河北医科大学学报 ›› 2023, Vol. 44 ›› Issue (2): 137-141.doi: 10.3969/j.issn.1007-3205.2023.02.003

• • 上一篇    下一篇

COPD中MicRNA-320c-5p调节SERPINA1基因表达的研究

  

  1. 新疆维吾尔自治区喀什地区第一人民医院呼吸与危重症医学科， 新疆 喀什 844000

  • 出版日期:2023-02-25 发布日期:2023-02-28
  • 作者简介:任杰（1987-），男，安徽阜阳人，新疆维吾尔自治区喀什地区第一人民医院副主任医师， 医学硕士，从事呼吸与危重系统疾病诊治研究。
  • 基金资助:
    新疆维吾尔自治区自然科学基金（2019D01C006）

MicRNA-320c-5p regulates SERPINA1 gene expression in COPD

  1. Department of Respiratory and Critical Care Medicine, the First People′s Hospital of Kashgar, Xinjiang, Kashgar 844000, China

  • Online:2023-02-25 Published:2023-02-28

HTML

PDF (PC)

217

摘要： 目的 通过双荧光素酶报告系统验证miR-320c-5p对SERPINA1基因的靶向调控关系。
方法 从人正常肺上皮细胞基因组库中获取SERPINA1基因的3′UTR序列，并通过序列匹配拟定候选miRNA。利用3%香烟烟雾提取物（cigarette smoke extract,CSE）诱导人支气管上皮细胞（BEAS-2b细胞）建立慢性阻塞性肺疾病（chronic obstructive pulmonary disease，COPD）细胞模型作为实验组，正常培养的BEAS-2b细胞作为对照组，使用实时荧光定量聚合酶链反应（real time flurocent qualitative polymerase chain reaction,RT-qPCR）法检测两组SERPINA1和候选miRNA 的表达水平。构建野生型及突变型双荧光素酶报告基因载体，分别将双荧光素酶报告质粒(psiCHECK-2-SERPINA1-3′UTR和psiCHECK-2-SERPINA1-3′UTR mut)和miRNA质粒(miR-320-5p mimic)共转染到293T细胞，进行荧光活性测定。
结果 通过序列匹配，拟候选miR-26、miR-210、miR-320c和miR-96为目标miRNA。与对照组相比，实验组SERPINA1基因 mRNA表达水平显著上调（P＜0.05），四个候选miRNA表达水平均显著下调（P＜0.01），其中miR-320c和miR-96更为显著（P＜0.001），基于文献miR-320c与COPD的预后相关，挑选miR-320c用于后续双荧光素酶实验。携带micR-320c-5p mimic和SERPINA1野生型3，UTR 的构建体的相对荧光素酶活性明显下降(P＜0.001)，SERPINA1突变型3′UTR的构建体的相对荧光素酶活性无明显下降（P＞0.05）。
结论 SERPINA1在 3% CSE诱导的BEAS-2b细胞中表达上调，miR-320c-5p表达下调，miR-320c-5p可能通过负向调控SERPINA1的表达，在COPD的发生发展过程中起重要作用。


关键词: 肺疾病, 慢性阻塞性, 荧光素类, miR-320-5p

Abstract: Objective To verify the targeting regulatory relationship of MicR-320c-5p to SERPINA1 gene by dual luciferase reporter system. 
Methods The 3′UTR sequence of SERPINA1 gene was obtained from the genome of human normal lung epithelial cells. Candidate miRNAs were identified by sequence matching. 3% cigarette smoke extract （CSE） was used to induce human bronchial epithelial cells(BEAS-2b cells) to establish a chronic obstructive pulmonary disease （COPD） cell model as the experimental group, and the normally cultured BEAS-2b cells as the control group. The expression levels of SERPINA1 and candidate miRNAs in the two groups were detected by real time flurocent qualitative polymerase chain reaction (RT-qPCR). Wild-type and mutant double luciferase reporter gene vector was constructed. Dual-luciferase reporter plasmids (psiCHECK-2-SERPINA1-3′UTR and psiCHECK-2-SERPINA1-3′UTR ) and miRNA plasmids (miR-320-5p mimic) were co-transfected into 293 T cells respectively for fluorescence activity determination. 
Results By sequence matching, the candidate miR-26, miR-210, miR-320c and miR-96 were set as targeted miRNAs. Compared with the control group, the mRNA expression level of SERPINA1 gene in the experimental group was significantly increased (P＜0.05), while the expression levels of four candidate miRNAs were significantly decreased (P＜0.01), of which miR-320c and miR-96 were more significant (P＜0.001). Based on the correlation between miR-320c and the prognosis of COPD, miR-320c was selected for the subsequent double luciferase experiment. The relative luciferase activity of the construct carrying MicR-320c-5p mimic and SERPINA1 wild type 3, UTR was significantly decreased(P＜0.001), while the relative luciferase activity of the construct carrying SERPINA1 mutant 3′UTR was not significantly decreased (P＞0.05).
Conclusion SERPINA1 expression is up-regulated in BEAS-2b cells induced by 3% CSE, and miR-320c-5p expression is down-regulated. miR-320c-5p may play an important role in the occurrence and development of COPD by negatively regulating SERPINA1 expression.


Key words: pulmonary disease, chronic obstructive, luciferins, micR-320-5p

版权所有 © 《河北医科大学学报》编辑部
本系统由北京玛格泰克科技发展有限公司设计开发