| 王旭天,孙 亮,刘宇方,尚楚智,郑 鑫.miR-101a在乙型肝炎病毒相关性肝纤维化患者中的表达及对肝星状细胞的影响[J].,2021,(13):2415-2422 |
| miR-101a在乙型肝炎病毒相关性肝纤维化患者中的表达及对肝星状细胞的影响 |
| Expression of miR-101a in Patients with Hepatitis B Virus-related Liver Fibrosis and Its Effect on Hepatic Stellate Cells |
| 投稿时间：2021-01-23 修订日期：2021-02-18 |
| DOI：10.13241/j.cnki.pmb.2021.13.004 |
| 中文关键词: miR-101a 乙型肝炎病毒 肝纤维化 肝星状细胞 上皮间质转化 |
| 英文关键词: miR-101a Hepatitis B virus Liver fibrosis Hepatic stellate cells Epithelial-mesenchymal transition |
| 基金项目:中国红十字基金会INTACT科研基金（XM_HR_INTACT_2020_10_01） |
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| 中文摘要: |
| 摘要 目的：探究miR-101a在乙型肝炎病毒（HBV）相关性肝纤维化患者中的表达及对肝星状细胞（HSC）的影响。方法：根据肝纤维化程度将HBV相关性肝纤维化患者进行分组（S0组、S1组、S2组、S3组和S4组），健康受试者作为健康对照组。通过RT-PCR检测肺组织中miR-101a的表达，并分析miR-101a与疾病严重程度的关系。使用重组人TGF-β1处理人肝星状细胞系LX-2，并对LX-2细胞转染阴性对照 miRNA模拟物（NC-mimic组）、miR-101a模拟物（miR-101a-mimic组）、阴性对照 miRNA抑制剂（NC-inhibitor组）或miR-101a抑制剂（miR-101a-inhibitor组），未转染的细胞作为对照组，然后通过RT-PCR或蛋白质印迹检测激活HSC及ECM产生的关键基因（α-SMA、COL1A1、COL1A2和COL3A1）和蛋白（a-SMA、collagen I和collagen III）的表达水平。将SD大鼠随机分为4组：对照组、CCl4组、Ad-control组和Ad-miR-101a组，对大鼠腹腔注射CCl4（1 mL/kg体重）诱导肝纤维化模型，每周3次，共4周。然后将5×109感染单位的携带miR-101a的重组腺病毒（Ad-miR-101a）或对照腺病毒（Ad-control）经尾静脉注射到大鼠中。4周后，通过苏木精和伊红（H＆E）和Masson三色染色评估肝脏形态和纤维化，通过免疫组化染色评估肝脏α-SMA、E-cadherin、vimentin、Smad4或p-Smad2/3的表达。结果：与健康受试者相比，HBV相关肝纤维化患者肝组织中miR-101a的表达水平明显降低，并且miR-101a的表达水平随着患者的严重程度升高而降低（P<0.05）。与未处理的细胞相比，miR-101a在TGF-β1处理的LX-2细胞中以浓度和时间依赖性方式显著下降（P<0.05）。与未处理的细胞相比，5 ng/mL TGF-β1处理LX-2细胞中的α-SMA、COL1A1、COL1A2和COL3A1 mRNA表达水平及a-SMA、collagen I和collagen III 蛋白表达水平均显著升高（P<0.05）。与对照组相比，miR-101a-mimic组的α-SMA、COL1A1、COL1A2、COL3A1和TGF-β1 mRNA和a-SMA、collagen I、collagen III、TGF-β1、Smad3和p-Smad3蛋白表达均下调（P<0.05）。与对照组相比，Ad-miR-101a组大鼠肝组织中E-cadherin的表达上调，但α-SMA、vimentin、Smad4和p-Smad2/3的表达下调（P<0.05）；Ad-miR-101a组大鼠的肝组织形态基本恢复正常，肝组织纤维化程度低于CCl4组。结论：miR-101a水平与乙型肝炎病毒相关性肝纤维化严重程度相关，上调miR-101a可能通过抑制HSC的活化及上皮间质转化发挥抗纤维化作用。 |
| 英文摘要: |
| ABSTRACT Objective: To reveal the expression of miR-101a in patients with hepatitis B virus (HBV) -related liver fibrosis and its effect on hepatic stellate cells (HSC). Methods: According to the degree of liver fibrosis, patients with HBV-related liver fibrosis were divided into S0 group, S1 group, S2 group, S3 group and S4 group, and healthy subjects were taken as healthy control group. The expression of miR-101a in the lung tissue of patients with HBV-related liver fibrosis was detected by RT-PCR, and the relationship between miR-101a and disease severity was analyzed. Human hepatic stellate cell line LX-2 was treated with recombinant human TGF-β1 and transfected with negative control miRNA mimics (NC-mimic group), miR-101a mimics (miR-101a-mimic group), negative control miRNA inhibitors(NC-inhibitor group) or miR-101a inhibitors(miR-101a-inhibitor group). Untransfected cells served as control groups. Then the expression levels of key genes (α-SMA, COL1A1, COL1A2 and COL3A1) and proteins (a-SMA, collagen I and collagen III) that activated HSC and ECM were detected by RT-PCR or Western blot. The SD rats were randomly divided into 4 groups: control group, CCl4 group, Ad-control group and Ad-miR-101a group. Rats were intraperitoneally injected with CCl4 (1 mL/kg body weight) to induce liver fibrosis model, 3 times a week for 4 weeks. Then, 5×109 infected units of recombinant adenovirus carrying miR-101a (Ad-miR-101a) or control adenovirus (Ad-control) were injected into rats via tail vein. After 4 weeks, liver morphology and fibrosis were evaluated by hematoxylin and eosin(H & E) and Masson trichrome staining, and liver α-SMA, E-cadherin, vimentin, Smad4 or p-Smad2/3 were evaluated by immunohistochemical staining expression. Results: Compared with healthy subjects, the expression level of miR-101a in liver tissues of patients with HBV-related liver fibrosis was significantly decreased, and the expression level of miR-101a decreased with the increase of patient severity(P<0.05). Compared with untreated cells, miR-101a decreased significantly in LX-2 cells treated with TGF-β1 in a concentration-and time-dependent manner(P<0.05). Compared with untreated cells, the expression of α-SMA, COL1A1, COL1A2 and COL3A1 mRNAs and a-SMA, collagen I and collagen III proteins in LX-2 cells treated with 5 ng/mL TGF- β1 were significantly increased(P<0.05). Compared with control group, the expressions of α-SMA, COL1A1, COL1A2, COL3A1 and TGF- β1 mRNA and a-SMA, collagen I, collagen III, TGF- β1, Smad3 and p-Smad3 proteins in miR-101a-mimic group were all down-regulated. Compared with control group, the expression of E-cadherin in Ad-miR-101a group was up-regulated, but the expression of α-SMA, vimentin, Smad4 and p-Smad2/3 was down-regulated. The liver tissue morphology of rats in the Ad-miR-101a group returned to normal, and the degree of liver fibrosis was lower than that of the CCl4 group. Conclusion: The level of miR-101a is related to the severity of hepatitis B virus-related liver fibrosis. Up-regulation of miR-101a may play an anti-fibrotic effect by inhibiting the activation of HSC and epithelial-mesenchymal transformation. |
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