左西孟旦通过调控EGOT/微小RNA-641对缺氧/复氧心肌细胞纤维化的影响

黄泓轲; 罗健玮; 冉华

doi:10.16098/j.issn.0529-1356.2022.04.011

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解剖学报 ›› 2022, Vol. 53 ›› Issue (4) : 479-487. DOI: 10.16098/j.issn.0529-1356.2022.04.011

左西孟旦通过调控EGOT/微小RNA-641对缺氧/复氧心肌细胞纤维化的影响

黄泓轲^1* 罗健玮² 冉华³

作者信息 +

Effects of levosimendan on the hypoxic/reoxygenated cardiomyocyte fibrosis by regulating long chain noncoding RNA/microRNA-641

HUANG Hong-ke^1* LUO Jian-wei² RAN Hua³#br#

Author information +

文章历史 +

摘要

目的探讨左西孟旦（Lev）是否通过调控长链非编码RNA（LncRNA）嗜酸性粒细胞个体发育转录本（EGOT）/微小RNA（miR）-641分子轴从而影响缺氧/复氧（H/R）诱导的心肌细胞增殖、凋亡及纤维化。方法体外培养大鼠心肌细胞系H9C2，H/R处理细胞建立细胞损伤模型，随机分为对照（control）组、H/R组、H/R+Lev 1 μmol/L（H/R+Lev-L）组、H/R+Lev 5 μmol/L（H/R+Lev-M）组和H/R+Lev 10 μmol/L（H/R+Lev-H）组，每组9例；MTT法检测细胞增殖；流式细胞术检测细胞凋亡率；Real-time PCR 检测EGOT、miR-641的mRNA表达水平；分别将pcDNA-EGOT、EGOT小分子干扰RNA（si-EGOT）转染至H9C2细胞，采用上述方法检测细胞增殖及凋亡率；双荧光素酶报告基因实验验证EGOT和miR-641的靶向关系；Western blotting检测Bax、Bcl-2、胶原蛋白Ⅰ型（ColⅠ）、胶原蛋白 Ⅲ型（ColⅢ）、组织金属蛋白酶抑制因子2（TIMP2）、基质金属蛋白酶 2（MMP-2）的表达。结果与control组比较，H/R组细胞存活率降低（P＜0.05），细胞凋亡率升高（P＜0.05），Bax、ColⅠ、ColⅢ、TIMP2和MMP-2蛋白水平升高（P＜0.05），Bcl-2蛋白水平降低（P＜0.05），EGOT的表达水平降低（P＜0.05），miR-641的表达水平升高（P＜0.05）；与H/R组比较，H/R+Lev-L组、H/R+Lev-M组及H/R+Lev-H组细胞存活率升高（P＜0.05），细胞凋亡率降低（P＜0.05），Bax、ColⅠ、ColⅢ、TIMP2和MMP-2蛋白水平降低（P＜0.05），Bcl-2蛋白水平升高（P＜0.05），EGOT的表达水平升高（P＜0.05），miR-641的表达水平降低（P＜0.05），且H/R+Lev-L组、H/R+Lev-M组、H/R+Lev-H组各指标比较差异有统计学意义（P＜0.05）；双荧光素酶报告基因实验证实EGOT可靶向结合miR-641；转染pcDNA-EGOT与Lev的作用相似；转染si-EGOT可降低Lev对H/R诱导的H9C2细胞增殖、凋亡及纤维化的作用。结论左西孟旦可能通过上调EGOT表达及下调miR-641表达而促进H/R诱导的H9C2细胞增殖及抑制细胞凋亡、纤维化。

Abstract

Objective To investigate whether levosimendan (Lev) affects hypoxia/reoxygenation (H/R)- induced cardiomyocyte proliferation, apoptosis and fibrosis by regulating the molecular axis of long chain noncoding RNA (LncRNA) eosinophil granule ontogeny transcript(EGOT)/microRNA(miR)-641. Methods Rat cardiomyocytes H9C2 were cultured in vitro, and H/R-treated cells were used to establish cell damage models, which were randomly divided into control group, H/R group, H/R+Lev 1 μmol/L (H/R + Lev-L) group, H/R+Lev 5 μmol/L (H/R + Lev-M) group, and H/R+Lev 10 μmol/L (H/R + Lev-H) group,9 samples per group. MTT method was used to detect cell proliferation. Flow cytometry was used to detect the apoptosis rate. Real-time PCR was used to detect the expression levels of EGOT and miR-641 mRNA. PcDNA-EGOT and EGOT small interfering RNA(si-EGOT) were transfected into H9C2 cells respectively, and the cell proliferation and apoptosis rates were detected by the above method. The dual luciferase report experiment verified the targeting relationship between EGOT and miR-641. Western blotting was used to detect the expression levels of Bax, Bcl-2, collagen Ⅰ(ColⅠ), collagen Ⅲ(ColⅢ), tissue inhibitor of matrix metalloproteinase 2(TIMP2), matrix metalloproteinase-2(MMP-2). Results Compared with the control group, the cell survival rate of the H/R group reduced significantly (P<0.05), the apoptosis rate increased significantly (P<0.05), and the protein levels of Bax, ColⅠ, Col Ⅲ, TIMP2, and MMP-2 increased significantly (P<0.05), the level of Bcl-2 protein reduced significantly (P<0.05), the expression level of EGOT reduced significantly (P<0.05), the expression level of miR-641 increased significantly (P<0.05). Compared with the H/R group, the cell survival rate of the H/R + Lev-L group, H/R + Lev-M group, and H/R + Lev-H group increased significantly (P<0.05), and the apoptosis rate decreased significant (P<0.05),the protein levels of Bax, ColⅠ, ColⅢ, TIMP2, MMP-2 reduced significantly (P<0.05), the level of Bcl-2 protein increased significantly (P<0.05), the expression level of EGOT increased significantly (P<0.05), the expression level of miR-641 reduced significantly (P<0.05), and each index of H/R + Lev-L group, H/R + Lev-M group, H/R + Lev-H group, the difference was statistically significant (P<0.05). The dual luciferase report experiment confirmed that EGOT ccould target and bind to miR-641. The effect of transfecting pcDNA-EGOT and Lev was similar. Transfection of si-EGOT could reduce the effect of Lev on H/R-induced proliferation, apoptosis and fibrosis of H9C2 cells. Conclusion Levosimendan may promote H/R-induced H9C2 cell proliferation and inhibit apoptosis and fibrosis by up-regulating EGOT expression and down-regulating miR-641 expression.

导出引用

黄泓轲罗健玮冉华. 左西孟旦通过调控EGOT/微小RNA-641对缺氧/复氧心肌细胞纤维化的影响[J]. 解剖学报. 2022, 53(4): 479-487 https://doi.org/10.16098/j.issn.0529-1356.2022.04.011

HUANG Hong-ke LUO Jian-wei RAN Hua. Effects of levosimendan on the hypoxic/reoxygenated cardiomyocyte fibrosis by regulating long chain noncoding RNA/microRNA-641[J]. Acta Anatomica Sinica. 2022, 53(4): 479-487 https://doi.org/10.16098/j.issn.0529-1356.2022.04.011

中图分类号： R349.1

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