微小RNA-141靶向表皮生长因子受体调控乳腺癌上皮间质转化的作用机制研究

Mechanism of microRNA-141 regulates epithelial-mesenchymal transition in breast cancer by targeting epidermal growth factor receptor

目的通过生物信息学筛选并探讨微小RNA-141(miR-141)、表皮生长因子受体(EGFR)在乳腺癌细胞中的表达水平及其对上皮间质转化、细胞迁移以及侵袭的影响。方法应用微阵列分析筛选差异表达的基因;采用加权基因共表达网络分析(WGCNA)和基因的蛋白质-蛋白质相互作用网络找出乳腺癌的关键基因;通过实时荧光定量聚合酶链式反应(qRT-PCR)检测乳腺癌组织和细胞中的miR-141和EGFR表达;应用Targetscan和Miranda预测miR-141与EGFR的靶标关系,并通过双荧光素酶报告基因检测验证二者之间的靶向关系;使用Transwell实验测定评估细胞迁移能力;通过CCK-8法测定细胞活力;采用免疫印迹法(Western blot)检测E-钙黏蛋白(E-cadherin)和波形蛋白(Vimentin)的表达。结果基因表达热图及WGCNA分析结果显示,EGFR在肿瘤组织中显著高表达。经分子生物学验证乳腺癌组织和细胞中miR-141的表达下调,而EGFR的表达上调,差异有统计学意义(P<0.05)。双荧光素酶报告基因检测发现EGFR是miR-141的靶基因,在乳腺癌细胞中受miR-141表达的调控。细胞实验显示,miR-141过表达组可以抑制乳腺癌细胞迁移和增殖,而EGFR组则呈现出相反的结果,差异均有统计学意义(P<0.05)。miR-141过表达能够促进E-cadherin表达,抑制Vimentin的表达,差异有统计学意义(P<0.05),提示miR-141过表达能抑制肿瘤细胞的上皮间质转化的进程。结论miR-141通过靶向EGFR抑制乳腺癌细胞增殖并调节上皮间质转化的进展。

Objective To screen and investigate the expression levels of microRNA-141(miR-141)and epidermal growth factor receptor(EGFR)in breast cancer cells and their effects on epithelial-mesenchymal transition,cell migration and invasion through bioinformatics.Methods Microarray analysis was used to screen differentially expressed genes;the weighted gene co-expression network analysis(WGCNA)and protein-protein interaction network of genes were used to identify key genes of breast cancer;the real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to detect the expressions of miR-141 and EGFR in breast cancer tissues and cells;the Targetscan and Miranda were used to predict the target relationship between miR-141 and EGFR,and the target relationship between them was verified by dual luciferase reporter gene detection;the Transwell assay was used to evaluate migration ability of cells;the cell viability was detected by CCK-8 method;the western blot was used to detect the expressions of E-cadherin and vimentin.Results The results of gene expression heat map and WGCNA analysis showed that EGFR was significantly highly expressed in tumor tissues.It was verified by molecular biology that the expression of miR-141 was significantly down-regulated and the expression of EGFR was significantly up-regulated in breast cancer tissues and cells(P<0.05).Dual luciferase reporter gene detection found that EGFR was a target gene of miR-141,and was regulated by miR-141 expression in breast cancer cells.Cell experiments showed that the migration and proliferation of breast cancer cells were inhibited in miR-141 overexpression group,while the opposite results were observed in the EGFR group,and there were significant differences between two groups(P<0.05).Overexpression of miR-141 was able to significantly promote the expression of E-cadherin and significantly inhibit the expression of Vimentin(P<0.05),indicating that overexpression of miR-141 can inhibit the process of epithelial-mesenchymal transition of tumor cells.Conclusion The miR-141 can inhibit the proliferation of breast cancer cells and regulate progress of epithelial-mesenchymal transition by targeting EGFR.