下调miR-199a-5p抑制脂多糖诱导的大鼠Ⅱ型肺泡上皮细胞系AECⅡ凋亡

Down-regulation of miR-199a-5p inhibits lipopolysaccharide-induced apoptosis of rat typeⅡalveolar epithelial cell line AECⅡ

目的探讨miR-199a-5p在慢性阻塞性肺疾病(COPD)患者血清中的表达及其对脂多糖(LPS)诱导的大鼠Ⅱ型肺泡上皮细胞系(AECⅡ)凋亡的影响和可能机制。方法选取2017年4月至2018年12月于湖南中医药大学第一附属医院就诊的45例COPD患者和同时期45名健康体检者。体外培养AECⅡ,分为对照组(control)、LPS组(100 ng/mL LPS)、anti-miR-199a-5p+LPS组(转染miR-199a-5p抑制剂后用100 ng/mL LPS培养)、anti-miR-NC+LPS组(转染miR-199a-5p抑制剂阴性对照序列)、anti-miR-199a-5p+si-FZD4+LPS组(同时转染miR-199a-5p抑制剂与FZD4小干扰RNA)和anti-miR-199a-5p+si-NC+LPS组(同时转染miR-199a-5p抑制剂与FZD4乱序无意义阴性序列)。RT-qPCR检测血清和细胞中miR-199a-5p和FZD4 mRNA表达,流式细胞计量术检测细胞凋亡率,Western blot检测细胞中FZD4、B淋巴细胞瘤-2(Bcl-2)和B淋巴细胞瘤-2相关蛋白(Bax)表达。双荧光素酶报告基因实验验证miR-199a-5p与FZD4靶向关系。结果与对照组比较,COPD患者血清中miR-199a-5p表达水平显著升高(P<0.05),而FZD4 mRNA表达水平显著降低(P<0.05)。与对照组比较,LPS组细胞中miR-199a-5p表达水平显著升高(P<0.05),而FZD4 mRNA和蛋白表达水平显著降低(P<0.05),细胞凋亡率和Bax蛋白表达水平显著升高(P<0.05),Bcl-2蛋白表达水平显著降低(P<0.05)。与anti-miR-NC+LPS组比较,anti-miR-199a-5p+LPS组细胞凋亡率和Bax蛋白表达水平显著降低(P<0.05),而Bcl-2蛋白表达水平显著升高(P<0.05)。miR-199a-5p靶向调控FZD4,与anti-miR-199a-5p+si-NC+LPS组比较,anti-miR-199a-5p+si-FZD4+LPS组细胞凋亡率和Bax蛋白表达水平显著升高(P<0.05),而Bcl-2蛋白表达水平显著降低(P<0.05)。结论miR-199a-5p在COPD患者血清及LPS诱导的AECⅡ中表达升高,下调miR-199a-5p表达可能通过上调FZD4表达抑制LPS诱导的AECⅡ凋亡。

Objective To investigate the expression of miR-199a-5p in the serum of patients with chronic obstructive pulmonary disease(COPD)and its effect on lipopolysaccharide(LPS)-induced apoptosis of rat typeⅡalveolar epithelial cells(AECⅡ)and possible mechanism.Methods Forty-five patients with COPD and 45 controls were enrolled in the first affiliated hospital of Hunan University of Traditional Chinese Medicine from April 2017 to December 2018.AECⅡcells were cultured in vitro and were divided into control group,LPS group,anti-miR-199a-5p+LPS group,anti-miR-NC+LP group,anti-miR-199a-5p+si-FZD4+Lgroup and anti-miR-199a-5p+si-NC+LPS group.RT-qPCR was used to detect the expression of miR-199a-5p and FZD4 mRNA in serum and cells,flow cytometry was used to detect the apoptosis rate and Western blot was used to detect FZD4,B-lymphoma-2(Bcl-2)and B-lymphoma-2 related proteins(Bax)in cells.The dual luciferase reporter gene experiment verified the targeting relationship between miR-199a-5p and FZD4.Results Compared with the control,the expression of miR-199a-5p in serum of COPD patients significantly increased(P<0.05),and the expression of FZD4 mRNA significantly reduced(P<0.05).Compared with the control group,the expression of miR-199a-5p in the LPS group significantly increased(P<0.05),the mRNA and protein expression of FZD4 significantly reduced(P<0.05),the apoptosis rate and the expression level of Bax protein significantly increased(P<0.05),and the expression of Bcl-2 protein significantly reduced(P<0.05).Compared with anti-miR-NC+LPS group,cell apoptosis and the expression of Bax protein in the anti-miR-199a-5p+LPS group significantly reduced(P<0.05),while the expression of Bcl-2 protein significantly increased(P<0.05).There was no significant change in the detection indexes between the LPS group and the anti-miR-NC+LPS group(P>0.05).miR-199a-5p targets FZD4,compared with anti-miR-199a-5p+si-NC+LPS group,cell apoptosis rate and the expression level of Bax protein in the anti-miR-199a-5p+si-FZD4+LPS group all significantly increased(P<0.05),while the expression of Bcl-2 protein significantly reduced(P<0.05).Conclusions The expression of miR-199a-5p is increased in serum of COPD patients and in LPS-induced AECⅡcells.Down-regulating miR-199a-5p expression may inhibit LPS-induced AECⅡcell apoptosis by up-regulating FZD4 expression.