circ_0114427靶向微小RNA-330-5p调控脂多糖诱导的肺泡上皮细胞凋亡及炎症反应

Circ_0114427 targets microRNA-330-5p to regulate apoptosis and inflammatory response in lipopolysaccharide-induced alveolar epithelial cells

目的探讨circ_0114427对脂多糖(LPS)诱导的肺泡上皮细胞凋亡和炎症的影响及其机制。方法体外培养人肺泡上皮细胞,分别转染si-circ_0114427、微小RNA(miR)-330-5p mimics或共转染si-circ_0114427与anti-miR-330-5p后,用10 mg/L LPS处理24 h。采用实时荧光定量聚合酶链反应(qRT-PCR)法检测细胞中circ_0114427和miR-330-5p表达量;采用蛋白免疫印迹评估活化的天冬氨酸特异性半胱氨酸蛋白酶-3(Cleaved-caspase3)和活化的天冬氨酸特异性半胱氨酸蛋白酶-9(Cleaved-caspase9)蛋白水平;采用酶联免疫吸附试验(ELISA)检测白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)表达;采用流式细胞术分析细胞凋亡。采用双荧光素酶报告实验验证circ_0114427和miR-330-5p的互作关系。结果肺泡上皮细胞经LPS处理后,细胞中circ_0114427表达升高,miR-330-5p表达降低(P<0.05)。敲减circ_0114427或上调miR-330-5p可以抑制LPS诱导的肺泡上皮细胞凋亡、Cleaved-caspase3、Cleaved-caspase9表达以及IL-6和TNF-α水平(P<0.05)。circ_0114427靶向结合并负调控miR-330-5p。miR-330-5p下调可以逆转circ_0114427敲低对LPS诱导的细胞凋亡和炎症的抑制作用。结论敲减circ_0114427可抑制LPS诱导的肺泡上皮细胞凋亡和炎症,这可能与miR-330-5p上调有关。

Objective To investigate the effect of circ_0114427 on apoptosis and inflammation of alveolar epithelial cells induced by lipopolysaccharide(LPS)and its mechanism.Methods Human alveolar epithelial cells were cultured in vitro and transfected with si-circ_0114427,microRNA(miR)-330-5p mimics or co-transfected with si-circ_0114427 and anti-miR-330-5p,and the cellswere then treated with 10 mg/L LPS for 24 hours.The expression levels of circ_0114427 and miR-330-5p in the cells were detected using real-time fluorescent quantitative polymerase chain reaction(qRT-PCR).The protein levels of Cleaved-caspase3 and Cleaved-caspase9 were evaluated using western blot.Enzyme-linked immunosorbent assay(ELISA)was used to assess the expression of interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α).Cell apoptosis was analyzed using flow cytometry.The interaction between circ_0114427 and miR-330-5p was validated using a dual luciferase reporter assay.Results After LPS treatment of alveolar epithelial cells,the expression of circ_0114427 was upregulated,while miR-330-5p expression was downregulated in in the cells(P<0.05).Knockdown of circ_0114427 or upregulation of miR-330-5p could inhibit LPS-induced apoptosis,Cleaved-caspase3,Cleaved-caspase9,IL-6,and TNF-αlevels in alveolar epithelial cells(P<0.05).Circ_0114427 directly interacted with and negatively regulated miR-330-5p.Downregulation of miR-330-5p reversed the inhibitory effect of circ_0114427 knockdown on LPS-induced cell apoptosis and inflammation.Conclusion Knockdown of circ_0114427 can inhibit LPS-induced apoptosis and inflammation in alveolar epithelial cells,possibly through upregulation of miR-330-5p.