【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。更多还原

【Abstract】 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, 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 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 ofa-SMA, collagen Ⅰ and LC3B Ⅱ/Ⅰ, and inhibited the level of P62(P<0.05). Compared with the TGF-β1+siNC group,a-SMA, collagen Ⅰ and LC3B Ⅱ/Ⅰ were 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.更多还原