天津医药

天津医药 ›› 2022, Vol. 50 ›› Issue (6): 571-578.doi: 10.11958/20212642

• 细胞与分子生物学 • 上一篇    下一篇

circ_0007762通过miR-18a-5p调节肺成纤维细胞自噬的机制研究

黄彬1，张军2，郑金旭1△，丁慢玲1，吴妍1   

  1. 1江苏大学附属医院呼吸与危重症医学科（邮编212001）；2江苏大学附属澳洋医院呼吸与重症医学科
  • 收稿日期:2021-11-29 修回日期:2022-01-22 出版日期:2022-06-15 发布日期:2023-12-20
  • 通讯作者: 郑金旭 E-mail:jxuzh135@163.com
  • 基金资助:
    镇江市重大（社会发展）项目（SH2018048）；苏州市社会发展（民生医疗）项目（SYSD2020010）

The study on the mechanism of circ_0007762 regulating autophagy of lung fibroblasts through miR-18a-5p

HUANG Bin1, ZHANG Jun2, ZHENG Jinxu1△, DING Manling1, WU Yan1   

  1. 1 Department of Respiratory and Critical Care Medicine, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China;
    2 Department of Respiratory and Critical Care Medicine, Aoyang Hospital Affiliated to Jiangsu University
  • Received:2021-11-29 Revised:2022-01-22 Published:2022-06-15 Online:2023-12-20

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摘要： 摘要：目的　探索circ_0007762和miR-18a-5p的相互作用及在肺成纤维细胞中的作用。方法　利用生物信息学分析circ_0007762在特发性肺纤维化（IPF）患者的表达并筛选其miRNA靶标，在转化生长因子（TGF）-β1干预的肺成纤维细胞HFL1中验证其表达。双荧光素酶报告基因实验验证miR-18a-5p mimics与报告基因载体共转染后的荧光素酶活性。CCK-8法检测circ_0007762、miR-18a-5p敲低及TGF-β1、自噬抑制剂3-甲基腺嘌呤（3-MA）干预后的细胞活力。Western blot检测circ_0007762、miR-18a-5p敲低及TGF-β1、3-MA干预后的α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原（collagenⅠ）、P62及微管相关蛋白1轻链3β（LC3B）水平。结果　HFL1细胞中，TGF-β1组较Control组circ_0007762表达水平上调，miR-18a-5p表达下调（P＜0.05）。在circ_0007762野生型载体中，过表达miR-18a-5p抑制了荧光素酶活性（P＜0.05）。circ_0007762敲低后细胞增殖活力下降，并由miR-18a-5p的抑制而恢复，同时3-MA可逆转TGF-β1诱导的成纤维细胞增殖（P＜0.05）。TGF-β1促进α-SMA、collagenⅠ及LC3BⅡ/Ⅰ表达，而抑制P62水平（P＜0.05）。相较于TGF-β1+si-NC组，TGF-β1+si-circ_0007762组P62表达上调，其余蛋白下调，并能由miR-18a-5p的抑制而逆转（P＜0.05）。此外，3-MA增强了P62的表达而降低了α-SMA、collagenⅠ的表达及LC3BⅡ/Ⅰ水平（P＜0.05）。结论　circ_0007762可与miR-18a-5p相互作用，通过激活自噬促进肺成纤维细胞增殖，诱导纤维化相关表型。

关键词: 特发性肺纤维化, 自噬, 细胞增殖, 成纤维细胞, 转化生长因子β, circ_0007762, miR-18a-5p

Abstract: Abstract: Objective　To explore the interaction between the role of circ_0007762 and miR-18a-5p changes in lung fibroblasts. Methods　Bioinformatics was used to analyze the expression of circ_0007762 in patients with idiopathic pulmonary fibrosis (IPF) and screen its miRNA targets. Circ_0007762 level was verified in HFL1 cells with transforming growth factor (TGF)-β1 intervention. The luciferase activity after co-transfection of miR-18a-5p mimics with reporter gene vectors was verified by dual luciferase reporter assay. CCK8 assay was used to detect the cell viability after the knockdown of circ_0007762 or miR-18a-5p and the intervention of TGF-β1 or 3-MA. Western blot assay was applied to determine the expression of a-SMA, collagen Ⅰ, P62 and LC3B Ⅱ/Ⅰ after the knockdown of circ_0007762 or miR-18a-5p and the intervention of TGF-β1 or 3-MA. Results　In contrast with the control group, the level of circ_0007762 in HFL1 was significantly up-regulated in the TGF-β1 group, and the level of miR-18a-5p was significantly down-regulated (P＜0.05). Overexpression of miR-18a-5p inhibited the luciferase activity in circ_0007762 wild-type vector (P＜0.05). The cell proliferation activity was decreased after knocking-down circ_0007762, while the effect could be rescued by the inhibition of miR-18a-5p. Meanwhile, 3-MA reversed TGF-β 1-induced fibroblast proliferation (P＜0.05). TGF-β1 promoted the levels of a-SMA, collagen Ⅰ and LC3B Ⅱ/Ⅰ, and inhibited the level of P62 (P＜0.05). Compared with the TGF-β1+si-NC group, a-SMA, collagen Ⅰ and LC3B Ⅱ/Ⅰ were down-regulated in the TGF-β1+si-circ_0007762 group, while P62 expression was increased, and the effect could be rescued by the suppression of miR-18a-5p (P＜0.05). Besides, 3-MA enhanced the expression of P62 and suppressed the expression levels of other proteins (P＜0.05). Conclusion　The circ_0007762 can interact with miR-18a-5p to accelerate the proliferation of lung fibroblasts and induce the fibrotic phenotype by activating autophagy.

Key words: idiopathic pulmonary fibrosis, autophagy, cell proliferation, fibroblasts, transforming growth factor beta, circ_0007762, miR-18a-5p