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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (5): 8-16    DOI: 10.13523/j.cb.2012039
研究报告     
miR-29a通过调控PTEN表达对脂多糖诱导人肺微血管内皮细胞损伤的保护作用研究*
陈玉琼1,2,谭文华2,刘海峰1,陈根1,2,**()
1 湖南省郴州市第一人民医院 郴州 423000
2 南华大学药物药理研究所 衡阳 421001
Protective Effect of miR-29a on Lipopolysaccharide-induced Human Pulmonary Microvascular Endothelial Cells Injury by Targeting PTEN Expression
CHEN Yu-qiong1,2,TAN Wen-hua2,LIU Hai-feng1,CHEN Gen1,2,**()
1 The First People’s Hospital of Chenzhou, Chenzhou 423000, China
2 Institute of Pharmacy and Pharmacology, University of South China, Hengyang 421001, China
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摘要：

目的:探讨miR-29a在脂多糖(LPS)诱导人肺微血管内皮细胞(HPMVECs)损伤中的作用及机制。方法:构建LPS损伤HPMVECs模型。RT-qPCR检测miR-29a表达变化;试剂盒测乳酸脱氢酶(LDH)释放量;MTT和流式细胞术分别检测细胞存活率及凋亡率;Western blot测蛋白质表达水平;Microcosm、starBase、Pictar、TargetScan软件预测 miR-29a的可能靶基因,双萤光素酶实验验证miR-29a和PTEN的靶向关系。结果:使用LPS处理HPMVECs,显著降低细胞中miR-29a的表达和细胞存活率,诱导LDH释放量和HPMVECs凋亡率增加,上调细胞中PTEN、Bim蛋白表达,下调p-Akt/Akt、p-FOXO3a/FOXO3a表达 (P<0.05);过表达miR-29a逆转LPS对HPMVECs的损伤作用。萤光素酶报告基因实验证实miR-29a 靶向PTEN,转染miR-29a mimics显著下调PTEN蛋白表达,转染miR-29a inhibitors明显上调PTEN蛋白表达 (P<0.05),但PTEN mRNA表达水平差异均无统计学意义(P>0.05)。结论:过表达miR-29a可能通过抑制PTEN蛋白的表达水平、激活Akt/FOXO3a/Bim信号通路对LPS致HUVECs的损伤发挥保护作用。
关键词： MiR-29a;  脂多糖;  人肺微血管内皮细胞;  PTEN    
Abstract:

Aim: To investigate the effect of miR-29a on lipopolysaccharide-induced injury in human pulmonary microvascular endothelial cells and its potential mechanism. Methods: The LPS-induced HPMVECs injury model was constructed. The expression level of miR-29a was detected by RT-qPCR. The concentration of LDH was measured by ELISA. MTT assay was used to detect the cell proliferation. The flow cytometry was applied to determine the HPMVECs apoptosis. The protein levels of PTEN, p-Akt, Akt, p-FOXO3a, FOXO3a and Bim were determined by Western blot. The targeting relationship between miR-29a and PTEN was predicted by Microcosm, starBase, Pictar, TargetScan and confirmed by luciferase test. Results: LPS treatment of HPMVECs significantly reduced the expression level of miR-29a and cell viability,induced the increase of LDH release amount and cell apoptic rate, upregulated the expression of PTEN and Bim protein, and down regulated the expression of p-Akt/Akt and p-FOXO3a/FOXO3a (P<0.05). Overexpression of miR-29a reversed the injury of LPS to HPMVECs. Dual luciferase reporter gene assay confirmed that PTEN was a negative regulatory target gene of miR-29a. The protein expression of PTEN was significantly down regulated by miR-29a mimics, and up-regulated by miR-29a inhibitors(P<0.05). However, the expression level of PTEN mRNA had no statistically significant difference (P>0.05). Conclusion: Overexpression of miR-29a, which targets inhibition of PTEN protein expression, protects against LPS-induced HUVECs injury by activating the Akt/FOXO3a/Bim pathway.
Key words: MicroRNA-29a    Lipopolysaccharide    Human pulmonary microvascular endothelial cells    PTEN
收稿日期: 2020-12-22 出版日期: 2021-06-01
ZTFLH:  R563.9  
基金资助: * 郴州市科技局(jsyf2017024);郴州市科技局(ZDYF2020028)
通讯作者: 陈根     E-mail: myth3878@aliyun.com
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引用本文:

陈玉琼,谭文华,刘海峰,陈根. miR-29a通过调控PTEN表达对脂多糖诱导人肺微血管内皮细胞损伤的保护作用研究*[J]. 中国生物工程杂志, 2021, 41(5): 8-16.

CHEN Yu-qiong,TAN Wen-hua,LIU Hai-feng,CHEN Gen. Protective Effect of miR-29a on Lipopolysaccharide-induced Human Pulmonary Microvascular Endothelial Cells Injury by Targeting PTEN Expression. China Biotechnology, 2021, 41(5): 8-16.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2012039    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I5/8

基因名称 引物序列
GAPDH F:5'-GGAGCGAGATCCCTCCAAAAT-3'
R:5'-GGCTGTTGTCATACTTCTCATGG-3'
PTEN F:5'-CGACGGGAAGACAAGACAAGTT-3'
R:5'-GCTAGCCTCTGGATTTGACG-3'
U6 F:5'-GCTTCGGCAGCACATATACTAAAAT-3'
R:5'-CGCTTCACGAATTTGCGTGTCAT-3'
miR-29a F:5'-GACTCGTAGCACCATCTG-3'
R:5'-GTGCAGGGTCCGAGGTAT-3'
表1  引物序列
图1  LPS处理对HPMVECs中miR-29a表达和LDH释放的影响
图2  过表达miR-29a逆转LPS致HPMVECs的损伤作用
图3  miR-29a与PTEN靶向关系的验证
图4  过表达miR-29a可能通过PTEN/Akt/FOXO3a/Bim信号通路对LPS致HPMVECs损伤作用的影响
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