miR-541-3p靶向SENP1调控皮肤黑色素瘤细胞增殖、迁移、侵袭机制的研究

The mechanism of miR-541-3p regulates proliferation, migration,and invasion of skin melanoma cell by targeting SENP1

目的:探讨微小RNA-541-3p(miR-541-3p)对皮肤黑色素瘤细胞增殖、迁移、侵袭的影响及其分子机制。方法:采用实时荧光定量聚合酶链反应(qRT-PCR)与蛋白免疫印迹法(Western blotting)分别检测皮肤黑色素瘤组织中miR-541-3p、SENP1的表达水平;以人皮肤黑色素瘤细胞A375为研究对象,分别将miR-NC、miR-541-3p mimics、si-NC、si-SENP1、miR-541-3p mimics与pcDNA、miR-541-3p mimics与pcDNA-SENP1转染至A375细胞;甲基噻唑基四唑(MTT)检测细胞增殖;Transwell小室实验检测细胞迁移及侵袭能力;双荧光素酶报告实验验证miR-541-3p过表达对野生型和突变型SENP1荧光素酶活性的影响;Western blotting检测细胞周期蛋白1(CyclinD1)、p21、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)蛋白表达量。结果:与正常皮肤组织比较,皮肤黑色素瘤组织中miR-541-3p的表达量显著降低(P<0.05),SENP1 mRNA及蛋白表达水平显著升高(P<0.05);与miR-NC组比较,miR-541-3p组A375细胞活力显著降低(P<0.05),迁移细胞数与侵袭细胞数显著减少(P<0.05),CyclinD1、MMP-2、MMP-9蛋白水平显著降低(P<0.05),p21蛋白水平显著升高(P<0.05);与si-NC组比较,siSENP1组A375细胞活力显著降低(P<0.05),迁移细胞数与侵袭细胞数显著减少(P<0.05),CyclinD1、MMP-2、MMP-9蛋白水平显著降低(P<0.05),p21蛋白水平显著升高(P<0.05);双荧光素酶报告实验证实miR-541-3p能够抑制SENP1的3′-UTR区荧光素酶活性;与miR-541-3p+pcDNA组比较,miR-541-3p+pcDNA-SENP1组细胞活力显著升高(P<0.05),迁移细胞数与侵袭细胞数显著增多(P<0.05),CyclinD1、MMP-2、MMP-9蛋白水平显著升高(P<0.05),p21蛋白水平显著降低(P<0.05)。结论:miR-541-3p过表达可靶向抑制SENP1表达从而减弱皮肤黑色素瘤细胞增殖、迁移及侵袭能力。

Objective: To investigate the effect and its molecular mechanism of micro RNA-541-3p(miR-541-3p) on the proliferation, migration, and invasion of cutaneous melanoma cells. Methods: qRT-PCR and Western blotting were used to detect the expression levels of miR-541-3p and SENP1 in skin melanoma tissues. Taking human skin melanoma cells A375 as the research object, miR-NC, miR-541-3p mimics, si-NC, siSENP1, miR-541-3p mimics, and pcDNA, miR-541-3p mimics and pcDNA-SENP1 were transfected into A375 cells. MTT was used to detect cell proliferation. Transwell chamber experiment was used to detect cell migration and invasion ability.The double luciferase reporting experiment was used to verifythe effect of miR-541-3p overexpression on wild-type and mutant SENP1 luciferase activity. Western blotting was used to detect the protein expression of CyclinD1, p21, MMP-2, and MMP-9. Results: Compared with normal skin tissue, the expression of miR-541-3p in skin melanoma tissue was significantly decreased(P<0.05), and the expression levels of SENP1 mRNA and protein were significantly increased(P<0.05). Compared with the miR-NC group, in the miR-541-3p group, the cellular viability was significantly decreased(P<0.05), and the number of migrating cells and invasive cells were significantly decreased(P<0.05);the proteins levels of CyclinD1, MMP-2, and MMP-9 were significantly decreased(P<0.05), and the protein level of p21 was significantly increased(P<0.05). Compared with the si-NC group, in the siSENP1 group,the cellular viability was significantly decreased(P<0.05), the number of migrating cells and invasive cells weresignificantly decreased(P<0.05), and the protein levels of CyclinD1, MMP-2, and MMP-9 were decreased significantly(P<0.05), and the protein level of p21 was increased significantly(P<0.05). The double luciferase reporting experiment confirmed that miR-541-3p could inhibit the luciferase activity in the 3’-UTR region of SENP1. Compared with the miR-541-3p + pcDNA group, in the miR-541-3p + pcDNA-SENP1 group, the cell viability was significantly increased(P<0.05),and the number of migrating cells and invasive cells weresignificantly increased(P<0.05), the protein levels of CyclinD1,MMP-2, and MMP-9 were increased significantly(P<0.05),the protein levels of p21 was decreased significantly(P<0.05). Conclusion: Overexpression of miR-541-3p can inhibit the expression of SENP1 and attenuate proliferation, migration, and invasion ability of skin melanoma cell.