微小RNA⁃526b⁃3p通过JAK/STAT3信号通路对黑色素瘤细胞侵袭迁移的影响

Effects of microRNA-526b-3p on the invasion and migration of melanoma cells through the JAK/STAT3 signaling pathway

目的探讨微小RNA-526b-3p(miR-526b-3p)对黑素瘤细胞侵袭与迁移的影响及潜在机制。方法实时定量PCR(qPCR)检测黑色素瘤组织和A2058细胞中miR-526b-3p和信号转导子与激活子3(STAT3)的表达水平,双荧光素酶报告基因实验验证miR-526b-3p和STAT3的靶向关系。成功构建miR-526b-3p模拟物(mimics)和过表达STAT3的质粒pcDNA3.1-STAT3后进行转染,将A2058细胞分为miR-NC组(对照)、miR-526b-3p mimics组(过表达miR-526b-3p)、miR-526b-3p mimics+pcDNA3.1-STAT3组(过表达miR-526b-3p和STAT3)。采用CCK-8法检测细胞增殖,划痕实验和Transwell小室实验检测各组细胞迁移和侵袭;运用qPCR和Western blot方法检测miR-526b-3p和JAK/STAT3信号通路相关基因STAT3和p-JAK1的表达水平。结果黑色素瘤组织和细胞中miR-526b-3p表达下调,而STAT3表达上调(P<0.05)。相较于miR-NC组,miR-526b-3p mimics组A2058细胞增殖、迁移和侵袭能力下调(P<0.05),同时STAT3、p-STAT3和p-JAK1水平降低(P<0.05)。miR-526b-3p mimics能够抑制STAT3表达。相较于miR-526b-3p mimics组,miR-526b-3p mimics+pcDNA3.1-STAT3组A2058细胞增殖、迁移和侵袭能力上调(P<0.05)。结论miR-526b-3p可能通过靶向STAT3基因,下调JAK/STAT3信号活性,抑制恶性黑色素瘤细胞增殖、迁移和侵袭,miR-526b-3p/STAT3轴有望成为黑色素瘤的治疗靶点。

Objective To explore the effect and potential mechanism of microRNA-526b-3p(miR-526b-3p)on the invasion and migration of melanoma cells.Methods Expressions of miR-526b-3p and signal activator of transcription 3(STAT3)were detected in melanoma tissues and cells via quantitative PCR(qPCR).The targeting relationship between miR-526b-3p and STAT3 was verified by double luciferase reporter gene experiment.MiR-526b-3p mimics and eukaryotic expression plasmid pcDNA3.1-STAT3 were successfully constructed.A2058 cells were transfected and assigned into miR-NC group(control),miR-526b-3p mimics group(over-expression of miR-526b-3p)and miR-526b-3p mimics+pcDNA3.1-STAT3 group(over-expression of miR-526b-3p and STAT3).Cell proliferation was detected by CCK-8 method.Cell migration and invasion were determined by wound-healing and Transwell chamber assays.The expressions of STAT3 and p-JAK1 were detected by qPCR or Western blot.Results MiR-526b-3p was down-regulated in melanoma tissues and A2058 cells,while STAT3 were up-regulated(P<0.05).Compared with miR-NC group,A2058 cells abilities of proliferation,migration and invasion were inhibited,and the levels of STAT3,p-STAT3 and p-JAK1 were decreased in miR-526b-3p mimics group(P<0.01).MiR-526b-3p negatively regulated the STAT3 expression.Over-expression of STAT3 can reverse the inhibition of miR-526b-3p on proliferation,migration and invasion of A2058 cell(P<0.05).Conclusion MiR-526b-3p may target the STAT3 gene,downregulate JAK/STAT3 signaling activity,and inhibit the proliferation,migration and invasion of malignant melanoma cells.The miR-526b-3p/STAT3 axis is expected to become a therapeutic target for melanoma.