Dataset Information

Circ_0030235 knockdown protects H9c2 cells against OGD/R-induced injury via regulation of miR-526b

ABSTRACT:

Backgrounds

Acute myocardial infarction (MI) is the common clinical manifestation of coronary heart disease. Circular RNAs (circRNAs) act key roles in cardiomyocytes growth and angiogenesis. However, their functions in MI are not entirely clear. This research intended to investigate the role and underlying mechanisms of circ_0030235 in H9c2 cells.

Methods

H9c2 cells were conducted to oxygen glucose deprivation/reperfusion (OGD/R) inducement to establish the MI model. Circ_0030235 and miR-526b expression was tested and altered by qRT-PCR and transfection. Cell viability, apoptosis and reactive oxygen species (ROS) injury were tested by CCK-8 assay, TUNEL assay kit, and ROS Detection Assay Kit, respectively. Assessment of cell injury-related factors was performed by employing ELISA, Mitochondrial Viability Staining and the JC-1-Mitochondrial Membrane Potential Assay Kit. The relationship between circ_0030235 and miR-526b was analyzed by dual luciferase reporter assay. The expression of key proteins was analyzed by western blot.

Results

Circ_0030235 was highly expressed in OGD/R-induced H9c2 cells. OGD/R inducement cell viability, while accelerated apoptosis. Besides, the level ROS, cell injury-related factors, mitochondrial membrane potential were notably elevated by OGD/R inducement, while mitochondrial viability was remarkably declined. Whereas, these impacts were all noticeably remitted by circ_0030235 knockdown. miR-526b was a target of circ_0030235. Circ_0030235 knockdown-induced impacts were all notably abrogated by miR-526b inhibition, including the activating impacts on PI3K/AKT and MEK/ERK pathways.

Conclusions

This research implied that circ_0030235 knockdown might remit OGD/R-induced impacts via activation of PI3K/AKT and MEK/ERK pathways and regulation of miR-526b.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC8603820 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Few studies have addressed the mechanism by which circ_0010729 regulates hypoxia-induced cell injury in cardiovascular diseases. However, its role and its regulatory mechanism in myocardial infarction remain to be explored. Cell viability, cycle, apoptosis, and migration were analyzed using cell counting kit-8 assay, flow cytometry, caspase-3 activity assay kit and transwell assay, respectively. Tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) concentrations were examined by enzyme-linked immunosorbent assay. Glucose metabolism was calculated by detecting ATP production, glucose uptake and lactate production. Levels of circ_0010729, miR-370-3p and TNF Receptor Associated Factor 6 (TRAF6) were detected using quantitative real-time polymerase chain reaction or western blot. The direct interaction between circ_0010729 and TRAF6 or miR-370-3p was verified using dual-luciferase reporter assay and RNA immunoprecipitation assay. Under hypoxia condition, cardiomyocytes suffered from cell viability suppression, cell cycle arrest, cell apoptosis promotion, migration reduction, increase of inflammatory factor IL-6 and TNF-α, as well as glycolysis inhibition. Circ_0010729 expression was up-regulated in the cardiomyocytes at different hypoxia-exposed time points. Circ_0010729 knockdown protected cardiomyocytes against hypoxic dysfunction, while circ_0010729 overexpression showed inverse effects. MiR-370-3p was confirmed to directly bind to circ_0010729 or TRAF6. MiR-370-3p inhibition attenuated the protective effects of circ_0010729 knockdown on hypoxia-modulated cardiomyocyte dysfunction. MiR-370-3p restoration protected cardiomyocytes against hypoxic injury via targeting TRAF6. Besides, circ_0010729 indirectly regulated TRAF6 expression via miR-370-3p. This study demonstrated that circ_0010729 knockdown attenuated hypoxia-induced cardiomyocyte dysfunction via miR-370-3p/TRAF6 axis, indicating a potential therapeutic target for myocardial infarction.