摘要/Abstract

摘要： 目的·探讨miR-214在肝星状细胞（hepatic stellate cells，HSCs）活化过程及肝纤维化进程中的生物学作用及其可能的作用机制。方法·采用实时荧光定量PCR（quantitative real-time PCR，qPCR）检测HSCs活化过程及CCl4诱导大鼠（肝纤维化模型）的纤维化进程中miR-214的表达。采用慢病毒转染法处理HSC-T6细胞，并将其分为miR-214过表达组、miR-214敲除组和阴性对照慢病毒组即mock组。采用qPCR和蛋白质印迹法（Western blotting）分别检测各组1型胶原蛋白（collagen type 1，COL1）及α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）等HSCs活化标志物的基因及蛋白表达水平，通过Transwell细胞迁移实验检测不同组细胞的迁移能力，采用流式细胞仪检测各组细胞的凋亡率。通过双荧光素酶报告基因实验检测缺氧诱导因子-1α抑制剂（hypoxia-inducible factor-1α inhibitor，Hif1an）是否为miR-214的靶基因，而后采用qPCR和Western blotting分别检测3组细胞、原代HSCs活化过程及肝纤维化模型中HIF1AN的表达水平。结果·miR-214在HSCs活化过程和肝纤维化进程中表达显著增加（均P<0.05）。与mock组对比，miR-214过表达组的COL1及α-SMA的基因及蛋白表达增加，细胞迁移能力增强，细胞凋亡率降低（均P<0.05）；miR-214敲除组的COL1及α-SMA表达下降，细胞迁移能力减弱（均P<0.05）。双荧光素酶报告基因实验显示，Hif1an为miR-214的靶基因。与mock组对比，miR-214过表达组中HIF1AN的基因及蛋白表达下降（均P<0.05），而miR-214敲除组中则增加（均P<0.05）。且在原代HSCs活化过程和肝纤维化进程中HIF1AN的表达下调（均P<0.05）。结论·miR-214可能通过靶向调控Hif1an促进HSCs的迁移及活化，继而促进肝纤维化进程，提示其或可作为治疗肝纤维化的新的标志物和潜在靶点。
关键词: miR-214, 肝星状细胞活化, 肝纤维化, 迁移
Abstract:
Objective · To explore the biological role of miR-214 in the activation of hepatic stellate cells (HSCs) and the procession of liver fibrosis and its possible mechanism. Methods · Quantitative real-time PCR (qPCR) was used to detect the expression of miR-214 in the activation of HSCs and the progression of liver fibrosis in rats induced by CCl4 (liver fibrosis model). HSC-T6 cells were treated with lentivirus infection and divided into miR-214 overexpression group, miR-214 knockout group and negative control lentivirus group (mock group). qPCR and Western blotting were used to detect the gene and protein expression levels of collagen type 1 (COL1) and α-smooth muscle actin (α-SMA) in the three groups, respectively. Transwell assay and flow cytometry were used to detect the migration and apoptosis of HSCs in the three groups, respectively. Double luciferase reporter gene assay was used to detect weather Hif1an was the target gene of miR-214, and then qPCR and Western blotting were used to detect the expression levels of HIF1AN in the three groups, the activation of HSCs and the liver fibrosis model. Results · miR-214 was upregulated during HSCs activation and the progression of liver fibrosis (both P<0.05). Compared with the mock group, the gene and protein expressions of COL1 and α-SMA in the miR-214 overexpression group were increased, and HSCs migration ability was increased and apoptosis rate was decreased (all P<0.05); the expressions of COL1 and α-SMA in the miR-214 knockdown group were decreased, and HSCs migration ability was decreased (all P<0.05). Double luciferase reporter gene assay showed that Hif1an was the target gene of miR-214. Compared with the mock group, the gene and protein expressions of HIF1AN in the miR-214 overexpression group were decreased (both P<0.05), and those in the miR-214 knockdown group were increased (both P<0.05). The expression of HIF1AN was decreased during HSCs activation and the progression of liver fibrosis (all P<0.05). Conclusion · miR-214 may promote the migration and activation of HSCs by targeting Hif1an, and then promote the progression of liver fibrosis, suggesting that miR-214 may be a new marker and potential target for the treatment of liver fibrosis.
Key words: miR-214, hepatic stellate cell activation, liver fibrosis, migration


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