miRNA-4465过表达对乳腺癌MDA-MB-231细胞迁移侵袭的影响及其机制

Effect and mechanism of miRNA-4465 overexpression in the migration and invasion of breast cancer MDA-MB-231 cells

目的探讨miRNA-4465的过表达对乳腺癌MDAMB-231细胞迁移侵袭能力的影响及机制。方法将miRNA-4465的模拟物(minics)转染入MDA-MB-231细胞,通过实时定量PCR检测miRNA-4465的表达变化;运用Transwell实验检测转染后细胞迁移和侵袭能力的改变。采用生物信息学技术预测miRNA-4465的潜在靶基因,并通过荧光报告载体实验及Western blot加以证实。结果与阴性对照组相比,转染miRNA-4465 minics组miRNA-4465表达水平明显升高(P=0.001)。Transwell迁移实验结果显示转染miRNA-4465 minics组细胞迁移能力减弱(P=0.001);Transwell侵袭实验结果显示转染miRNA-4465 minics组细胞侵袭能力降低(P=0.010)。通过生物信息学技术预测了EZH2为miRNA-4465的潜在靶基因;荧光报告载体实验结果显示转染miRNA-4465 minics+psi CHECK2-EZH2 3’UTR(野生型)后荧光素酶活性较转染阴性对照序列(NC)+psi CHECK2-EZH2 3’UTR(野生型)降低66%(P=0.001)。此外将miRNA-4465转染入MDA-MB-231细胞后EZH2蛋白表达降低。结论 miRNA-4465能降低MDA-MB-231细胞的迁移和侵袭能力,这个过程可能是通过调控EZH2基因的表达实现的。

Objective To investigate the effect of overexpression of miRNA-4465 on invasion and metastasis of breast cancer MDA-MB-231 cells and its mechanism. Methods MiRNA-4465 minics was transfected into the MDA-MB-231 cells,and real-time PCR was used to detect the expression of miRNA-4465. Transwell assay was used to investigate the migration and invasion capability of cells after being transfected. Bioinformatics analysis were performed to predict the potential targets of miRNA-4465,and finally,luciferase reporter plasmids assay and western blot was used to confirm the potential target of miRNA-4465. Results Comparing with negative control group,the expression of miRNA-4465 in miRNA-4465 minics group was increased significantly. Transwell migration assay showed that migration capability of cells in miRNA-4465 minics group was decreased（ P = 0. 001）; transwell invasion assay showed that invasion capability of cells in miRNA-4465 minics group was decreased（ P = 0. 010）. Luciferase reporter plasmids assay showed that comparing with the negative control ＋ psi CHECK2- EZH2 3 ＇UTR（ wild type）,the fluorescence activity of miRNA-4465 minics ＋ psi CHECK2- EZH2 3＇UTR（ wild type） was decreased by66%（ P = 0. 001）. Moreover,comparing with the negative control group,the expression of EZH2 protein was decreased after transfecting miRNA-4465 minics into MDA-MB-231 cells. Conclusion MiRNA-4465 can suppress the invasion and metastasis of breast cancer MDA-MB-231 cells,which may be related to the regulation of EZH2.