Li Wenting, Yu Xiaolan, Chen Xiliu, Wang Zheng, Yin Ming, Zhao Zonghao, Zhu Chuanwu
3rd Liver Unit, Department of Infectious Disease, Anhui Provincial Hospital, Hefei, Anhui 230001, P.R. China.
The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230000, P.R. China.
Exp Ther Med. 2021 Feb;21(2):169. doi: 10.3892/etm.2020.9600. Epub 2020 Dec 25.
MicroRNA (miR)-21-5p is a newly discovered factor that mediates TGF-β1 signaling. The present study was designed to investigate the role of TGF-β1/miR-21-5p in hepatitis B virus (HBV)-induced liver fibrosis. HBV-infected sodium taurocholate co-transporting polypeptide (NTCP)-transfected Huh7.5.1 cells were co-cultured with LX2 cells to simulate HBV infection in the present study. A total of 29 patients with chronic HBV infection were enrolled. Cells were transfected with miR-21-5p mimic or inhibitor with or without TGF-β1 stimulation. The demographic, biochemical and virological data from the 29 patients were analyzed and liver tissues were collected. miR-21-5p levels and the mRNA and protein expression of α-smooth muscle actin (SMA), collagen type 1 α 1 (CoL1A1), tissue inhibitor of metalloproteinase (TIMP)-1 and Smad from liver cells or tissues were detected by quantitative PCR analysis and western blotting, respectively. Cell viability was observed, and the liver fibrosis score was evaluated. The association between miR-21-5p and liver fibrosis was evaluated by correlation analysis. HBV infection upregulated TGF-β1/miR-21-5p mRNA expression in NTCP-Huh7.5.1 cells compared with mock infection (P<0.05). TGF-β1 incubation significantly increased miR-21-5p levels, as well as the mRNA and protein expression of α-SMA, CoL1A1 and TIMP-1, and reduced Smad7 expression in LX2 cells compared with the normal group, and these effects were counteracted by miR-21-5p inhibitor (P<0.05). miR-21-5p overexpression also contributed to TGF-β1-induced α-SMA, CoL1A1 and TIMP-1 expression in LX2 cells (P<0.05). Co-culture with HBV-infected NTCP-Huh7.5.1 cells upregulated TGF-β1/miR-21-5p activity and CoL1A1 expression in LX2 cells compared with normal control, which were significantly reduced by miR-21-5p inhibitor (P<0.05). miR-21-5p levels were significantly correlated with the liver fibrosis score (r=0.888; P<0.05). These data demonstrated that HBV induced liver fibrosis via the TGF-β1/miR-21-5p pathway and suggested that miR-21-5p may be an effective anti-fibrosis target.
微小RNA（miR）-21-5p是一种新发现的介导转化生长因子-β1（TGF-β1）信号传导的因子。本研究旨在探讨TGF-β1/miR-21-5p在乙型肝炎病毒（HBV）诱导的肝纤维化中的作用。在本研究中，将感染HBV的牛磺胆酸钠共转运多肽（NTCP）转染的Huh7.5.1细胞与LX2细胞共培养以模拟HBV感染。共纳入29例慢性HBV感染患者。细胞分别用miR-21-5p模拟物或抑制剂转染，同时有或无TGF-β1刺激。分析了这29例患者的人口统计学、生化和病毒学数据，并收集了肝组织。分别通过定量PCR分析和蛋白质印迹法检测肝细胞或组织中miR-21-5p水平以及α-平滑肌肌动蛋白（SMA）、Ⅰ型胶原α1（CoL1A1）、金属蛋白酶组织抑制剂（TIMP）-1和Smad的mRNA及蛋白表达。观察细胞活力并评估肝纤维化评分。通过相关性分析评估miR-21-5p与肝纤维化之间的关联。与mock感染相比，HBV感染使NTCP-Huh7.5.1细胞中TGF-β1/miR-21-5p mRNA表达上调（P<0.05）。与正常组相比，TGF-β1孵育显著增加了LX2细胞中miR-21-5p水平以及α-SMA、CoL1A1和TIMP-1的mRNA及蛋白表达，并降低了Smad7表达，而miR-21-5p抑制剂可抵消这些作用（P<0.05）。miR-21-5p过表达也促进了TGF-β1诱导的LX2细胞中α-SMA、CoL1A1和TIMP-1的表达（P<0.05）。与正常对照相比，与感染HBV的NTCP-Huh7.5.1细胞共培养上调了LX2细胞中TGF-β1/miR-21-5p活性和CoL1A1表达，而miR-21-5p抑制剂可使其显著降低（P<0.05）。miR-21-5p水平与肝纤维化评分显著相关（r=0.888；P<0.05）。这些数据表明，HBV通过TGF-β1/miR-21-5p途径诱导肝纤维化，并提示miR-21-5p可能是一个有效的抗纤维化靶点。
