MiR-183-5p inhibitor promotes mitoxantrone-induced immunogenic death of hepatoma cells by targeting STC1.

Abstract

This study aimed to unravel the regulatory mechanism of the microRNA (miR)-183-5p/STC1 axis in regulating mitoxantrone (MIT)-induced immunogenic cell death (ICD) within hepatocellular carcinoma (HCC) cells and assess its therapeutic potential. Dual-luciferase reporter assays were employed to validate that miR-183-5p directly interacts with the 3'-untranslated region (3'-UTR) of STC1 mRNA. HepG2 cells were subjected to treatment with MIT, either in the presence of miR-183-5p inhibitors or STC1 knockout. The expression levels of ICD markers, namely adenosine triphosphate (ATP), high mobility group box 1 (HMGB1), calreticulin (CALR), along with cellular proliferation and apoptosis, were subsequently evaluated. The dual-luciferase assays confirmed that miR-183-5p specifically binds to a defined sequence (5'-GUGCCAU-3') within the 3'-UTR of STC1 mRNA. In MIT-treated HepG2 cells, inhibition of miR-183-5p resulted in a significant upregulation of both STC1 mRNA (+0.78-fold) and protein levels (+0.21-fold). This was accompanied by an enhanced the expression of ICD markers, including a 0.26-fold increase in CALR membrane exposure, a 0.27-fold rise in ATP secretion, and a 0.44-fold elevation in HMGB1 release. Notably, the effects induced by miR-183-5p inhibition were partially mitigated by STC1 gene knockout. Further investigations demonstrated that combination of miR-183-5p inhibitors with MIT synergistically boosted STC1 expression by 5.08-fold. Conversely, STC1 knockout triggered a 184.1% surge in miR-183-5p expression, indicating a negative feedback loop between them. Functional cellular assays revealed that miR-183-5p inhibition significantly augmented MIT's anti-proliferative efficacy, increasing the inhibition rate from 41.9% to 68.0%, and enhanced its pro-apoptotic effects, elevating the apoptosis rate from 30.57% to 36.08%. However, STC1 knockout attenuated these effects. Collectively, our finding indicate that the miR-183-5p/STC1 axis modulates MIT's cytotoxicity through targeted regulation and a negative feedback mechanisms, thereby influencing HCC cell proliferation, apoptosis, and ICD. These insights offer noval strategies for synergistic therapy that integrate epigenetics and the immune microenvironment in HCC treatment.