摘要
目的 探讨miR-378b通过ALX4调节甲状腺癌细胞的上皮-间质转化以及侵袭和迁移能力。方法 MSP法检测甲状腺癌细胞ALX4基因甲基化状态,用去甲基化药物5-aza-dc处理甲状腺癌细胞后,通过MSP法、RT-PCR和Western blot探究ALX4表达与启动子甲基化的关系;将miR-378b mimics/inhibitors转染甲状腺癌细胞后,通过MSP法检测miR-378b对ALX4甲基化的影响,通过RT-PCR、Western blot检测miR-378b对ALX4以及上皮-间质转化相关因子HMGA1、N-cadherin、Vimentin表达的影响,通过TranswellTM实验检测甲状腺癌细胞的侵袭迁移情况;荧光素酶活性分析实验验证ALX4是miR-378b的作用靶点;pcDNA3.1-ALX4质粒过表达甲状腺癌细胞后,检测ALX4对细胞上皮-间质转化以及侵袭迁移能力的影响。结果 与正常的甲状腺细胞比较,甲状腺癌细胞中ALX4基因发生了甲基化,导致ALX4表达受抑制;miR-378b mimics转染甲状腺癌细胞后,抑制ALX4甲基化发生,促进ALX4表达,抑制甲状腺癌细胞上皮-间质转化相关因子HMGA1、N-cadherin、Vimentin的表达,抑制细胞的侵袭迁移能力;miR-378b靶向作用ALX4基因的3′非翻译区(3′UTR);过表达pcDNA3.1-ALX4后,抑制HMGA1、N-cadherin、Vimentin的表达,抑制细胞侵袭迁移能力。结论 miR-378b通过抑制ALX4甲基化的发生从而抑制甲状腺癌细胞上皮-间质的转化以及细胞的侵袭和迁移能力。
Objective To investigate the ability of miR-378b to regulate epithelial-mesenchymal transition,invasion and migration of thyroid cancer cells through ALX4.Methods The methylation status of ALX4 gene in thyroid cancer cells was detected by MSP method,the thyroid cancer cells were treated with the demethylation drug 5-aza-dc,and the relationship between ALX4 expression and promoter methylation was investigated by MSP,RT-PCR and Western blot.miR-378b mimics/inhibitors were transfected into thyroid cancer cells,and the effect of miR-378b on ALX4 methylation was detected by MSP.The effects of miR-378b on the expression of ALX4 and epithelial-mesenchymal factors HMGA1,N-cadherin,and Vimentin were detected by RT-PCR and Western blot,and the invasion and migration of thyroid cancer cells were detected by TranswellTM The targeting relationship between miR-378b and ALX4 was determined by double luciferase activity analysis experiments.After pcDNA3.1-ALX4 plasmid was overexpressed,the effect of ALX4 on cell epithelial-mesenchymal transition and its ability to invade and migrate was examined.Results Compared with normal thyroid cells,ALX4 gene was methylated in thyroid cancer cells,and the expression of ALX4 was suppressed due to the occurrence of methylation.MiR-378b mimics inhibited the occurrence of ALX4 methylation,promoted the expression of ALX4,inhibited the expression of epithelial-mesenchymal transition-related factors HMGA1,N-cadherin and Vimentin,and inhibited cell invasion and migration ability.MiR-378b targets the 3'untranslated region(3'UTR)of the ALX4 gene.Overexpression of pcDNA3.1-ALX4 inhibited the expression of HMGA1,N-cadherin and Vimentin,and inhibited the invasion and migration ability of the cells.Conclusion miR-378b inhibits the epithelial-mesenchymal transition and cell invasion and migration of thyroid cancer cells via inhibiting the occurrence of ALX4 methylation.
作者
宋婷婷
刘江豪
SONG Ting-ting;LIU Jiang-hao(Department of General Surgery,the Eighth People's Hospital of Xinjiang Uygur Autonomous Region,Urumqi 830000,China)
出处
《解剖科学进展》
CAS
2024年第1期90-94,共5页
Progress of Anatomical Sciences
基金
新疆维吾尔自治区自然科学基金(2019D01C215)。
