miR-29b通过调控Bax基因抑制LPS诱导的肺泡上皮细胞系A549凋亡

目的探讨miR-29b对脂多糖(LPS)诱导的肺泡上皮细胞系A549凋亡的影响及其机制。方法将体外培养的A549细胞分为对照组、LPS组(给予10 mg/L的LPS处理)、LPS+miR-NC组(转染miR-29b mimics阴性对照后给予LPS处理)、LPS+miR-29b组(转染miR-29b mimics后给予LPS处理);用RT-qPCR检测细胞中miR-29b的表达水平;MTT法检测细胞存活率;流式细胞仪检测细胞凋亡率;Western blot检测Bcl-2、Bax和cleaved caspase-3蛋白的表达;双荧光素酶报告基因实验检测Bax和miR-29b的靶向关系。结果与对照组相比,LPS组、LPS+miR-NC组和LPS+miR-29b组细胞中miR-29b、Bcl-2蛋白的表达水平和细胞存活率均明显降低,而细胞凋亡率和Bax、cleaved caspase-3蛋白的表达水平均明显升高(P<0.05);与LPS组相比,LPS+miR-29b组细胞中miR-29b、Bcl-2蛋白的表达水平和细胞存活率均明显升高,而细胞凋亡率和Bax、cleaved caspase-3蛋白的表达水平均明显降低(P<0.05)。双荧光素酶报告基因实验证实Bax是miR-29b的潜在靶基因。结论miR-29b可抑制LPS诱导的A549细胞凋亡,其作用机制可能与靶向调控Bax表达有关。

miR-29b inhibits LPS-induced apoptosis of alveolar epithelial cell line A549 through regulating Bax gene

Objective To investigate the effect of miR-29 b on LPS-induced apoptosis of alveolar epithelial cell line A549 and its mechanism.Methods A549 cells were divided into control group,LPS group(treated with 10 mg/L LPS),LPS+miR-NC group(treated with LPS after transfection of miR-29 b mimics negative control),LPS+miR-29 b group(treated with LPS after transfection of miR-29 b mimics),the expression level of miR-29 b in each group was detected by RT-qPCR,cell viability was detected by MTT assay,and apoptosis rate was detected by flow cytometry.The expressions of Bcl-2,Bax and cleaved caspase-3 protein were detected by Western blot,and the dual luciferase reporter gene assay was used to detect the targeting relationship between Bax and miR-29 b.ResultsCompared with the control group,the expression levels of miR-29 b and Bcl-2 proteins and cell viability in the LPS group,LPS+miR-NC group and LPS+miR-29 b group were significantly decreased,while the apoptosis rate wasdecreased.The expression of Bax and cleaved caspase-3 proteins significantly increased(P<0.05).Compared with LPS group,the expression of miR-29 b and Bcl-2 proteins and cell survival rate in LPS+miR-29 b cells were significantly higher,while the apoptotic rate,the expression of Bax and cleaved caspase-3 protein were all significantly lower(P<0.05).Dual luciferase reporter assays confirmed that Bax could be a potential target gene for miR-29 b.Conclusions miR-29 b can inhibit the apoptosis of alveolar epithelial cells A549 induced by LPS,and its mechanism may be related to the targeted regulation of Bax expression.