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

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

作　　者：黄彬 张军[2] 郑金旭[1] 丁慢玲 吴妍 HUANG Bin;ZHANG Jun;ZHENG Jinxu;DING Manling;WU Yan(Department of Respiratory and Critical Care Medicine,Affiliated Hospital of Jiangsu University,Zhenjiang 212001,China;Department of Respiratory and Critical Care Medicine,Aoyang Hospital Affiliated to Jiangsu University)

机构地区：[1]江苏大学附属医院呼吸与危重症医学科,212001 [2]江苏大学附属澳洋医院呼吸与重症医学科

出　　处：《天津医药》2022年第6期571-578,共8页Tianjin Medical Journal

基　　金：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)。

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。Objective To explore the interaction between the role of circ_0007762 and miR-18a-5p changes in lung fibroblasts.Methods 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 ofa-SMA,collagenⅠ,P62 and LC3BⅡ/Ⅰafter the knockdown of circ_0007762 or miR-18a-5p and the intervention of TGF-β1 or 3-MA.Results 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-β1-induced fibroblast proliferation(P<0.05).TGF-β1 promoted the levels ofa-SMA,collagenⅠand LC3BⅡ/Ⅰ,and inhibited the level of P62(P<0.05).Compared with the TGF-β1+siNC group,a-SMA,collagenⅠand LC3BⅡ/Ⅰwere down-regulated in the TGF-β1+si-circ_0007762 group,while P62expression 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.

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 

分 类 号：R363.2[医药卫生—病理学] R563.9[医药卫生—基础医学]