miR-6775-3p在乳腺癌细胞中的表达及其对乳腺癌细胞生物学行为的影响

Expression of miR-6775-3p in breast cancer cells and its effect on biological behavior of breast cancer cells

背景与目的:微小RNA(microRNA,miRNA)与肿瘤的发生、发展过程密切相关。探讨miR-6775-3p在乳腺癌细胞系中的表达及其对乳腺癌细胞生物学行为的影响。方法:通过实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)检测4种乳腺癌细胞系MDA-MB-231、MDA-MB-453、MDA-MB-468和BT-549中miR-6775-3p的表达水平,选取miR-6775-3p表达水平最低的乳腺癌细胞系过表达miR-6775-3p后,采用细胞计数试剂盒(cell counting kit-8,CCK-8)法检测细胞的增殖情况,同时采用transwell迁移和侵袭实验分别检测细胞迁移和侵袭能力的变化。通过RTFQ-PCR和蛋白[质]印迹法(Western blot)检测miR-6775-3p过表达的乳腺癌细胞系中细胞周期蛋白依赖性蛋白激酶4(cyclin-dependent protein kinase 4,CDK4)和CDK6,以及侵袭转移标志物基质金属蛋白酶(matrix metalloproteinase,MMP)17和MMP24 mRNA以及蛋白的表达变化。结果:RTFQ-PCR结果显示,乳腺癌细胞系MDA-MB-453中miR-6775-3p的表达最低,在MDA-MB-453细胞中转染miR-6775-3p mimics后,miR-6775-3p的表达水平明显升高(P<0.001)。CCK-8实验结果显示,MDA-MB-453细胞过表达miR-6775-3p后,细胞的增殖能力明显降低(P<0.01)。Transwell迁移和侵袭实验结果显示,MDA-MB-453细胞过表达miR-6775-3p后,细胞的迁移(P<0.001)和侵袭能力(P<0.01)明显降低。RTFQ-PCR和Western blot实验结果显示,CDK4、CDK6及MMP17、MMP24的mRNA表达和蛋白水平均显著降低(P<0.01)。结论:miR-6775-3p可能抑制乳腺癌细胞的增殖、迁移和侵袭能力。

Background and purpose:MicroRNA(miRNA)is closely related to the occurrence and development of tumors.This study aimed to investigate the expression of miR-6775-3p in breast cancer cells and its effect on the biological characteristics of breast cancer cells.Methods:Real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)was used to detect the expression level of miR-6775-3p in four breast cancer cell lines MDA-MB-231,MDA-MB-453,MDA-MB-468 and BT-549.After miR-6775-3p was over-expressed in breast cancer cells with the lowest expression level of miR-6775-3p,the proliferation of cells was detected by cell counting kit-8(CCK-8)assay,and the migration and invasion ability of cells were detected by transwell migration and invasion assay.To further explore the molecular mechanism of miR-6775-3p in breast cancer,the expressions of cyclin-dependent protein kinase 4(CDK4),CDK6 and invasion and metastasis markers matrix metalloproteinase 17(MMP17)and MMP24 in miR-6775-3p overexpressing breast cancer cells were detected by RTFQ-PCR and Western blot.Results:RTFQ-PCR results showed that the expression of miR-6775-3p was the lowest in breast cancer cell line MDA-MB-453,and the expression level of miR-6775-3p was significantly increased after transfection of miR-6775-3p mimics in MDA-MB-453 cells(P<0.001).CCK-8 assay results showed that the cell proliferation activity was significantly decreased after miR-6775-3p was overexpressed in MDAMB-453 cells(P<0.01).Transwell migration and invasion assay results showed that the cell migration(P<0.001)and invasion abilities(P<0.01)were significantly decreased after miR-6775-3p was over-expressed in MDA-MB-453 cells.RTFQ-PCR and Western blot results showed that the expressions of cyclin-dependent protein kinases CDK4 and CDK6,as well as the mRNA and protein levels of cell invasion and metastasis markers MMP17 and MMP24 were significantly decreased(P<0.01).Conclusion:miR-6775-3p may inhibit the proliferation,migration and invasion of breast cancer cells.