长链非编码RNA牛磺酸上调基因靶向作用于微小RNA-145对甲状腺癌细胞侵袭的影响及其机制

Effect and mechanism of long non coding RNA taurine upregulated gene 1 targeting microRNA-145 on the invasion of thyroid cancer cells

目的观察甲状腺乳头状癌(PTC)细胞中长链非编码RNA(lncRNA)牛磺酸上调基因(TUG1)靶向作用于微小RNA(microRNA,miR)-145/锌指E-盒结合同源异形盒-1(ZEB1)对细胞侵袭能力的影响。方法实验标本选自2018年4月至2019年12月间河北医科大学第二医院甲状腺乳腺外科收集的甲状腺乳头状癌及癌旁标本共30例,采用实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)的方法观察乳头状甲状腺癌及其癌旁组织中lncRNA TUG1及miR-145的表达水平;将lncRNA TUG1 shRNA和miR-145 mimics分别转染到甲状腺乳头状癌TPC-1细胞中,利用Transwell细胞侵袭实验观察lncRNA TUG1和miR-145对甲状腺癌细胞侵袭能力的影响。应用生物信息学及双荧光素酶报告基因分析lncRNA TUG1和miR-145的关系及miR-145的靶基因。同时,利用蛋白质印迹法(Western blot)实验检测lncRNA TUG1和miR-145对靶蛋白表达水平的影响。两组间统计学差异通过双尾t检验进行分析。结果实时荧光定量PCR实验结果显示,lncRNA TUG1在甲状腺乳头状癌组织中的表达水平(4.43±0.07)高于癌旁组织(1.13±0.06),差异有统计学意义(t=3.124,P<0.05),miR-145的表达水平(0.39±0.08)低于癌旁组织(1.08±0.04),差异有统计学意义(t=2.937,P<0.05);甲状腺乳头状癌TPC-1细胞中转染lncRNA TUG1 shRNA组及miR-145 mimics组侵袭细胞数[(292.4±48.2)、(234.2±42.4)个]低于各自对照组侵袭细胞数[(452.9±50.8)、(439.4±39.8)个],差异有统计学意义(t=3.205,P<0.05;t=3.186,P<0.05);生物信息学及双荧光素酶报告基因分析结果显示,lncRNA TUG1可以与miR-145互补结合,ZEB1是miR-145的靶基因;lncRNA TUG1 shRNA组细胞ZEB1蛋白表达水平(0.33±0.12)低于lncRNA对照组细胞中ZEB1蛋白表达水平(1.17±0.12),差异有统计学意义(t=3.320,P<0.05),miR-145 mimics转染组细胞蛋白表达水平(0.46±0.11)低于miRNA对照组细胞中ZEB1蛋白表达水平(1.05±0.12),差异有统计学意义(t=3.450,P<0.05)。结论lncRNA TUG1能够靶向作用于miR-145/ZEB1调控甲状腺癌细胞的侵袭能力。

Objective To observe the effect of long non-coding RNA(lncRNA)taurine upregulated gene 1(TUG1)targeting microRNA(miR)-145/zinc-finger E-box binding homeobox 1(ZEB1)on the invasion of papillary thyroid carcinoma(PTC)cells.Methods Real-time polymerase chain reaction(PCR)method was used to detect the expression level of lncRN TUG1 and miR-145 in PTC and the adjacent tissues.The lncRNA TUG1 shRNA and miR-145 mimics were transfected into papillary thyroid carcinome cells-1(TPC-1)cells,respectively,and the effect of lncRNA TUG1 and miR-145 on the invasion ability of PTC cells was observed by the Transwell assay.The bioinformatics and dual luciferase reporter gene used to analyze the relationship between lncRNA TUG1 and miR-145 and the target gene of miR-145.Western blotting was used to verify the influence of lncRNA TUG1 and miR-145 on the expression level of target protein.Two-tailed Student′s t-test was used to evaluate the differences between two groups.Results Real-time PCR showed that the expression level of lncRNA TUG1 in PTC tissues(4.43±0.07)was significantly higher than that in adjacent tissues(1.13±0.06,t=3.124,P<0.05),and the expression level of miR-145(0.39±0.08)was lower than that in adjacent tissues(1.08±0.04,t=2.937,P<0.05);The number of invasive cells in TPC-1 cells transfected with lncRNA TUG1 shRNA(292.4±48.2)and miR-145 mimics(234.2±42.4)was lower than that in control group(452.9±50.8,439.4±39.8,t=3.205,P<0.05;t=3.186,P<0.05);The results of bioinformatics and dual-luciferase reporter gene analysis showed that lncRNA TUG1 could bind to miR-145,and ZEB1 was the target gene of miR-145.Compared with the respective control cells(1.17±0.12,1.05±0.12),the expression level of ZEB1 protein in lncRNA TUG1 shRNA group was down-regulated(0.33±0.12),and the difference was statistically significant(t=3.320,P<0.05),and the protein expression level in miR-145 mimics transfected group was down-regulated(0.46±0.11),and the difference was statistically significant(t=3.450,P<0.05).Conclusion lncRNA TUG1 can target miR-145/ZEB1 to regulate the invasion ability of PTC cells.