miR-30a-3p靶向NOD1对心肌细胞缺氧复氧损伤的影响

doi:10.13418/j.issn.1001-165x.2019.04.011

中国临床解剖学杂志 ›› 2019, Vol. 37 ›› Issue (4): 414-420.doi: 10.13418/j.issn.1001-165x.2019.04.011

miR-30a-3p靶向NOD1对心肌细胞缺氧复氧损伤的影响

刘建花，　雷大洲，　周凡，　齐俊杰，　李世勋

新乡市中心医院心内二科，河南新乡 453000

收稿日期:2019-01-07 出版日期:2019-07-25 发布日期:2019-08-01
作者简介:刘建花（1978-），女，河南淇县人，硕士，主治医师，研究方向：心血管内科，E-mail：shige134448@sina.com，xinnei2liu@163.com
基金资助:
河南省自然科学基金面上项目（162300410220）

The effects of miR-30a-3p on Hypoxia/Reoxygenation induced injury of cardiomyocytes by targeting NOD1

LIU Jian-hua, LEI Da-zhou, ZHOU Fan, QI Jun-jie, LI Shi-xun

Department of Internal Medicine-Cardiovascular, Xinxiang Central Hospital, Xinxiang 453000, Henan Province, China

Received:2019-01-07 Online:2019-07-25 Published:2019-08-01

摘要/Abstract

摘要： 目的　探究miR-30a-3p与NOD1的靶向关系，及其对心肌细胞缺氧复氧损伤的影响和机制。方法　将细胞分为Ctrl组、H/R组、miR-30a-3p mimic组和miR-30a-3p inhibitor组，经过缺氧复氧处理后，转染相应的miRNA处理细胞，RT-PCR检测miR-30a-3p和NOD1基因表达水平，荧光素酶报告检测miR-30a-3p和NOD1靶向关系，Western blot检测NOD1、p-RPI2、p38 MAPK、NF-κB（p65）、cl-caspase-3蛋白表达水平，CCK8法检测细胞活性，Hoechst检测细胞凋亡，试剂盒检测LDH、CK、MDA、T-SOD水平。结果　miR-30a-3p表达水平随缺氧复氧处理时间增长而下降，NOD1基因表达水平随缺氧复氧处理时间增长而升高。在荧光素酶报告实验中，NOD1 WT+miR-30a-3p mimic荧光素酶活性显著低于NOD1 WT+NC组。与Ctrl组比较，H/R组miR-30a-3p基因表达水平、细胞活性、T-SOD水平降低，NOD1、p-RPI2、p38 MAPK、NF-κB（p65）、cl-caspase-3蛋白表达水平、LDH、CK、MDA水平、细胞凋亡率升高；与H/R组比较，miR-30a-3p mimic组miR-30a-3p基因表达水平、细胞活性、T-SOD水平升高，NOD1、p-RPI2、p38 MAPK、NF-κB（p65）、cl-caspase-3蛋白表达水平、LDH、CK、MDA水平、细胞凋亡率降低，miR-30a-3p inhibitor组miR-30a-3p基因表达水平、细胞活性、T-SOD水平降低，NOD1、p-RPI2、p38 MAPK、NF-κB（p65）、cl-caspase-3蛋白表达水平、LDH、CK、MDA水平、细胞凋亡率升高。结论　miR-30a-3p可靶向作用于NOD1，缓解心肌细胞缺氧复氧造成的损伤，其作用机制可能与调控NF-κB信号通路有关。

关键词: 缺氧复氧损伤, 　心肌缺血再灌注, 　NOD1, 　miR-30a-3p

Abstract: Objective　To explore the targeting relationship between miR-30a-3p and NOD1, and its effect on Hypoxia/Reoxygenation induced injury of cardiomyocytes and its mechanism.　Methods　Cells were divided into Ctrl, H/R, miR-30a-3p mimic and miR-30a-3p inhibitor groups. After treatment with Hypoxia/Reoxygenation, cells in each group were transfected with corresponding miRNA. RT-PCR was performed for measuring the gene levels of miR-30a-3p and NOD1, luciferase reporter assay was performed for measuring the relationship between miR-30a-3p and NOD1, Western blot was used to determine the protein levels of NOD1, p-RPI2, p38 MAPK, NF-κB (p65), cl-caspase-3, cell viability was measured by CCK8, cell apoptosis was determined by Hoechst, and LDH, CK, MDA, T-SOD levels were detected by kit. Results　The expression level of miR-30a-3p decreased with the hypoxia-reoxygenation treatment time, and the expression level of NOD1 gene increased with the hypoxia-reoxygenation treatment time. In the luciferase reporter assay, the luciferase activity of NOD1 WT+miR-30a-3p mimic group was significantly lower than that of the NOD1 WTR+NC group. Compared with the Ctrl group, the gene level of miR-30a-3p, cell viability, T-SOD level were decreased, and the protein levels of NOD1, p-RPI2, p38 MAPK, NF-κB (p65), cl-caspase-3, expression levels of LDH, CK, MDA, cell apoptosis rate were increased in the H/R group. Compared with the H/R group, the gene level of miR-30a-3p, cell viability, T-SOD level were increased, and the protein levels of NOD1, p-RPI2, p38 MAPK, NF-κB（p65）, cl-caspase-3, expression levels of LDH, CK, MDA, and cell apoptosis rate were decreased in the miR-30a-3p mimic group. Meanwhile, the gene level of miR-30a-3p, cell viability, and T-SOD level were decreased, and the protein levels of NOD1, p-RPI2, p38 MAPK, NF-κB（p65）, cl-caspase-3, expression levels of LDH, CK, MDA, cell apoptosis rate were increased in the miR-30a-3p inhibitor group.　Conclusion　miR-30a-3p can target NOD1, alleviate the Hypoxia/Reoxygenation induced injury of carddiomyocytes, and the mechanism may be related to regulation of the NF-κB signaling pathway.

Key words: Hypoxia/Reoxygenation induced injury, 　Myocardial ischemia reperfusion, 　NOD1, 　miR-30a-3p

中图分类号:

R541

刘建花, 雷大洲, 周凡, 齐俊杰, 李世勋. miR-30a-3p靶向NOD1对心肌细胞缺氧复氧损伤的影响[J]. 中国临床解剖学杂志, 2019, 37(4): 414-420.

LIU Jian-hua, LEI Da-zhou, ZHOU Fan, QI Jun-jie, LI Shi-xun . The effects of miR-30a-3p on Hypoxia/Reoxygenation induced injury of cardiomyocytes by targeting NOD1[J]. Chinese Journal of Clinical Anatomy, 2019, 37(4): 414-420.

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miR-30a-3p靶向NOD1对心肌细胞缺氧复氧损伤的影响

The effects of miR-30a-3p on Hypoxia/Reoxygenation induced injury of cardiomyocytes by targeting NOD1

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