恶性黑素瘤细胞miR-508-3p与叉头框转录因子C1的关系及对增殖、迁移和侵袭的影响

The relationships between miR-508-3p and forkhead box transcription factor C1 in malignant melanoma cells and their effects on proliferation, migration and invasion

目的探讨miR-508-3p与叉头框转录因子C1(forkhead box C1,FOXC1)的关系及其在恶性黑素瘤中的表达和作用机制。方法通过实时荧光定量聚合酶链反应(RT-qPCR)检测mi R-508-3p和FOXC1在HEM正常黑素细胞及A375恶性黑素瘤细胞中的表达;将体外培养的A375细胞分为mimics NC组、mi R-508-3p mimics组、inhibitor NC组和mi R-508-3p inhibitor组,RT-qPCR检测其沉默效果及对FOXC1 mRNA水平的影响;免疫印迹法(Western blot)分别检测FOXC1蛋白的表达量;MTT法检测A375细胞的增殖能力;Transwell实验检测转染后A375细胞侵袭及迁移能力的变化;荧光素酶报告基因技术检测FOXC1是否为mi R-508-3p的直接靶基因。结果与人正常黑素细胞相比,A375细胞中mi R-508-3p表达明显降低,而FOXC1表达明显升高;与inhibitor NC组相比,转染mi R-508-3p inhibitors的A375细胞中mi R-508-3p的表达明显下调,FOXC1 mRNA和蛋白表达、细胞的增殖、侵袭及迁移能力明显升高;与mimics NC组相比,转染mi R-508-3p mimics的A375细胞中mi R-508-3p的表达明显升高,FOXC1 mRNA和蛋白表达、细胞的增殖、侵袭及迁移能力明显降低;mi R-508-3p mimics可降低野生型FOXC1 3’-UTR的荧光素酶报告基因相对活性。结论 mi R-508-3p通过负向调控FOXC1抑制恶性黑素瘤的增殖、迁移和侵袭。

Objective To investigate the expression, relationship, function, mechanism of miR-508-3 p and forkhead box C1(FOXC1) in malignant melanoma. Methods The expression of miR-508-3 p and FOXC1 in HEM normal melanocytes and A375 malignant melanoma cells was detected by RT-qPCR. The A375 cells cultured in vitro were divided into mimics NC group, miR-508-3 p mimics group, inhibitor NC group and miR-508-3 p inhibitor group. RT-qPCR was used to detect the expression levels of miR-508-3 p and FOXC1;Western blot was used to detect the protein expression of FOXC1;MTT was used to detect the proliferative capacity of A375 cells;the migration and invasion of malignant melanoma were detected by Transwell chamber test;luciferase assays were performed to investigate whether FOXC1 was the direct target of miR-508-3 p in A375 cells. Results Compared with normal human melanocytes, the expression of miR-508-3 p was significantly decreased in A375 cells, while the expression of FOXC1 was significantly increased in A375 cells. Compared with the inhibitor NC group, the expression of miR-508-3 p was down-regulated in A375 cells transfected with miR-508-3 p inhibitors, while FOXC1 mRNA and protein expression, cell proliferation, invasion and migration ability significantly increased. Compared with the mimics NC group, the expression of miR-508-3 p was significantly increased in A375 cells transfected with miR-508-3 p mimics, while FOXC1 mRNA and protein expression, cell proliferation, invasion and migration ability significantly reduced. MiR-508-3 p mimics reduced the relative activity of the luciferase reporter gene of wild-type FOXC1 3’-UTR. Conclusion MiR-508-3 p inhibits the proliferation, migration and invasion of malignant melanoma by negatively regulating FOXC1.