LncRNA-MIAT通过抑制miR-519d-3p激活EZH2促进甲状腺癌的恶性行为

LncRNA-MIAT Promotes Malignant Behavior of Thyroid Cancer by Inhibiting miR-519d-3p to Activate EZH2

目的:探讨长链非编码RNA MIAT(LncRNA-MIAT)对甲状腺癌细胞增殖、凋亡、迁移和侵袭的调控作用与分子机制。方法:用癌症基因组图谱(TCGA)和甲状腺癌(THCA)mRNA-seq数据分析LncRNA-MIAT和组蛋白甲基化转移酶2(EZH2)在甲状腺癌中的表达。用人正常甲状腺细胞系Nthy-ori 3-1和人甲状腺癌细胞系FTC-133作为靶细胞。qPCR法检测LncRNA-MIAT、miR-519d-3p、EZH2 mRNA的表达水平差异。RNA荧光原位杂交(FISH)检测LncRNA-MIAT在FTC-133细胞中的定位,Western blot检测EZH2蛋白的表达。荧光素酶报告基因检测LncRNA-MIAT和miR-519d-3p以及miR-519d-3p与EZH2的直接作用。将FTC-133细胞分为6组[空白对照组(control组)、空载体组(Empty vector组)、过表达LncRNA-MIAT组(LncRNA-MIAT组)、miR-519d-3p的抑制剂组(miR-519d-3p inhibitor组)、EZH2的抑制剂EPZ-6438处理组(EPZ-6438组)、miR-519d-3p抑制剂联合过表达LncRNA-MIAT组(LncRNA-MIAT+miR-519d-3p inhibitor组)]。用Edu试剂盒和平板克隆法检测细胞增殖。用流式细胞术检测细胞凋亡。用Transwell细胞迁移和侵袭实验检测细胞的行为。结果:LncRNA-MIAT和EZH2在甲状腺癌组织和细胞中的表达均上调,二者具有正相关性(r=0.466,P<0.05),miR-519d-3p在FTC-133细胞中表达下调(P<0.05)。经过双荧光素酶报告基因实验检测发现miR-519d-3p的靶基因是EZH2,且LncRNA-MIAT靶向抑制miR-519d-3p激活EZH2。与Empty vector组相比,LncRNA-MIAT组和miR-519d-3p inhibitor组的Edu阳性细胞百分比、细胞克隆数、细胞侵袭和迁移数均显著上调(P<0.05),细胞凋亡率均减少(P<0.05)。EZH2的抑制剂EPZ-6438则减少Edu阳性细胞百分比、细胞克隆数、细胞侵袭和迁移数(P<0.05),增加细胞凋亡率(P<0.05)。分别与LncRNA-MIAT组或miR-519d-3p inhibitor组比,LncRNA-MIAT+miR-519d-3p inhibitor组的Edu阳性细胞百分比、细胞克隆数、细胞侵袭和迁移数均显著上调(P<0.05),细胞凋亡率降低(P<0.05)。结论:甲状腺癌细胞中LncRNA-MIAT通过抑制miR-519d-3p激活EZH2促进甲状腺癌的恶性行为。

Objective:To investigate the regulation and molecular mechanism of long non-coding RNA MIAT(LncRNA-MIAT)on proliferation,apoptosis,migration and invasion of thyroid cancer cells.Methods:The expression of LncRNA-MIAT and histone methyltransferase 2(EZH2)in thyroid cancer was analyzed by Cancer Genome Atlas(TCGA)and thyroid cancer(THCA)mRNA-seq data.Human normal thyroid cell line Nthy-ori 3-1 and human thyroid cancer cell line FTC-133 were used as target cells.The mRNA expression levels of LncRNA-MIAT,miR-519d-3p and EZH2 were detected by qPCR.RNA fluorescence in situ hybridization(FISH)was used to detect the localization of LncRNA-MIAT in FTC-133 cells,and Western blot was used to detect the expression of EZH2 protein.Luciferase reporter gene was used to detect LncRNA-MIAT and miR-519d-3p and the direct effect of miR-519d-3p on EZH2.FTC-133 cells were divided into 6 groups(control group,Empty vector group,LncRNA-MIAT group(LncRNA-MIAT group),miR-519d-3p inhibitor group(miR-519d-3p group),EZH2 inhibitor EPZ-6438 treatment group(EPZ-6438 group),miR-519d-3p inhibitor combined with overexpression of LncRNA-MIAT group(LncRNA-MIAT+miR-519d-3p inhibitor group)].Cell proliferation was detected by Edu kit and plate cloning.Apoptosis was detected by flow cytometry.Cell behavior was detected by Transwell cell migration and invasion assay.Results:The expressions of LncRNA-MIAT and EZH2 in thyroid carcinorna tissues and cells were up-regulated,and there was a positive correlation between the two(r=0.466,P<0.05).The expression of miR-519d-3p was down-regulated in FTC-133 cells(P<0.05).Double luciferase reporter assay showed that the target gene of miR-519d-3p was EZH2,and LncRNA-MIAT targeted inhibition of miR-519d-3p to activate EZH2.Compared with Empty vector group,the percentage of Edu positive cells,the number of cell clones,the number of cell invasion and migration in LncRNA-MIAT group and miR-519d-3p inhibitor group were significantly up-regulated(P<0.05),and the cell apoptosis rate was decreased(P<0.05).EZH2 inhibitor EPZ-6438 decreased the percentage of Edu positive cells,the number of cell clones,the number of cell invasion and migration(P<0.05),and increased the rate of apoptosis(P<0.05).Compared with LncRNA-MIAT group or miR-519d-3p inhibitor group,the Edu positive cell percentage,cell clone number,cell invasion and migration number of LncRNA-MIAT+miR-519d-3p inhibitor group were significantly up-regulated(P<0.05),respectively.The apoptosis rate was decreased(P<0.05).Conclusion:LncRNA-MIAT can promote the malignant behavior of thyroid cancer by inhibiting the activation of EZH2 by miR-519d-3p.