵袭转移中的研究相对较少。本研究通过网上在线microRNA分析工具下载乳腺癌组织及正常乳腺组织表达比较的数据集,分析发现在GSE4589、GSE38167、GSE61438等3个数据库中,在乳腺癌组织中存在26个相同的microRNA,其中8个上调,而我们实验验证发现hsa-miR-182在8例病理组织中的表达上调差异最显著(P=0.001),选定目的基因hsa-miR-182;qRT-PCR检测细胞中miR-182-5p的表达,结果显示,与MCF-10A相比,miR-182-5p在MDA-MB-231、T47D、MDA-MB-453、MCF-7中表达上调(P<0.05);转染miR-182-5p干扰质粒,qRT-PCR检测细胞中miR-182-5p的表达情况。结果显示,miR-182-5p表达显著降低(P=0.003),提示转染成功;Transwell侵袭结果显示,MDAMB-231细胞敲低miR-182-5p,与对照组相比,体外侵袭能力明显降低(P=0.002);Western印迹检测转染miR-182-5p干扰质粒时,MDA-MB-231中上皮-间质转化(epithelial-mesenchymal transition,EMT)相关标志物的表达情况,结果显示,与对照组相比,敲低miR-182-5p使细胞中上皮-钙黏着蛋白(E-cadherin)表达上调,神经-钙黏着蛋白(N-cadherin)、波形蛋白(vimentin)表达下调。为研究探讨miR-182-5p的靶蛋白,采用在线预测软件预测可能与miR-182-5p结合的靶蛋白,cytoscape构建蛋白质互作网络图并筛选出hub基因;双荧光素酶结果证实,miR-182-5p可与EP300靶向结合(P=0.001);采用qRT-PCR、Western印迹检测转染miR-182-5p干扰质粒后EP300在mRNA及蛋白质水平的表达,结果显示,与对照组相比,在敲低miR-182-5p组中EP300在mRNA及蛋白质的表达上调(P=0.001)。综上所述,miR-182-5p可靶向调节EP300,促进乳腺癌细胞的侵袭与转移。

miR-182-5p Promoted the Invasion and Metastasis of Breast Cancer Cells by Targeting EP300

Recent studies have shown that miR-182-5p plays an important role in the invasion and metastasis of a variety of cancers,but reports regarding to its roles in the invasion and metastasis of breast cancer is relatively rare.This study analyzed 3 GEO data sets through online miRNAs analysis tools,and the results found in 26 miRNAs were commonly expressed in breast cancer tissues in these 3 data sets,eight of them was up-regulated,and hsa-miR-182 was the most significantly up-regulated one in breast cancer tissues(P=0.001)and was selected in this study.As compared with MCF-10 A cells,miR-182-5p was up-regulated in MDA-MB-231,T47D,MDA-MB-453,and MCF-7 breast cancer cells by qRTPCR analysis(P<0.05).When the cells were transfected with miR-182-5p interfering plasmid,the expression of miR-182-5p was significantly decreased(P=0.003),suggesting that transfection was successful.Results from transwell invasion test showed that after miR-182-5p was knocked down,the invasion ability of MDA-MB-231 cells was significantly reduced as compared with the control group(P=0.002).Western blot assay detected the expression of epithelial-mesenchymal transition(EMT)related markers in MDA-MB-231 after transfection of miR-182-5p interfering plasmid.The results showed that when compared with the control group,the suppression of miR-182-5p up-regulated the expression of Ecadherin and down-regulated the expression of N-cadherin and Vimentin.To analyze the targets of miR-182-5p,online prediction software was used to predict the possible binding target proteins with miR-182-5p.Cytoscape was applied to construct the protein interaction network and screen the hub genes.The results of the double luciferase experiment confirmed that miR-182-5p could bind to EP300(P=0.001).qRT-PCR and Western blot analysis were used to detect EP300 expression at mRNA and protein levels after transfection of miR-182-5p interfering plasmids.The results showed that compared with the control group,EP300 expression at mRNA and protein levels were up-regulated in the miR-182-5p knockdown group(P=0.001).In conclusion,miR-182-5p can target EP300 and promote the invasion and metastasis of breast cancer cells.