Supplementary file 1_Long noncoding RNA X-inactive-specific transcript promotes hepatic fibrosis by suppressing ferroptosis in hepatic stellate cells via the miR-663a/GPX4 axis.docx

AimHepatic fibrosis (HF) is a critical pathological stage in the progression of chronic liver diseases, where hepatic stellate cell (HSC) activation is a key event. Ferroptosis regulates the fate of HSCs and represents a potential anti-fibrotic target. Long non-coding RNA XIST (lncRNA-XIST) is involved in fibrosis-related diseases. This study investigated how lncRNA-XIST promotes HF by regulating ferroptosis through the microRNA-663 (miR-663a)/GPX4 axis.
MethodsLX-2 HSCs were activated using ethanol at varying concentrations for different durations to determine optimal conditions. HSCs were intervened with small interfering RNA against lncRNA-XIST, and Liproxstatin-1 was applied. RT-qPCR, Western blotting, CCK-8, colony formation, LDH release, and biochemical assays assessed gene/protein expression, cell viability, proliferation, ferroptosis markers (Fe2+, MDA, GSH), and cell death. Dual-luciferase assays validated interactions among lncRNA-XIST, miR-663a, and GPX4. In vivo, an HF mouse model was established and treated with sh-XIST or miR-663a antagonists. Liver fibrosis was evaluated by histology, immunohistochemistry, and serum liver injury markers (ALT, AST, HYP).
ResultsEthanol promoted LX-2 activation and upregulated lncRNA-XIST in a time- and dose-dependent manner (optimal: 100 mM, 24 h). LncRNA-XIST knockdown reduced α-SMA, CoL1A1, GPX4 levels, and cell proliferation while increasing ferroptosis markers (indicative of enhanced ferroptosis) and miR-663a expression. Mechanistically, lncRNA-XIST was found to act as a competing endogenous RNA (ceRNA) to sponge miR-663a, thereby upregulating GPX4 and inhibiting ferroptosis. In vivo, lncRNA-XIST was shown to promote HF progression via the miR-663a/GPX4 axis.
ConclusionLncRNA-XIST promotes HF by acting as a ceRNA for miR-663a, regulating GPX4, and suppressing ferroptosis to activate HSCs.