Silencing of SNHG6 alleviates hypoxia/reoxygenation-induced cardiomyocyte apoptosis by modulating miR-135a-5p/HIF1AN to activate Shh/Gli1 signalling pathway.

To examine the effects of small nucleolar RNA host gene 6 (SNHG6) on apoptosis during myocardial ischemic/reperfusion (I/R) injury and its potential molecular mechanisms. In vitro model of I/R was built through exposing mouse HL-1 cardiomyocytes to hypoxia/reoxygenation (H/R) treatment. Quantitative real-time polymerase chain reaction assays were performed to determine gene expression. Cell Counting Kit-8, flow cytometric and western blot assays were conducted to detect cell viability, apoptosis and protein expression. Lactate dehydrogenase (LDH) activity was examined by a commercial detection kit. Dual-luciferase gene reporter and RNA immunoprecipitation experiments were applied for determining the interaction between the molecules. SNHG6 expression was increased in H/R-challenged cardiomyocytes. Depletion of SNHG6 protected against H/R-induced cardiomyocytes apoptosis. SNHG6 could sponge miR-135a-5p to inhibit its expression. Down-regulation of miR-135a-5p reversed the anti-apoptotic effect caused by SNHG6 knockdown in H/R-induced cardiomyocytes. Hypoxia inducible factor 1 subunit alpha inhibitor (HIF1AN) was identified as a direct target of miR-135a-5p, and knockdown of HIF1AN relieved H/R-induced cardiomyocytes apoptosis. Silencing of SNHG6 activated Shh/Gli1 signalling pathway by regulating miR-135a-5p/HIF1AN. Furthermore, inactivation of Shh/Gli signalling abolished the anti-apoptotic effects of SNHG6 knockdown in H/R-induced cardiomyocytes. SNHG6 serves as a sponge for miR-135a-5p to promote HIF1AN expression and inactivate Shh/Gli1 signalling, eventually aggravating H/R-induced apoptosis in cardiomyocytes.

Lang Z ，Fan X ，Lin H ，Qiu L ，Zhang J ，Gao C ... -  《-》

Long Noncoding RNA Taurine-Upregulated Gene 1 Knockdown Protects Cardiomyocytes Against Hypoxia/Reoxygenation-induced Injury Through Regulating miR-532-5p/Sox8 Axis.

Cai X ，Wang S ，Hong L ，Yu S ，Li B ，Zeng H ，Yang X ，Zhang P ，Shao L ... -  《-》

Mesenchymal Stem Cell-Originated Exosomal Lnc A2M-AS1 Alleviates Hypoxia/Reperfusion-Induced Apoptosis and Oxidative Stress in Cardiomyocytes.

Mesenchymal stem cell (MSC)-derived exosomes play significant roles in ameliorating cardiac damage after myocardial ischemia-reperfusion (I/R) injury. Long non-coding RNA alpha-2-macroglobulin antisense RNA 1 (Lnc A2M-AS1) was found that might protect against myocardial I/R. However, whether Lnc A2M-AS1 delivery via MSC-derived exosomes could also regulate myocardial I/R injury remains unknown. Exosomes were isolated by ultracentrifugation, and qualified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Hypoxia/reoxygenation (H/R) treatment in human cardiomyocytes was used to mimic the process of myocardial I/R in vitro. The viability and apoptosis of cardiomyocytes were detected using cell counting kit-8, flow cytometry, and Western blot assays. The contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were evaluated using corresponding commercial kits. The quantitative real-time polymerase chain reaction and Western blot were used to determine the expression levels of Lnc A2M-AS1, microRNA (miR)-556-5p, and X-linked inhibitor of apoptosis protein (XIAP). The binding interaction between miR-556-5p and Lnc A2M-AS1 or XIAP was confirmed by the dual-luciferase reporter, RIP and pull-down assays. Exosomes isolated from hMSCs (hMSCs-exo) attenuated H/R-induced apoptosis and oxidative stress in cardiomyocytes. Lnc A2M-AS1 was lowly expressed in AMI patients and H/R-induced cardiomyocytes. Besides, Lnc A2M-AS1 was detectable in hMSCs-exo, exosomes derived from Lnc A2M-AS1-transfected hMSCs weakened H/R-induced apoptosis and oxidative stress, and enhanced the protective action of hMSCs-exo on H/R-induced cardiomyocytes. Further mechanism analysis showed that Lnc A2M-AS1 acted as a sponge for miR-556-5p to increase XIAP expression level. Importantly, miR-556-5p overexpression or XIAP knockdown reversed the action of exosomal Lnc A2M-AS1 on H/R-induced cardiomyocytes. Lnc A2M-AS1 delivery via MSC-derived exosomes ameliorated H/R-induced cardiomyocyte apoptosis and oxidative stress via regulating miR-556-5p/XIAP, opening a new window into the pathogenesis of myocardial I/R injury.

Yu H ，Pan Y ，Dai M ，Wang X ，Chen H ... -  《-》

Sevoflurane protects cardiomyocytes against hypoxia/reperfusion injury via LINC01133/miR-30a-5p axis.

Previous studies failed to elucidate the detailed mechanisms of anesthetic preconditioning as a protective approach against ischemic/reperfusion (I/R) injury in cells. The present study mainly centered on discovering the mechanisms of Sevoflurane (Sev) in preventing cardiomyocytes against I/R injury. Human cardiomyocyte AC16 cell line was used to simulate I/R injury based on a hypoxia/reperfusion (H/R) model. After Sev treatment, cell viability and apoptosis were detected by MTT assay and flow cytometry, respectively. Lactate dehydrogenase (LDH) content was measured using an LDH Detection Kit. Relative mRNA and protein expressions of LINC01133, miR-30a-5p and apoptosis-related proteins were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. Target gene of miR-30a-5p and their potential binding sites were predicted using Starbase and confirmed by dual-luciferase reporter assay. Cell behaviors were assessed again after miR-30a-5p and LINC01133 transfection. Sev could improve cell viability, reduce LDH leakage, and down-regulate the expressions of apoptosis-related proteins (Bax, cleaved caspase-3 and cleaved caspase-9) and LINC01133 as well as up-regulate miR-30a-5p and Bcl-2 expressions in H/R cells. MiR-30a-5p was the target of LINC01133, and up-regulating miR-30a-5p enhanced the effects of Sev in H/R cells, with a suppression on H/R-induced activation of the p53 signaling pathway. However, up-regulating LINC01133 reversed the enhancing effects of miR-30a-5p on Sev pretreatment in H/R cells. Sev could protect cardiomyocytes against H/R injury through the miR-30a-5p/LINC01133 axis, which may provide a possible therapeutic method for curing cardiovascular I/R injury.

Yu Z ，Ren Q ，Yu S ，Gao X ... -  《-》

Pinoresinol diglucoside ameliorates H/R-induced injury of cardiomyocytes by regulating miR-142-3p and HIF1AN.

This study is aimed to investigate the effect of pinoresinol diglucoside (PDG) in ameliorating myocardial ischemia-reperfusion injury (MIRI). Hypoxia/reperfusion (H/R)-induced H9c2 cardiomyocytes were used to establish an in-vitro ischemia-reperfusion injury model of cardiomyocytes. Cells were treated with 1 μmol/L of PDG. Reactive oxygen species (ROS) level was detected by a 2',7'-dichlorofluorescein-diacetate assay. The release of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) was examined by enzyme-linked immunosorbent assay. The viability and apoptosis of H9c2 cells were probed by MTT assay and flow cytometry. Besides this, Western blot and quantitative real-time PCR were used to detect microRNA-142-3p (miR-142-3p) and hypoxia-inducible factor 1 subunit alpha inhibitor (HIF1AN) expression levels. The binding sequence between miR-142-3p and HIF1AN 3'-untranslated region was validated by a dual-luciferase reporter gene assay. PDG treatment significantly reduced the level of ROS, LDH, and CK-MB, promoted viability, and inhibited the apoptosis of H9c2 cells. PDG treatment promoted miR-142-3p expression and inhibited HIF1AN expression in H9c2 cells. MiR-142-3p overexpression enhanced the effects of PDG on ROS, LDH, CK-MB levels, cell viability, and apoptosis in H9c2 cardiomyocytes, while overexpression of HIF1AN reversed the above effects. PDG ameliorates H/R-induced injury of cardiomyocytes by regulating miR-142-3p and HIF1AN.

Wei Y ，Xiao L ，Yingying L ，Haichen W ... -  《-》