miR-582-5p通过靶向SOCS1基因调节皮肤黑色素瘤细胞迁移、侵袭和EMT的机制研究  

The effects of miR-582-5p in regulating the migration,invasion and EMT of melanoma cells of skin by targeting SOCS1 gene

作　　者：李静 刘琴[2] 柯锦 LI Jing;LIU Qin;KE Jin(Department of Dermatology,The Nineth Hospital of Wuhan City,Hubei,Wuhan 430081,China)

机构地区：[1]湖北省武汉市第九医院皮肤科,430081 [2]武汉大学中南医院皮肤科 [3]复旦大学附属浦东医院皮肤科

出　　处：《河北医药》2021年第2期165-170,共6页Hebei Medical Journal

摘　　要：目的研究微小RNA-582-5p(miR-582-5p)对细胞因子信号转导抑制因子1(SOCS1)基因的靶向关系及对皮肤黑色素瘤细胞迁移、侵袭和上皮间充质转化(EMT)的影响。方法实时荧光定量PCR(qPCR)检测人黑色素瘤细胞株A375、G361、WM35、M14和人类皮肤黑色素细胞NHEM中miR-582-5p、SOCS1 mRNA表达,蛋白质印迹法(Western blot)检测SOCS1蛋白表达。构建miR-582-5p过表达的A375细胞,噻唑蓝(MTT)和流式细胞术分别检测细胞增殖与凋亡,Transwell小室法检测细胞迁移和侵袭,Western blot检测N-钙黏蛋白(N-cadherin)、E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)表达,生物信息学结合双荧光素酶报告实验分析miR-582-5p和SOCS1的靶向关系。miR-582-5p和pcDNA-SOCS1共转染入A375细胞,评价过表达SOCS1对miR-582-5p过表达诱导的细胞增殖、凋亡、迁移、侵袭和EMT的影响。结果与NHEM细胞相比,A375、G361、WM35、M14细胞中miR-582-5p表达明显降低(P<0.05),SOCS1 mRNA和SOCS1蛋白表达显著提高(P<0.05)。miR-582-5p过表达明显降低A375细胞48 h、72 h的细胞活性、迁移细胞数、侵袭细胞数、N-cadherin、Vimentin蛋白表达(P<0.05),显著提高细胞凋亡率、E-cadherin蛋白表达(P<0.05)。miR-582-5p靶向调控SOCS1的表达。过表达SOCS1逆转了miR-582-5p过表达对A375细胞增殖、迁移、侵袭、SOCS1、N-cadherin和Vimentin蛋白表达的抑制作用,及对细胞凋亡、E-cadherin蛋白表达的促进作用。结论miR-582-5p直接靶向SOCS1调控皮肤黑色素瘤细胞增殖、迁移、侵袭、凋亡和EMT过程。Objective To investigate the targeting correlation of miR-582-5p with SOCS1 gene and its effects on the migration,invasion and EMT of melanoma cells of skin.Methods The qPCR was used to detect the expression levels of miR-582-5p and SOCS1 mRNA in human melanoma cell lines-A375,G361,WM35,M14 and human skin melanocytes-NHEM.The expression levels of SOCS1 protein were detected by Western Blot.The A375 cells overexpressing miR-582-5p were constructed,and the cell proliferation and apoptosis were detected by MTT assay and flow cytometry,and the cell migration and invasion were detected by Transwell Chamber assay,and the expression levels of N-cadherin,E-cadherin and Vimentin protein were measured by Western Blot.The bioinformatics combined with dual luciferase reporter assay were used to analyze the targeting relationship between miR-582-5p and SOCS1.Moreover the miR-582-5p and pcDNA-SOCS1 were co-transfected into A375 cells,and the effects of overexpression of SOCS1 on cell proliferation,apoptosis,migration,invasion and EMT induced by miR-582-5p overexpression were evaluated.Results As compared with those in NHEM cells,the expression levels of miR-582-5p in A375,G361,WM35 and M14 cells were significantly decreased(P<0.05),however,the expression levels of SOCS1 mRNA and SOCS1 protein were significantly increased(P<0.05).The overexpression of miR-582-5p significantly decreased the cell viability,migration number,number of invasive cells,N-cadherin and Vimentin protein expression in A375 cells at 48h and 72h(P<0.05),which significantly increased the apoptosis rate and E-cadherin protein expression(P<0.05).In addition the miR-582-5p targeted to regulate the expression of SOCS1.And the overexpression of SOCS1 reversed the inhibitory effects of miR-582-5p overexpression on A375 cell proliferation,migration,invasion,SOCS1,N-cadherin and Vimentin protein expression,as well as the promotion effects on cell apoptosis and E-cadherin protein expression.Conclusion The miR-582-5p directly targets SOCS1 to regulate the proliferatio

关 键 词：miR-582-5p SOCS1 黑色素瘤 迁移 侵袭 EMT 

分 类 号：R739.5[医药卫生—肿瘤]