Hsa-miR-423-5p selectively loaded in hypoxic exosomes reduces the sensitivity of normoxic hepatocellular carcinoma to sorafenib via autophagy.

BACKGROUND: For patients with advanced hepatocellular carcinoma, sorafenib is considered a highly effective targeted molecular drug; however, sorafenib resistance limits its therapeutic efficacy. Hepatocellular carcinoma, a type of solid tumour, contains hypoxic and oxygen-rich regions within its internal structure. This study investigated the mechanism by which hypoxic hepatocellular carcinoma cells influence the sorafenib sensitivity of normoxic hepatocellular carcinoma cells through the secretion of hypoxic exosomes containing miRNA signals. METHODS: Experiments like CCK-8, IC50, and flow cytometry were used to determine how hypoxic exosomes affect the sorafenib sensitivity of normoxic hepatocellular carcinoma cells. A high-throughput sequencing approach was employed to identify the target of miR-423-5p selectively loaded in hypoxic exosomes. Subsequently, RNA pull-down, RIP, and other experiments were conducted to investigate the mechanism by which hsa-miR-423-5p is selectively loaded into hypoxic exosomes through the RNA-binding proteins (RBPs) acting upstream. Next, Western blot, quantitative real-time PCR, apoptosis flow cytometry, and dual-luciferase reporter assays were performed to investigate how hsa-miR-423-5p affects downstream direct targets and modulates the sorafenib sensitivity of normoxic hepatocellular carcinoma cells through an autophagy mechanism. Finally, the experimental results were validated in an orthotopic hepatocellular carcinoma mouse model by constructing lipid nanoparticles (LNPs) that carry hsa-miR-423-5p inhibitors. RESULTS: Hypoxic hepatocellular carcinoma cells secreted hypoxic exosomes, which could be taken up by normoxic hepatocellular carcinoma cells. These exosomes subsequently reduced the sorafenib sensitivity of normoxic hepatocellular carcinoma cells through an autophagy mechanism. High-throughput sequencing revealed that hsa-miR-423-5p was selectively loaded into hypoxic exosomes via HNRNPA1. In normoxic hepatocellular carcinoma cells, hsa-miR-423-5p promoted autophagy by targeting the downstream target TAB2, thereby decreasing the sorafenib sensitivity of normoxic hepatocellular carcinoma cells to sorafenib. CONCLUSIONS: In response to the upstream factor HNRNPA1, hsa-miR-423-5p is selectively loaded into hypoxic exosomes, which then target the downstream protein TAB2 to regulate the autophagy pathway and reduce the sorafenib sensitivity of normoxic hepatocellular carcinoma cells.