基础医学与临床 ›› 2021, Vol. 41 ›› Issue (4): 521-527.

• 研究论文 • 上一篇    下一篇

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

王伟*, 赵四林, 范伏元, 金朝晖, 李妲, 符艳   

  1. 湖南中医药大学第一附属医院 呼吸内科, 湖南 长沙 410007
  • 收稿日期:2020-04-24 修回日期:2020-09-02 出版日期:2021-04-05 发布日期:2021-04-05
  • 通讯作者: *qingqhong163yx@163.com
  • 基金资助:
    湖南省中医药科研计划项目(201838)

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

WANG Wei*, ZHAO Si-lin, FAN Fu-yuan, JIN Zhao-hui, LI Da, FU Yan   

  1. Department of Respiratory Medicine, the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine, Changsha 410007, China
  • Received:2020-04-24 Revised:2020-09-02 Online:2021-04-05 Published:2021-04-05
  • Contact: *qingqhong163yx@163.com

摘要: 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。

关键词: COPD, AEC, miR-199a-5p, FZD4, 细胞凋亡

Abstract: 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 type Ⅱ alveolar 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 . AEC Ⅱ cells 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 AECⅡ cell apoptosis by up-regulating FZD4 expression.

Key words: COPD, AEC, miR-199a-5p, FZD4, cell apoptosis

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