miR-590靶向SIP1抑制胆管癌细胞上皮间充质转化的实验研究

目的 探讨miR-590在胆管癌细胞上皮间充质转化（EMT）中的作用及可能机制。方法 采用荧光定量PCR（QPCR）检测人正常肝内胆管上皮细胞系HIBEC以及人胆管癌细胞系HUCCT1、HCCC-9810、RBE中miR-590的表达情况。双荧光素酶报告试验评价miR-590对SIP1的靶向调控作用。向HUCCT1细胞转染miR-590 mimics（转染组）和无义核苷酸序列（NC组），Western blotting 检测两组EMT相关蛋白的表达。向HUCCT1细胞转染SIP1 siRNA，Western blotting 检测干扰SIP1后EMT相关蛋白的表达。结果 HUCCT1、HCCC-9810、RBE细胞系中miR-590水平分别为0.37±0.084、0.31±0.071和0.53±0.089，显著低于人正常肝内胆管上皮细胞系HIBEC的1.12±0.201，差异具有统计学意义（P＜0.05）。miR-590可抑制野生型SIP1 3’UTR报告基因载体的荧光素酶活性，而对突变型SIP1 3’UTR-MUT的荧光素酶活性无影响。转染miR-590 mimics可以下调HUCCT1细胞中Vimentin、N-cadherin、ZEB1、ETS1、SNAIL1及TWIST1蛋白表达，上调E-cadherin蛋白表达。SIP1沉默能上调上皮标志物E-cadherin蛋白表达，并下调间充质蛋白Vimentin和N-cadherin蛋白表达。结论 miR-590通过靶向SIP13’-UTR阻断其翻译，最终抑制胆管癌细胞上皮间充质转化。

miR-590 targeting SIP1 inhibits epithelial-mesenchymal transitionin cholangiocarcinoma cells

Objective To investigate the role and possible mechanism of miR-590in epithelial-mesenchymal transition （EMT） of cholangiocarcinoma cells. Methods  Fluorescence quantitative PCR （QPCR） wasused to detect the expression of miR-590 in human normal intrahepatic bile ductepithelial cell line HIBEC，human cholangiocarcinoma cell line HUCCT1，HCCC-9810 and RBE. Dual luciferase reporter assay was used to evaluate theregulatory effect of miR-590 on SIP1. MiR-590 mimics （transfected group） andnonsense nucleic aicd sequence （NCgroup） weretransfected into HUCCT1 cells， andWestern blotting was used to detect the expression of EMT related protein in twogroups. SIP1 siRNA was transfected into HUCCT1 cells and Western blotting wasused to detect EMT related protein expression after interfering SIP1. ResultsThe expression levels of miR-590 in HUCCT1，HCCC-9810 and RBE cell lines were 0.37±0.084，0.31±0.071and 0.53±0.089， which was significantly lower than that of normal intrahepaticbile duct epithelial cell line HIBEC （1.12±0.201）， and thedifference was statistically significant （P＜0.05）. MiR-590 mimics significantly reduced luciferase expression in thepsiCHECK2-SIP1-3’UTRgroup （0.37±0.041， P＜0.001），whilenot affecting the expression of the psiCHECK2-SIP1-3’UTR-MUT group （1.21±0.211， P＞0.05）.Overexpressed of miR-590 could down-regulated the mesenchymal proteins Vimentin， N-cadherin， ZEB1， ETS1， SNAIL1， TWIST1 and up-regulated the epithelial marker E-cadherin. SIP1silencing could up-regulate the expression of E-cadherin protein anddown-regulate the expression of Vimentin and N-cadherin protein. ConclusionMiR-590 blocked its translation by targeting SIP1-3’UTR and ultimately inhibited epithelial mesenchymal transition ofcholangiocarcinoma cells.