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

HUANG Hong-Ke; LUO Jian-Wei; RAN Hua

doi:10.16098/j.issn.0529-1356.2022.04.011

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Acta Anatomica Sinica ›› 2022, Vol. 53 ›› Issue (4) : 479-487. DOI: 10.16098/j.issn.0529-1356.2022.04.011

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#

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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.

Key words

Levosimendan / Molecular axis of long chain noncoding RNA eosinophil granule ontogeny transcript;Hypoxia/reoxygenation / Fibrosis / Real-time PCR / H9C2 cell line

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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

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