miR-126对皮肤黑色素瘤C8161迁移、侵袭作用机制的研究

A Study on the Mechanism of miR-126 on the Migration and Invasion of Cutaneous Melanoma C8161

本研究主要观察微小RNA-126-3p(miR-126)的差异性表达对人皮肤黑色素瘤(CM)细胞株C8161的迁移、侵袭性的影响,并探究了Janus蛋白酪氨酸激酶2/信号传导与转录激活因子3(JAK2/STAT3)通路和上皮-间质转化(EMT)过程在其中的角色。采用细胞转染法实现miR-126的差异性表达,用JAK2/STAT3通路抑制剂AG490、激动剂Coumermycin A1处理miR-126差异表达的细胞,并将C8161细胞分为对照组(不做转染,不做药物处理)、阴性对照(NC)组(转染模拟物对照,不做药物处理)、miR-126组(转染miR-126模拟物,不做药物处理)、miR-126+通路抑制剂组(转染miR-126模拟物后AG490处理)和miR-126+通路激动剂组(转染miR-126模拟物后Coumermycin A1处理)。采用实时荧光定量PCR(RT-qPCR)检测miR-126的表达水平;利用蛋白免疫印迹法测定EMT关键蛋白和JAK2/STAT3通路蛋白的表达水平;利用细胞计数试剂盒-8(CCK-8)、划痕愈合试验、Transwell小室联合基质胶法分别检测细胞的活力、迁移和侵袭能力。与NC组相比,miR-126模拟物转染使miR-126的表达水平在miR-126组显著提高(^(#)P<0.05),与此同时,相对细胞活力、细胞迁移率和细胞侵袭数均显著降低(^(#)P<0.05)。此外,miR-126组的上皮型钙黏蛋白(E-cadherin)高于NC组(^(#)P<0.05),而波形蛋白(vimentin)、神经钙黏蛋白(N-cadherin)、纤维连接蛋白(FN)、JAK2、STAT3、p-JAK2、p-STAT3蛋白的表达水平和p-JAK2/JAK2、p-STAT3/STAT3的比值均低于NC组(^(#)P<0.05)。更重要的是,与NC组和miR-126组相比,JAK2和STAT3蛋白的表达水平的上述指标在miR-126+通路抑制剂组中的变化更显著(^(#)P<0.05和^(&)P<0.05),而上述指标在miR-126+通路激活剂组中的变化趋势减弱(^(#)P<0.05和^(&)P<0.05),其中细胞侵袭数和FN、JAK2、STAT3蛋白的表达水平在miR-126+通路激动剂组和NC组之间无显著差异。过表达miR-126抑制人CM细胞的活力、迁移和侵袭能力,而该作用很可能是通过阻碍EMT进程、抑制JAK2/STAT3通路的活化来实现的。这些研究结果有助于为人CM的临床治疗提供新的理论依据,为其治疗方法提供新的策略。

This study focused on the effects of the differential expression of microRNA-126-3p(miR-126)on migration and invasion of human cutaneous melanoma(CM)cell line C8161,and the role of Janus tyrosine protein kinase 2/signaling and transcriptional activator 3(JAK2/STAT3)pathway and epithelial-mesenchymal transition(EMT)process were investigated.Dysregulation of miR-126 was achieved by cell transfection,and miR-126-deregulated cells were treated with the JAK2/STAT3 pathway inhibitor AG490 or the agonist Coumermycin A1.Thus,C8161 cells were divided into 5 groups:control group(without transfection or drug treatment),negative control(NC)group(with mimics control transfection,but no drug treatment),miR-126 group(with miR-126 mimics transfection,but no drug treatment),miR-126+pathway inhibitor group(with miR-126 mimics transfection and AG490 treatment),miR-126+pathway agonist group(with miR-126 mimics transfection and Coumermycin A1 treatment).Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression level of miR-126.The expression levels of EMT key proteins and JAK2/STAT3 pathway proteins were determined by Western blotting.Cell viability,migration and invasion abilities were determined by cell counting kit-8(CCK-8),scratch healing assay and Transwell chamber combined with matrix gel,respectively.Compared with the NC group,transfection with miR-126 mimics significantly increased the expression level of miR-126 in the miR-126 group(^(#)P<0.05),while the relative cell vitality,cell migration rate and cell invasion number significantly decreased(^(#)P<0.05),accompanied with higher protein level of E-cadherin(^(#)P<0.05),and lower protein levels of Vimentin,N-cadherin,fibronectin(FN),JAK2,STAT3,p-JAK2,and p-STAT3,and as well as lower ratios of p-JAK2/JAK2 and p-STAT3/STAT3(^(#)P<0.05).More importantly,compared with NC group and miR-126 group,the above indicators of JAK2 and STAT3 protein levels were further changed in miR-126+pathway inhibitor group(^(#)P<0.05 and ^(&)P<0.05),whereas all the above indexes were less changed in miR-126+pathway activator group(^(#)P<0.05 and ^(&)P<0.05),and cell invasion number and FN,JAK2 and STAT3 protein levels in miR-126+pathway activator group were little different from NC group without significance.Overexpression of miR-126 inhibits cell viability,migration and invasion of human CM cells presumably by blocking EMT process and inhibiting the activation of JAK2/STAT3 pathway.These results were helpful to provide a new theoretical basis for the clinical treatment of human CM and a new strategy for its treatment.