微小RNA-630靶向调控锌指转录因子影响甲状腺癌细胞侵袭能力

MicroRNA-630 targeted regulation Slug affects the invasion ability of thyroid cancer cells

目的观察甲状腺癌(TC)细胞中微小RNA-630(miR-630)对上皮-间充质转化(EMT)标志物锌指转录因子(Slug)表达的影响,及对细胞侵袭能力的影响。方法收集河北医科大学第二医院普外科2018年1月至2019年12月之间的40例乳头状甲状腺癌及其癌旁标本。采用荧光定量聚合酶链反应(PCR)的方法检测甲状腺癌及其癌旁组织中miR-630的表达水平;将miR-630模拟物(mimics)转染至人乳头瘤状甲状腺癌细胞(TPC-1)细胞中,应用荧光定量PCR的方法观察Slug基因的表达水平,使用双荧光素酶报告基因实验观察miR-630对Slug基因的调控作用;同时,将过表达Slug质粒与miR-630 mimics共转染至TPC-1细胞,应用Transwell细胞侵袭实验观察miR-630和Slug表达变化对甲状腺癌细胞侵袭能力的影响,组间比较采用t检验。结果miR-630在乳头状甲状腺癌组织中的表达水平为(1.13±0.07),其表达水平明显低于癌旁组织表达水平(5.33±0.08),差异有统计学意义(t=15.472,P<0.05);miR-630 mimics上调甲状腺乳头状癌TPC-1细胞中miR-630后,Slug mRNA表达水平(0.48±0.09)明显低于对照组细胞(1.03±0.07,t=7.132,P<0.05),差异有统计学意义;miR-630 mimics与Slug 3’非翻译区野生型共转染组荧光素酶活性明显低于对照组(0.38±0.03,t=13.021,P<0.05),差异有统计学意义。而miR-630 mimics与Slug 3’非翻译区突变型共转染组,荧光素酶活性却无明显变化(1.08±0.02比1.02±0.03,t=1.122,P>0.05),差异无统计学意义;miR-630 mimics组侵袭细胞数(241.3±42.4)明显低于对照组侵袭细胞数(467.5±51.7),差异有统计学意义(t=3.214,P<0.05);miR-630 mimics和Slug过表达质粒共转染组细胞的侵袭细胞数(411.6±28.3)与阴性对照组(NC,418.3±31.2)组比较,差异无统计学意义(t=1.131,P>0.05)。结论miR-630能够通过靶向抑制Slug的表达,抑制甲状腺癌细胞的侵袭能力。

Objective To observe the effect of microRNA(miRNA,miR)-630 in thyroid cancer(TC)cells on the expression of the epithelial-mesenchymal transition(EMT)marker Slug,and its influence on cell invasion.Methods A total of 40 cases of papillary TC and their para-cancerous specimens were collected from the Department of General Surgery of Hebei Medical University from January 2018 to December 2019.The real-time quantitative polymerase chain reaction(Real-time PCR)was used to detect the expression level of miR-630 in TC and its adjacent tissues.After transfecting miR-630 mimics into human TC cell line TPC-1,the expression level of Slug gene was detected by Real-time PCR,and the regulatory effect of miR-630 on Slug gene was observed by double luciferase reporter gene assay.The overexpression Slug plasmid and miR-630 mimics were co-transfected into TPC-1 cells.Transwell cell invasion assay was used to observe the effect of miR-630 and Slug expression on the invasion of TC cells.Results Real-time PCR results showed that the expression level of miR-630 in papillary TC tissues was(1.13±0.07),significantly lower than that in adjacent tissues(5.33±0.08)with the difference being statistically significant(t=15.472,P<0.05),and miR-630 mimics up-regulated miR-630 in papillary TC cells,and Slug mRNA expression level(0.48±0.09)was also significantly lower than that in control group(1.03±0.07,t=7.132,P<0.05).The luciferase activity of miR-630 mimics and Slug 3′wild-type co-transfection group was significantly lower than that of control group(0.38±0.03,t=13.021,P<0.05).However,in the co-transfected group of miR-630 mimics and Slug 3′mutant,the luciferase activity did not change significantly(1.08±0.02 vs.1.02±0.03,t=1.122,P>0.05).The number of invasive cells in mir-630 mimics group(241.3±42.4)was significantly less than that in control group(467.5±51.7,t=3.214,P<0.05).Compared with the negative control group(418.3±31.2),the invasion ability of the cells co-transfected with miR-630 mimics and Slug overexpression plasmids(411.6±28.3)was not statistically different(t=1.131,P>0.05).Conclusion miR-630 can inhibit the invasion ability of TC cells by targeting Slug.