miR-125在甲状腺癌组织中的表达及通过靶向负调控RAB11A促进甲状腺癌细胞活性的研究

Expression of miR-125 in thyroid carcinoma and its effect on the activity of thyroid cancer cells by targeting RAB11A

目的:探讨miR-125在甲状腺癌组织中的表达及促进甲状腺癌细胞增殖的相关机制。方法:采用RT-PCR法检测26例甲状腺乳头状癌(PTC)标本和4株PTC细胞株中miR-125的表达水平。采用CCK-8法、细胞集落形成实验、Transwell实验以及流式细胞法验证miR-125在PTC细胞中的作用。采用双荧光素酶实验验证RAB11A是否是miR-125的直接靶点。结果:miR-125在甲状腺癌组织中的相对表达水平显著高于癌旁正常组织(P<0.05);与Nthy-Ori3-1相比,甲状腺癌细胞株TPC-1、NPA、KTC-1、WRO中的miR-125表达水平显著升高(P<0.05)。与miRNA NC组相比,miR-125 mimics组的细胞克隆形成数量及侵袭细胞数量明显增多,miR-125 inhibitor组显著减少(P<0.05)。Transwell实验结果显示,miRNA NC组细胞的迁移速度明显比miR-125 mimics组慢,但显著快于miR-125 inhibitor组。流式细胞检测显示,miRNA NC组、miR-125 mimics组、miR-125 inhibitor组的凋亡细胞比例分别为(5.1±0.08)%、(1.5±0.06)%、(7.8±0.07)%,两两比较后发现,miR-125 mimics组凋亡细胞比例最低,miR-125 inhibitor组最高。荧光素酶报告显示,RAB11A是miR-125的直接靶点。将miR-125模拟物、抑制剂或miRNA NC(100 nmol/L)转染KTC-1细胞后,RAB11A mRNA水平均无明显改变。但Western blot检测显示,与对照组相比,miR-125 mimics组RAB11A蛋白表达显著降低,miR-125 inhibitor组RAB11A蛋白表达显著增加。表明miR-125不影响RAB11A mRNA的稳定性,但在转录后水平调节RAB11A蛋白的表达。结论:miR-125通过在转录后水平负调控RAB11A蛋白的表达而发挥促甲状腺癌的作用。miR-125有望成为PTC患者诊断和治疗的新靶点。

Objective:To investigate the expression of miR-125 in thyroid carcinoma tissues and its mechanism of promoting the proliferation of thyroid cancer cells.Methods:The expression of miR-125 in 26 cases of papillary thyroid carcinoma(PTC)and 4 PTC cell lines was detected by RT-PCR.CCK-8 assay,colony forming assay,Transwell assay and flow cytometry were used to verify the role of miR-125 in PTC cells.Double luciferase assay was used to verify whether RAB11A is a direct target of miR-125.Results:The relative expression level of miR-125 in thyroid carcinoma tissues was significantly higher than that in adjacent normal tissues(P<0.05).Compared with Nthy-Ori3-1,the expression levels of miR-125 in TPC-1,NPA,KTC-1 and WRO were significantly higher(P<0.05).Compared with miRNA NC group,the number of cell clones and invasive cells in miR-125 mimics group was significantly higher,and that in miR-125 inhibitor group was significantly reduced(P<0.05).Transwell results showed that the migration rate of miRNA NC group was significantly slower than that of miR-125 mimics group,but significantly faster than that of miR-125 inhibitor group.The percentage of apoptotic cells in miRNA NC group,miR-125 mimics group and miR-125 inhibitor group were(5.1±0.08)%,(1.5±0.06)%,and(7.8±0.07)%,respectively.After paired comparison,it was found that the apoptotic cell proportion of the miR-125 mimics group was the lowest and that of the miR-125 inhibitor group was the highest.Luciferase reports showed that RAB11A was a direct target of miR-125.After transfecting KTC-1 cells with miR-125 mimics,inhibitors or miRNA NC(100 nmol/L),RAB11A mRNA levels did not change significantly.Western blot analysis showed that RAB11A protein expression in miR-125 mimics group was significantly lower than that in control group,and RAB11A protein expression in miR-125 inhibitor group was significantly increased.The results showed that miR-125 did not affect the stability of RAB11A mRNA and regulated the post-transcriptional level expression of RAB11A protein.Conclusion:miR-125 plays a role in promoting thyroid cancer by negatively regulating RAB11A protein expression at the post transcriptional level.miR-125 is expected to become a new target for the diagnosis and treatment of PTC.