MicroRNA-219-5p靶向E-钙黏蛋白调控上皮间质转化抑制肝癌细胞侵袭转移

MicroRNA-219-5p regulates EMT and inhibits invasion and metastasis of hepatoma cells by targeting E-cadherin

摘要:

目的  研究MicroRNA-219-5p（miR-219-5p）靶向E-钙黏蛋白（E-Cadherin）调控上皮间质转化（EMT）抑制肝癌细胞侵袭转移的分子机制。方法  首先通过生物信息学方法，寻找与E-Cadherin结合特异性最好、稳定性强的miRNAs。在30例肝癌组织和20例癌旁肝组织中，通过蛋白免疫印迹法（Western blot）检测E-Cadherin的表达水平；实时荧光定量PCR检测（qRT-PCR）miR-219-5p表达，分析两者之间的相关性。在高转移和低转移的肝癌细胞株中，qRT-PCR检测miR-219-5p表达，Western blot检测E-Cadherin、N-cadherin表达。采用Lipofectamine 2000将miR-219-5p 模拟子（mimic）、抑制子（inhibitor）、阴性对照组（negative contral）转染到肝癌HepG2细胞系，qRT-PCR检测miR-219-5p表达，Western blot检测E-Cadherin、N-cadherin的表达；Transwell方法检测miR-219-5p表达改变对肝癌细胞侵袭转移能力的影响。结果  生物信息学发现miR-219-5p与E-Cadherin结合最稳定，且特异性最好。肝癌组织中miR-219-5p低表达、E-Cadherin高表达，而癌旁组织中miR-219-5p高表达、E-Cadherin低表达。高转移细胞株中miR-219-5p高表达、E-Cadherin低表达而N-cadherin高表达，在低转移细胞株中miR-219-5p低表达、E-Cadherin高表达而N-cadherin低表达（P <0.05）。miR-219-5p水平表达增高，引起E-Cadherin表达下调，N-cadherin高表达；miR-219-5p表达下调，可引起E-Cadherin表达上调，N-cadherin低表达（P <0.05）；HepG2-miR-219-5p 模拟子细胞株中侵袭细胞计数为（24±3）个/高倍镜视野，明显低于对照组细胞株（P <0.05）。结论  miRNA-219-5p可通过靶向结合E-Cadherin，调控EMT信号通路，抑制肝癌细胞的侵袭转移。

Abstract:

Objective To investigate the molecular mechanism of microRNA-219-5p (miR-219-5p) targeting E-cadherin (CDH1) in the regulation of epithelial mesenchymal trasition (EMT), thus inhibiting the invasion and metastasis of hepatoma cells. Methods Bioinformatics methods were used to determine miRNAs with the best specificity and stability of binding to E-cadherin. The correlation between E-cadherin expression detected by Western blot, and miR-219-5p level by qRT-PCR in 30 hepatoma tissues and 20 normal tissues, respectively, was analyzed. miR-219-5p level was detected by qRT-PCR. E-cadherin and N-cadherin levels were detected by Western blot in high and low metastatic hepatoma cell lines. miR-219-5p mimic, inhibitor and negative control were transfected into HepG2 cell line by Lipofectamine 2000, miR-219-5p expression was detected by qRT-PCR and the expressions of E-cadherin and N-cadherin were detected by Western blot. And the effects of miR-219-5p expression change on invasive and metastatic abilities were also tested by the transwell method. Results Bioinformatics methods showed that miR-219-5p was the best target miRNA with the highest specificity and stability for binding to E-cadherin. miR-219-5p had low expression and E-cadherin had high expression in the HCC, while miR-219-5p had high expression and E-cadherin had low expression in the paracancerous tissues. There were high expression of miR-219-5p, low expression of E-cadherin and high expression of N-cadherin in highly metastatic cell line MHCC97-H. There were low expression of miR-219-5p, high expression of E-cadherin and low expression of N-cadherin in low metastatic cell line MHCC97-L (P < 0.05). Increased miR-219-5p caused E-cadherin down-regulation and N-cadherin up-regulation. miR-219-5p down-regulation caused E-cadherin up-regulation and N-cadherin down-regulation in HepG2-miR-219-5p-inhibitor cell line (P < 0.05). The invasion cell count was (24 ± 3)/HP in the HepG2-miR-219-5p mimic cells which was significantly lower than that in the control group (P < 0.05). Conclusions miRNA-219-5p can specifically bind to E-Cadherin and regulate the EMT signaling pathway, thus suppress the invasion and metastasis of hepatoma cells.