微小RNA-204抑制E盒结合锌指蛋白1调控上皮-间充质转化途径影响甲状腺癌细胞侵袭能力的机制

Effect of microRNA-204 on invasion of thyroid cancer cells by inhibiting zinc finger E-box binding protein 1 mediated epithelial-mesenchymal transition pathway

目的观察甲状腺乳头状癌(PTC)细胞中微小RNA(miR)-204对E盒结合锌指蛋白1(ZEB1)表达的影响,及对上皮-间充质转化(EMT)及细胞侵袭能力的影响。方法采用实时荧光定量聚合酶链反应(PCR)法检测甲状腺乳头状癌及其癌旁组织中miR-204的表达;将miR-204 mimics转染到甲状腺乳头状癌TPC-1细胞中,采用荧光定量PCR的方法检测ZEB1、E-钙粘蛋白(E-cadherin)、N-钙粘蛋白(N-cadherin)及波形蛋白(vimentin)的表达水平,应用双荧光素酶报告基因分析实验观察miR-204对ZEB1基因表达的调控作用;同时,将miR-204 mimics与过表达ZEB1质粒共转染至人甲状腺乳头状癌细胞-1(TPC-1)细胞中,采用Transwell细胞侵袭实验观察miR-204和ZEB1表达变化对甲状腺乳头状癌细胞侵袭能力的影响。组间比较采用t检验。结果miR-204在甲状腺癌乳头状组织中的表达水平低于癌旁正常组织中的表达水平,差异有统计学意义(1.14±0.08比3.52±0.07,t=13.250,P<0.05);miR-204 mimics转染组侵袭细胞数低于对照组侵袭细胞数[(149.4±20.2)个比(268.6±30.2)个,t=3.402,P<0.05],差异有统计学意义;miR-204 mimics转染组细胞E-cadherin表达水平高于对照组细胞(0.98±0.17比0.35±0.12,t=2.903,P<0.05),差异有统计学意义,N-cadherin和vimentin表达量低于对照组细胞(0.29±0.11比0.89±0.10,t=2.974,P<0.05;0.30±0.13比0.93±0.15,t=2.859,P<0.05),差异有统计学意义;双荧光素酶报告基因分析结果显示,miR-204 mimics转染组ZEB1的相对荧光素酶活性低于对照组(0.23±0.06比1.12±0.04,t=12.460,P<0.05),差异有统计学意义;甲状腺癌乳头状癌TPC-1细胞中转染miR-204 mimics组ZEB1基因的表达水平低于对照组(0.41±0.06比1.10±0.07,t=6.783,P<0.05),差异有统计学意义;miR-204 mimics与ZEB1过表达质粒共转染后,甲状腺癌细胞的侵袭细胞数与对照组比较差异无统计学意义[(224.4±23.5)个比(230.0±28.5)个,t=1.140,P>0.05],差异有统计学意义。结论miR-204能够通过靶向抑制ZEB1的表达,从而抑制细胞发生上皮间质转化进而抑制甲状腺乳头状癌细胞的侵袭能力。

Objective To observe the effect of microRNA(miR)-204 on the expression of zinc finger E-box binding protein 1(ZEB1)in papillary thyroid carcinoma(PTC)cells,as well as the effect on epithelial-mesenchymal transition(EMT)and cell invasion ability.Methods Real-time polymerase chain reaction(PCR)method was used to detect the expression level of miR-204 in papillary thyroid carcinoma and its adjacent tissues.The miR-204 mimics were transfected into thyroid papillary carcinoma cells-1(TPC-1)cells of papillary thyroid carcinoma,the expression levels of ZEB1,E-cadherin,N-cadherin and vimentin were detected by real-time PCR,and the regulatory effect of miR-204 on ZEB1 gene was observed by double luciferase reporter gene assay.The miR-204 mimics and plasmid overexpressing ZEB1 were co-transfected into TPC-1 cells.Transwell cell invasion assay was used to observe the effect of miR-204 and ZEB1 expression on the invasion of thyroid cancer cells.Results Real-time PCR results found that the expression level of miR-204 in papillary thyroid carcinoma(1.14±0.08)was significantly lower than that in adjacent tissues(3.52±0.07,t=13.250,P<0.05).The number of invasive cells in the miR-204 mimics transfection group was significantly less than in the control group(149.4±20.2 vs.268.6±30.2,t=3.402,P<0.05).The expression level of E-cadherin in miR-204 mimics transfected cells was significantly higher than that in control cells(0.98±0.17 vs.0.35±0.12,t=2.903,P<0.05),and the expression of N-cadherin and vimentin was significantly lower(0.29±0.11 vs.0.89±0.10,t=2.974,P<0.05;0.30±0.13 vs.0.93±0.15,t=2.859,P<0.05).The relative luciferase activity of ZEB1 in the miR-204 mimics transfection group was significantly lower than that in the control group(0.23±0.06 vs.1.12±0.04,t=12.460,P<0.05).The expression level of ZEB1 in thyroid cancer papillary carcinoma TPC-1 cells transfected with miR-204 mimics was significantly lower than that in the control group(0.41±0.06 vs.1.10±0.07,t=6.783,P<0.05).After co-transfection with ZEB1-overexpressed plasmids,the number of invasive cells in thyroid cancer cells was not significantly different from that in the control group(224.4±23.5 vs.230.0±28.5,t=1.140,P>0.05).Conclusion MiR-204 can inhibit EMT and invasion of papillary thyroid carcinoma cells by targeting ZEB1 expression.