微小RNA-186调控E-钙黏蛋白对肾癌细胞增殖和转移的影响

Effect of miR-186 targeting E-cadherin on proliferation and metastasis of renal cell carcinoma

目的探讨微小RNA-186(miR-186)在肾癌中的作用及其靶向调控E-钙黏蛋白影响肾癌细胞增殖和转移的分子机制。方法收集2015年1月至2019年1月山西省人民医院肾癌组织标本40例,采用实时定量PCR检测miR-186在40例肾癌组织及4种肾癌细胞系中的表达。采用细胞计数试剂盒-8(CCK-8)、克隆形成、细胞划痕、Transwell实验和流式细胞学等方法检测miR-186过表达对肾癌786-O细胞增殖、侵袭迁移和凋亡的影响,裸鼠成瘤实验分析miR-186对肾肿瘤生长的影响。采用Western印迹分析miR-186对上皮-间充质转化(EMT)相关标记物如E-钙黏蛋白表达的影响,双荧光素酶报告基因验证miR-186与E-钙黏蛋白的靶向关系。结果40例患者中,男26例、女14例;年龄(58.4±9.2)岁。miR-186在肾癌组织和细胞中表达下调(组织:0.0052±0.0004比0.0155±0.0015,P<0.001;细胞:0.3343±0.0251、0.4570±0.0266、0.2298±0.0110、0.7411±0.0910比1.0000±0.0852,均P<0.001),miR-186在肿瘤直径大小(≥4 cm比<4 cm为0.0032±0.0034比0.0084±0.0072,P<0.001)、临床分期(≤Ⅱ期比>Ⅱ期为0.0078±0.0058比0.0027±0.0023,P=0.021)及组织学分级(<Ⅱ级比≥Ⅱ级为0.0088±0.0063比0.0046±0.0030,P<0.001)的组间差异有统计学意义。miR-186过表达可抑制肾癌786-O细胞增殖和侵袭迁移,诱导细胞凋亡,并抑制肿瘤生长。miR-186可以直接靶向调控E-钙黏蛋白并促进其表达。结论miR-186可能通过直接调控E-钙黏蛋白影响EMT并抑制肾癌细胞增殖和转移。

Objective To investigate the role of miR-186 in renal cell carcinoma(RCC)and its molecular mechanism of miR-186 targeting E-cadherin to inhibit cell proliferation and metastasis of RCC.Methods A total of 40 RCC samples which were collected in Shanxi Provincial People′s Hospital from January 2015 to January 2019 and four RCC cell lines were measured the expression of miR-186 by real-time quantitative polymerase chain reaction(qPCR).The effect of miR-186 overexpression on the proliferation,invasion,migration and apoptosis of 786-O cells were detected by cell counting kit-8(CCK-8),colony formation,wound healing and Transwell assay and flow cytometric analysis.The effect of miR-186 on the expression of epithelial-to-mesenchymal transition(EMT)related markers(E-cadherin,N-cadherin and Vimentin)was analyzed by Western blot,and the dual luciferase reporter was used to verify the miR-186 targeting E-cadherin.Results There were 26 males and 14 females with an age of(58.4±9.2)years.miR-186 expression levels decreased significantly in RCC tissues and cells(tissues:0.0052±0.0004 vs 0.0155±0.0015,P<0.001;cells:0.3343±0.0251,0.4570±0.0266,0.2298±0.0110,0.7411±0.0910 vs 1.0000±0.0852,all P<0.001).The expression of miR-186 had a negative correlation with tumor size(≥4 cm:0.0032±0.0034 vs<4 cm:0.0084±0.0072,P<0.001),TNM staging(≤Ⅱ:0.0078±0.0058 vs>Ⅱ:0.0027±0.0023,P=0.021)and Fuhrman grade(<Ⅱ:0.0088±0.0063 vs≥Ⅱ:0.0046±0.0030,P<0.001).The overexpression of miR-186 significantly inhibited cell proliferation and metastasis,and induced cell apoptosis.delivered.miR-186 overexpression can retard tumor growth in nude mice.Luciferase assay showed that E-cadherin was a direct target gene of miR-186.Conclusion miR-186 may affect EMT of RCC and inhibit the proliferation and metastasis of RCC by directly regulating E-cadherin.