Inhibition of circ_0073932 attenuates myocardial ischemia‒reperfusion injury via miR-493-3p/FAF1/JNK.

Oxidative stress and apoptosis play crucial roles in myocardial ischemia‒reperfusion injury (MIRI). In this study, we investigated the role of circ_0073932 in MIRI as well as its molecular mechanism. A hypoxia/reoxygenation (H/R) cardiomyocyte model was established with H9C2 cardiomyocytes, and RT-qPCR was used to measure gene expression. We observed that circ_0073932 expression was abnormally increased in the H/R cardiomyocyte model and in blood samples from MIRI patients. Inhibition of circ_0073932 suppressed H/R-induced cell apoptosis, oxidative stress (ROS, LDH and MDA), and p-JNK expression. Dual luciferase reporter assays showed that circ_0073932 targeted the downregulation of miR-493-3p, and miR-493-3p targeted the downregulation of FAF1. Furthermore, si-circ_0073932, an miR-493-3p inhibitor, oe-FAF1, or si-FAF1 were transfected into H9C2 cardiomyocytes to investigate the roles of these factors in MIRI. Our results showed that compared with the H/R group, si-circ_0073932 inhibited H/R-induced cell apoptosis, oxidative stress (ROS, LDH and MDA), and p-JNK expression. These results were reversed by the miR-493-3p inhibitor or oe-FAF1. Finally, a rat model of MIRI was established, and si-circ_0073932 was administered. Inhibition of circ_0073932 reduced the area of myocardial infarction and decreased the levels of apoptosis and oxidative stress by inhibiting the JNK signaling pathway. Our study indicated that circ_0073932 mediates MIRI via miR-493-3p/FAF1/JNK in vivo and in vitro, revealing novel insights into the pathogenesis of MIRI and providing a new target for the clinical treatment of MIRI.

Su Y ，Zhao L ，Lei D ，Yang X ... -  《-》

Circular RNA circ_0010729 Knockdown Attenuates Oxygen-Glucose Deprivation-Induced Human Cardiac Myocytes Injury by miR-338-3p/CALM2 Axis.

Circular RNAs have pivotal roles in cardiovascular disease. The injury of cardiac myocytes is associated with occurrence of cardiovascular disease. Circular RNA hsa_circ_0010729 (circ_0010729) is associated with cardiac myocytes injury. However, the mechanism of circ_0010729 in cardiac myocytes injury remains largely unclear. In our study, cardiac myocytes were treated by oxygen-glucose deprivation (OGD). The abundances of circ_0010729, microRNA-338-3p (miR-338-3p), and calmodulin 2 (CALM2) were detected by quantitative reverse transcription polymerase chain reaction or Western blot. OGD-induced damage in AC16 cells was assessed by cell viability, apoptosis, and autophagy using Cell Counting Kit-8, flow cytometry, and Western blot analyses. The target relationship of miR-338-3p and circ_0010729 or CALM2 was explored by starBase and dual-luciferase reporter analysis. Our results showed that the circ_0010729 level was enhanced in OGD-treated AC16 cells and murine primary cardiac myocytes. circ_0010729 silence weakened OGD-induced viability inhibition and promotion of apoptosis and autophagy in AC16 cells and murine primary cardiac myocytes. miR-338-3p was sponged by circ_0010729 and miR-338-3p knockdown alleviated the effect of circ_0010729 silence on OGD-induced damage. miR-338-3p directly targeted CALM2 to inhibit OGD-induced damage in AC16 cells. circ_0010729 could regulate CALM2 expression by sponging miR-338-3p. Collectively, circ_0010729 interference mitigated OGD-induced damage in cardiac myocytes through increasing cell viability and inhibiting apoptosis and autophagy by regulating miR-338-3p/CALM2 axis. This study indicated circ_0010729 might act as a target for treatment of cardiovascular disease.

Ma B ，Zhao M ，Guo Z 《-》

Silencing hsa_circ_0049271 attenuates hypoxia-reoxygenation (H/R)-induced myocardial cell injury via the miR-17-3p/FZD4 signaling axis.

This study sought to explore the role and molecular mechanism of circ_0049271 in hypoxia-reoxygenation (H/R)-induced cardiomyocyte injury. Significantly upregulated circular ribonucleic acids (circRNAs) in Gene Expression Omnibus (GEO) data sets were identified using a Venn diagram. A H9c2 (rat cardiomyocytes) cell model of acute myocardial infarction (AMI) was induced by 1% H/R. Quantitative reverse transcription-polymerase chain reaction was used to detect the expression levels of circ_0049271, miR-17-3p, and FZD4 in clinical blood samples and cells, and Cell Counting Kit-8 (CCK-8) was used to determine the proliferation rate of the cells in each group. Next, flow cytometry and Western blot were used to evaluate cell apoptosis. Biochemical tests and enzyme-linked immunosorbent assays (ELISAs) were then used to determine the activities/levels of the cell damage markers [i.e., creatine kinase (CK) and lactate dehydrogenase (LDH)], oxidative stress substances [i.e., malondialdehyde (MDA), reactive oxygen species (ROS), and superoxide dismutase (SOD)], and inflammatory factors [i.e., interleukin (IL)-1β, IL-6, and IL-8]. In addition, intermolecular interactions were verified using dual-luciferase reporter and RNA pull-down experiments. Circ_0049271 was significantly upregulated in both the blood of the AMI patients and the H/R-induced H9c2 cells. The knockdown of circ_0049271 increased the cell proliferation rate, decreased the apoptosis rate, inhibited oxidative stress (ROS and MDA were upregulated, and SOD was downregulated) and inflammatory responses (IL-1, IL-6, and IL-8 were downregulated), and relieved cell damage. However, the overexpression of circ_0049271 promoted H/R-induced H9c2 cell damage. Further experiments showed that miR-17-3p was a target of circ_0049271, and miR-17-3p was negatively correlated with circ_0049271 in the AMI blood samples. Additionally, miR-17-3p was found to target FZD4. A further exploration also revealed that miR-17-3p knockdown or FZD4 overexpression reversed the effects of si-circ_0049271 on the H/R-induced H9c2 cells; that is, miR-17-3p knockdown or FZD4 overexpression promoted H/R-induced injury in the H9c2 cells. Circ_0049271 promoted cellular function damage (e.g., proliferation inhibition, apoptosis, oxidative stress, and inflammation) in H/R-induced H9c2 cardiomyocytes via the miR-17-3p/FZD4 signaling axis.

Liao H ，Xiao C ，Li W ，Chen W ，Xiang D ... -  《-》

Sevoflurane exerts protection against myocardial ischemia-reperfusion injury and pyroptosis through the circular RNA PAN3/microRNA-29b-3p/stromal cell-derived factor 4 axis.

Sevoflurane is suggested to exert protective functions against myocardial ischemia-reperfusion injury (MIRI). However, the particular mechanism remains elusive. Therefore, this research explored the mechanism of sevoflurane in MIRI-induced damage and pyroptosis. Subsequent to gain-or loss-of-function assays or/and sevoflurane treatment, the MIRI model was developed in rats. Cardiac function and body and heart weight of rats were evaluated, followed by measurement of apoptosis and creatine kinase MB (CK-MB), lactate dehydrogenase (LDH), and pyroptosis-related protein levels. After loss-of-function assays or/and sevoflurane treatment in human cardiomyocytes (HCMs), the hypoxia/reoxygenation (H/R) model was constructed. In HCMs, cell viability, apoptosis, and pyroptosis-related proteins were detected. Circular RNA PAN3 (circPAN3), microRNA (miR)-29b-3p, and stromal cell-derived factor 4 (SDF4) expression was determined in rat myocardial tissues and HCMs. Mechanistically, interactions among circPAN3, miR-29b-3p, and SDF4 were analyzed. MIRI modeling increased miR-29b-3p expression and diminished circPAN3 and SDF4 expression in H/R-treated HCMs and MIRI rats, which was nullified by sevoflurane preconditioning. Mechanistically, circPAN3 negatively targeted miR-29b-3p to upregulate SDF4. Moreover, sevoflurane preconditioning reduced heart weight/body weight ratio, LDH, CK-MB, myocardial infarct size, left ventricular end-diastolic pressure, apoptosis, and pyroptosis, while elevating the increase and decrease of left ventricular pressure (±dp/dt max) and left ventricular systolic pressure in MIRI rats. In addition, sevoflurane preconditioning augmented viability while diminishing apoptosis and pyroptosis in H/R-treated HCMs. Moreover, circPAN3 silencing or miR-29b-3p overexpression abrogated alleviatory effects of sevoflurane on myocardial injury and pyroptosis in vitro. Sevoflurane treatment ameliorated myocardial injury and pyroptosis in MIRI via circPAN3/miR-29b-3p/SDF4 axis.

An L ，Zhong Y ，Tan J ，Liu Y ，Li A ，Yang T ，Wang S ，Liu Y ，Gao H ... -  《-》

miR-451-3p alleviates myocardial ischemia/reperfusion injury by inhibiting MAP1LC3B-mediated autophagy.

We aim to explore the molecular mechanism of myocardial ischemia-reperfusion injury (MIRI). The H9C2 cells were cultured under hypoxia/reoxygenation (H/R) condition to induce myocardial injury in vitro. The expression of miR-451-3p and MAP1LC3B was detected by RT-qPCR. Dual-luciferase reporter assay and RNA pull-down assay were performed to examine the relationship between microRNA (miR)-451-3p and MAP1LC3B. CCK8 was used to test cell viability. The level of LDH and CK was evaluated via ELISA. Immunofluorescence assay and flow cytometry were applied to detect autophagy and apoptosis, respectively. Autophagy-related protein expressions were determined by western blotting. Furthermore, an in vivo rat model of MIRI was established by subjection to 30 min ischemia and subsequently 24 h reperfusion for validation of the role of miR-451-3p in regulating MIRI in vivo. miR-451-3p was down-regulated in MIRI, and miR-451-3p mimics transfection alleviated autophagy and apoptosis induced by MIRI. miR-451-3p could target MAP1LC3B directly. Co-transfection miR-451-3p mimics and pcDNA 3.1 MAP1LC3B curbed the protected effects of miR-451-3p mimics on MIRI. miR-451-3p played a protective role in MIRI via inhibiting MAP1LC3B-mediated autophagy, which may provide new molecular targets for the treatment of MIRI and further improves the clinical outcomes of heart diseases.

Lv XW ，He ZF ，Zhu PP ，Qin QY ，Han YX ，Xu TT ... -  《-》