（淮安市第五人民医院皮肤性病美容科，淮安 223300）

摘　要：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。

关键词：皮肤黑色素瘤；微小RNA-126-3p；Janus蛋白酪氨酸激酶2/信号传导与转录激活因子3；迁移；侵袭

中图分类号：R739.5                           文献标志码：ADOI：10.3969/j.issn.1007-7146.2024.02.009

(Department of Dermatology and Aesthetic Surgery the Fifth People’s Hospital of Huai’an, Huai’an 223300, China)

Abstract: 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.

Key words: cutaneous melanoma; microRNA-126-3p; Janus tyrosine protein kinase 2/signal transduction and transcription activator 3; migration; invasion

（Acta Laser Biology Sinica, 2024, 33(2): 167-175）