摘要： 目的 探讨miR-34a-5p靶向刺猬（Hh）信号通路对肝星状细胞（HSC）增殖和凋亡的调控作用。方法 采用生物信息学分析筛选出LX2细胞中靶向Hh信号通路的差异miRNA，通过Cytoscape构建miRNA-mRNA网络。体外培养LX2细胞，通过转化生长因子-β1（TGF-β1）诱导建立LX2细胞活化模型。细胞活力检测试剂盒（CCK-8）检测miR-34a-5p过表达对LX2细胞增值活力的影响；流式细胞分析法（Annexin-V-FITC/PI）检测miR-34a-5p过表达对LX2细胞凋亡的影响；实时荧光定量PCR和Western blotting分析miR-34a-5p过表达对声波刺猬（Shh） 、脑胶质瘤相关癌基因1（Gli1） 、脑胶质瘤相关癌基因2（Gli2） 、Ⅰ型胶原蛋白（Col-Ⅰ）及α平滑肌肌动蛋白（α-SMA）mRNA和蛋白表达水平的影响。结果 LX2细胞经TGF-β1诱导活化后，细胞活力显著升高，Shh、Gli1、Gli2、Col-Ⅰ及 α-SMA表达均增高（P<0.01）。Hh信号通路特异性阻断剂环靶明脂可降低LX2细胞活力，下调Shh、Gli1、Gli2、Col-Ⅰ及α-SMA蛋白表达（P<0.01）。miR-34a-5p过表达可显著提高LX2细胞活力，上调Gli1、Gli2、Col-Ⅰ及α-SMA蛋白表达（P<0.05）。结论 miR-34a-5p靶向调控LX2细胞的Hh信号通路，过表达miR-34a-5p可以上调Hh信号通路相关蛋白表达，促进LX2细胞活化。

基于ｍiRNA-mRNA调控网络探讨miR-34a-5p靶向Hh通路对LX2细胞增殖的影响

任真¹, 马燕花², 王莉², 王爱娣², 赵秀萍²

1. 甘肃中医药大学 信息工程学院信息工程教研室, 兰州 730000;
2. 甘肃中医药大学 第一临床医学院内科学教研室, 兰州 730000

收稿日期:2022-09-09出版日期:2023-07-30发布日期:2023-07-08
通讯作者:马燕花E-mail:617747928@qq.com
作者简介:任真(1979-),男,副教授,硕士.
基金资助:国家自然科学基金（81860821）


关键词: miRNA-mRNA调控网络, 微RNA-34a-5p, 刺猬信号通路, 肝星状细胞, 肝纤维化
Abstract: Objective This study investigates the regulatory effect of microRNA-34a-5p（miR-34a-5p）on the proliferation and apoptosis of hepatic stellate cells（HSCs）by targeting the hedgehog（Hh）signaling pathway. Methods The differential miRNAs targeting the Hh signaling pathway in LX2 cells were screened using bioinformatics analysis，and the miRNA-mRNA network was constructed using Cytoscape. LX2 cells were cultured in vitro and induced using transforming growth factor-β1（TGF-β1）to establish a LX2 cell activation model. The cell viability assay kit（CCK-8）was used to detect the effect of miR-34a-5p overexpression on the proliferation of LX2 cells. Flow cytometry（Annexin-V-FITC/PI）was used to detect the effect of miR-34a-5p overexpression on the apoptosis of LX2 cells. Realtime fluorescent quantitative PCR and Western blotting were used to analyze the effects of miR-34a-5p overexpression on sonic hedgehog （Shh），glioma-related oncogenes 1（Gli1），glioma-related oncogenes 2（Gli2），collagen typeⅠ （Col-Ⅰ），and α-smooth muscle actin（α-SMA） mRNA and protein expression. Results After LX2 cells were activated using TGF-β1，the cell viability was significantly increased and the expressions of Shh，Gli1，Gli2，Col-Ⅰ，and α-SMA was increased（all P<0.01）. The specific inhibitor of the Hh signaling pathway cyclopamine reduced LX2 viability and down-regulated the expressions of Shh，Gli1，Gli2，Col-Ⅰ，and α-SMA proteins（all P<0.01）. Overexpression of miR-34a-5p significantly increased the viability of the LX2 cells and the protein expression levels of Gli1，Gli2，Col-Ⅰ， and α-SMA（all P<0.05）. Conclusion miR-34a-5p targeted the Hh signaling pathway in LX2 cells. Overexpression of miR-34a-5p up-regulates the expression of Hh signaling pathway-related proteins and promotes the activation of LX2 cells.
Key words: miRNA-mRNA regulatory network, miR-34a-5p, hedgehog signaling pathway, hepatic stellate cells, liver fibrosis
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