miR-218-5p通过靶向LIN28B调控COPD时气道上皮细胞凋亡和炎症反应

miR-218-5p regulates airway epithelial cell apoptosis and inflammatory response in chronic obstructive pulmonary disease by targeting LIN28B

目的探讨miR-218-5p通过靶向LIN28B调控慢性阻塞性肺疾病(COPD)时气道上皮细胞凋亡和炎症反应的作用及其机制。方法将人肺支气管上皮细胞BEAS-2B分为正常对照(NC)组、香烟烟雾提取物(CSE)组、miR-NC+CSE组、miR-218-5p+CSE组、si-NC+CSE组、si-LIN28B+CSE组、miR-218-5p+pcDNA-NC+CSE组和miR-218-5p+pcDNA-LIN28B+CSE组。RT-qPCR检测miR-218-5p的表达水平;流式细胞术检测细胞凋亡;酶联免疫吸附实验(ELISA)检测白细胞介素6(IL-6)和转化生长因子-β1(TGF-β1)的表达水平;蛋白质印记(Western blot)检测LIN28B、裂解的天冬氨酸半胱氨酸蛋白酶(Cleaved-caspase-3)的表达水平;双荧光素酶报告基因实验验证miR-218-5p对LIN28B的靶向作用。结果与NC组比较,CSE组BEAS-2B细胞miR-218-5p表达降低,细胞凋亡率、LIN28B、Cleaved-caspase-3、IL-6和TGF-β1表达显著增加(均P<0.05)。与miR-NC+CSE组比较,miR-218-5p+CSE组BEAS-2B细胞凋亡率、Cleaved-caspase-3、IL-6和TGF-β1表达显著降低(均P<0.05)。与si-NC+CSE组比较,si-LIN28B+CSE组BEAS-2B细胞凋亡率、Cleaved-caspase-3、IL-6和TGF-β1表达显著降低(均P<0.05)。与miR-218-5p+pcDNA-NC+CSE组比较,miR-218-5p+pcDNA-LIN28B+CSE组BEAS-2B细胞细胞凋亡率、Cleaved-caspase-3、IL-6和TGF-β1表达显著增加(均P<0.05)。结论miR-218-5p通过靶向LIN28B可抑制CSE诱导的气道上皮细胞凋亡和炎症反应。因此,miR-218-5p可能是慢性阻塞性肺疾病的潜在治疗靶点。

Objective To investigate the role of miR-218-5p in chronic obstructive pulmonary disease and its mechanism.Methods BEAS 2B cells were divided into normal control(NC)group,cigarette smoke extract(CSE)group,Mir NC+CSE group,Mir-218-5p+CSE group,Si NC+CSE group,Si-LIN28B+CSE group,Mir-218-5p+pcDNA-NC+CSE group and Mir-218-5p+pcDNA-LIN28B+CSE group.The expression of Mir-218-5p was detected by RT-qPCR and apoptosis was detected by flow cytometry.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression of IL-6 and TGF-β1.The expression levels of LIN28B and cleaved caspase 3 were detected by Western blot.The targeting effect of Mir-218-5p on LIN28B was verified by double luciferase reporter gene assay.Results Compared with the NC group,the expression of miR-218-5p in BEAS-2B cells of CSE group was reduced,while the apoptosis rate,LIN28B,Cleaved-caspase-3,IL-6 and TGF-β1 expression were significantly increased(P<0.05).Compared with the miR-NC+CSE group,the apoptosis rate,Cleaved-caspase-3,IL-6 and TGF-β1 expression in BEAS-2B cells of miR-218-5p+CSE group were significantly reduced(P<0.05).Compared with the si-NC+CSE group,the apoptosis rate,Cleaved-caspase-3,IL-6,and TGF-β1 expression in BEAS-2B cells of si-LIN28B+CSE group were significantly reduced(P<0.05).Compared with miR-218-5p+pcDNA-NC+CSE group,the apoptosis rate,Cleaved-caspase-3,IL-6,and TGF-β1 expression in BEAS-2B cells of miR-218-5p+pcDNA-LIN28B+CSE group were significantly increased(P<0.05).Conclusion miR-218-5p could inhibit CSE-induced airway epithelial cell apoptosis and inflammatory response by targeting LIN28B.Therefore,miR-218-5p may be a potential therapeutic target for chronic obstructive pulmonary disease.