皮肤黑素瘤中miRNA-34c的表达对促血管新生因子VEGF-A、VEGFR-1的影响

Effects of miRNA-34c in Cutaneous Malignant Melanoma on Angiogenic Factors VEGF-A and VEGFR-1

目的探讨皮肤黑素瘤中miRNA-34c的表达以及其对促血管新生因子VEGF-A和VEGFR-1的影响。方法 Northern blotting检测原代人黑素瘤细胞、黑素瘤细胞株A375和原代人表皮黑素细胞miRNA-34c表达。构建miRNA-34c基因过表达与基因敲低的黑素瘤细胞株A375细胞系即高表达miRNA-34c组和低表达miRNA-34c组,以空载体转染黑素瘤细胞株A375作为空载体对照组。CCK-8法检测各组细胞增殖活性。qRT-PCR和Western blotting分别检测各组VEGF-A、VEGFR-1 mRNA和蛋白表达。结果与原代人表皮黑素细胞相比,黑素瘤细胞株A375和原代人黑素瘤细胞中miRNA-34c的表达降低(P<0.05)。高表达miRNA-34c组、低表达miRNA-34c组和空载体对照组细胞增殖活性无明显差异(P>0.05)。高表达miRNA-34c组VEGF-A、VEGFR-1的mRNA和蛋白表达较空载体对照组降低(P<0.05)。低表达miRNA-34c组VEGF-A、VEGFR-1的mRNA和蛋白表达较空载体组升高(P<0.05)。结论 miRNA-34c在皮肤黑素瘤中呈现特异性下调,miRNA-34c可能通过对促血管新生因子VEGF-A、VEGFR-1的下调参与皮肤黑素瘤的发生与发展。

Objective To investigate the expression of miRNA-34 c and its effect on angiogenic factors VEGF-A and VEGFR-1 in cutaneous malignant melanoma.Methods Northern blotting was performed to detect the expression of miRNA-34 c in primary human melanoma cells,malignant melanoma A375 cells and primary human epidermal melanocytes respectively.Using the method of transient gene transfection,malignant melanoma A375 cells with high expression of miRNA-34 c group and malignant melanoma A375 cells with low expression of miRNA-34 c group were establish ed.Malignant melanoma A375 cells transfected with empty vectors were used as empty vector control group.CCK-8 assay was performed to evaluate the proliferation and activity of cells in each group.VEGF-A mRNA and VEGFR-1 mRNA were detected by qRT-PCR and VEGF-A protein and VEGFR-1 protein were detected by Western blotting respectively.Results Compared to primary human epidermal melanocytes,the expression of miRNA-34 c was significantly decreased in primary human melanoma cells and malignant melanoma A375 cells(P<0.05).There were no significant differences of cell proliferation and activity in the empty vector control group,high miRNA-34 c expression group and low miRNA-34 c expression group(P>0.05).The expression of VEGF-A mRNA,VEGF-A protein,VEGFR-1 mRNA and VEGFR-1 protein in the high miRNA-34 c expression group was lower than that in the empty vector control group(P<0.05).The expression of VEGF-A mRNA,VEGF-A protein,VEGFR-1 mRNA and VEGFR-1 protein in the low miRNA-34 c expression group was higher than that in empty vector control group(P<0.05).Conclusion The expression of miRNA-34 c is down-regulated in cutaneous malignant melanoma.miRNA-34 c may play a role in development and progression of malignant melanoma by attenuating the expression of angiogenic factors VEGF-A and VEGFR-1.