MiR-24-3p modulates cardiac function in doxorubicin -induced heart failure via the Sp1/PI3K signaling pathway

Purpose

The goal of this research was to explore the role of miR-24-3p in heart failure (HF), with a focus on its impact on the specificity protein 1 (Sp1)/phosphoinositide 3-kinase (PI3K) pathway.

Methods

HF rat and HF cell models were established using doxorubicin(Dox). Cardiac function was assessed through echocardiography, while histological changes were observed via hematoxylin-eosin (HE) staining. To further investigate the underlying mechanisms, HF cell models were treated with either an Sp1 inhibitor or a PI3K inhibitor. Additionally, models with miR-24-3p overexpression or silencing were constructed. N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were determined by ELISA. Cell apoptosis was evaluated using TUNEL staining, and lactate dehydrogenase (LDH) levels were measured by colorimetry. Reactive oxygen species (ROS) production was analyzed using flow cytometry. Related gene and protein expressions were assessed via qRT-PCR and Western blotting. Finally, the relationship between miR-24-3p and Sp1 was confirmed through dual-luciferase assays.

Results

Dox treatment increased the left ventricular internal diameter (LVIDd) while decreasing ejection fraction (EF) and fractional shortening (FS), leading to disorganized cardiomyocyte arrangement, cellular edema, and necrosis in rats. In HF rats, NT-proBNP, Caspase-3, and miR-24-3p expression levels were elevated, whereas Sp1 and PI3K mRNA and protein expression levels were decreased. Similarly, Dox-induced damage in H9c2 cardiomyocytes resulted in increased NT-proBNP, apoptosis, Caspase-3, LDH, ROS, and miR-24-3p expression, along with decreased Sp1 and PI3K expression. Treatment with either Sp1 or PI3K inhibitors exacerbated the Dox-induced cardiomyocyte damage, further elevating NT-proBNP, apoptosis, Caspase-3, LDH, ROS, and miR-24-3p expression levels. Notably, Sp1 inhibition reduced PI3K expression, and PI3K inhibition, in turn, suppressed Sp1 expression. Overexpression of miR-24-3p worsened Dox-induced cardiomyocyte damage, characterized by increased NT-proBNP, apoptosis, Caspase-3, LDH, and ROS expression, alongside reduced Sp1 and PI3K expression. In contrast, silencing miR-24-3p mitigated these detrimental effects and increased Sp1 and PI3K expression. Dual-luciferase assays confirmed that miR-24-3p directly targets Sp1.

Conclusion

Dox induces cardiomyocyte damage, impairs cardiac function, and promotes cardiomyocyte apoptosis and oxidative stress. Silencing miR-24-3p offers a protective effect by activating the Sp1/PI3K signaling pathway in heart failure.