miR-383-5p调控MAL2表达影响三阴性乳腺癌肿瘤细胞侵袭及转移机制的研究

Study on the mechanism of miR-383-5p regulating MAL2 expression to affect the invasion and metastasis of triple negative breast cancer cells

目的 探讨微小RNA-383-5p(miR-383-5p)是否可靶向T细胞分化蛋白2(MAL2)调控三阴性乳腺癌(TNBC)肿瘤细胞侵袭及转移。方法 RT-qPCR技术检测TNBC细胞系(MDA-MB-468、MDA-MB-453、BT-549)和人永生化乳腺上皮细胞系(MCF-10A)中miR-383-5p表达水平;MDA-MB-453细胞随机分为对照组、miR-383-5p NC组、miR-383-5p mimics组,转染48 h后,划痕愈合实验评估细胞迁移能力,Transwell实验评估细胞侵袭能力;双荧光素酶报告基因实验验证miR-383-5p与MAL2的靶向关系;RT-qPCR技术检测细胞中miR-383-5p和MAL2 mRNA表达水平;Western blotting法检测MAL2、基质金属蛋白酶2(MMP-2)、MMP-9、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、E-钙黏附蛋白(E-cadherin)相对表达水平。结果 与MCF-10A细胞比较,MDA-MB-468、BT-549和MDA-MB-453细胞中miR-383-5p相对表达量均降低(P <0.05),其中MDA-MB-453细胞中miR-383-5p相对表达水平最低(P<0.05);对照组与miR-383-5p NC组间差异无统计学意义(P>0.05);与对照组比较,miR-383-5p mimics组细胞划痕愈合率、细胞侵袭数、MAL2 mRNA和蛋白相对表达水平、MMP-2、MMP-9、N-cadherin、Vimentin蛋白相对表达水平降低(P<0.05),miR-383-5p相对表达水平及E-cadherin蛋白相对表达水平升高(P<0.05);经生物信息学分析,MAL2被预测为miR-383-5p的潜在靶基因。结论 miR-383-5p可靶向下调MAL2表达进而抑制TNBC肿瘤细胞的侵袭及转移。

Objective To investigate whether microRNA-383-5p(miR-383-5p) can target T cell differentiation protein 2(MAL2) to regulate the invasion and metastasis of triple negative breast cancer(TNBC) cells.Methods The expression of miR-383-5p in TNBC cell lines(MDA MB-468,MDA MB-453,BT-549) and human immortalized breast epithelial cell lines(MCF-10A) was detected by RT-qPCR.MDA-MB-453 cells were randomly divided into the control group,miR-383-5p NC group and miR-383-5p mimics group.After 48 h of transfection,the cell migration ability was evaluated by scratch healing test,and the cell invasion ability was evaluated by Transwell test.Double luciferase reporter gene experiment was used to verify the targeting relationship between miR-383-5p and MAL2.The expression of miR-383-5p and MAL2 mRNA was detected by RT-qPCR.The expression of MAL2,MMP-2,MMP-9,N-cadherin,Vimentin and E-cadherin were detected by western blotting.Results Compared with MCF-10A cells,the expression of miR-383-5p in MDA-MB-468,BT-549 and MDA-MB-453 cells was decreased(P<0.05),and the expression of miR-383-5p in MDA-MB-453cells was the lowest(P<0.05).There was no significant difference between the control group and miR-383-5p NC group(P>0.05).Compared with the control group,the cell scratch healing rate,the number of cell invasions,the expression of MAL2 mRNA and protein,and the expression of MMP-2,MMP-9,N-cadherin and Vimentin protein were decreased in the miR-383-5p mimics group(P<0.05),and the expression of miR-383-5p and E-cadherin protein was increased(P<0.05).Based on bioinformatics analysis,MAL2 was predicted to be a potential target gene of miR-383-5p.Conclusion miR-383-5p could target and lower the expression of MAL2 to inhibit the invasion and metastasis of TNBC cells.