Environmental DINCH promotes the progression and EMT of hepatocellular carcinoma by upregulating NEDD1.

BACKGROUND: Di(isononyl) cyclohexane-1,2-dicarboxylate (DINCH), a phthalate substitute widely detected in environmental and biological samples, has raised growing concerns regarding its potential health risks. However, its role in the pathogenesis and progression of hepatocellular carcinoma (HCC), particularly in epithelial-mesenchymal transition (EMT), remains largely unknown. METHODS: The effects of DINCH on HCC cells (LM3, Huh7) and immortalized normal hepatocytes (THLE-2) were assessed by evaluating proliferation (CCK-8, colony formation, and EdU assays), migration (wound healing and Transwell assays), EMT marker expression by Western blotting, and chemokine/cytokine secretion by ELISA. Bioinformatic analyses, including LASSO regression and immune infiltration profiling of the TCGA cohort, were performed to identify DINCH-related genes. Functional assays were combined with genetic manipulation of neural precursor cell expressed, developmentally down-regulated protein 1 (NEDD1), including knockdown and overexpression experiments, to elucidate its regulatory role. RESULTS: DINCH significantly promoted HCC cell proliferation and migration, accompanied by enhanced EMT characterized by decreased E-cadherin expression and increased N-cadherin, Vimentin, SNAI2, and ZEB1 expression. Notably, immortalized normal THLE-2 hepatocytes exhibited similar pro-tumorigenic responses to DINCH exposure, indicating that DINCH-induced oncogenic effects extend to non-malignant hepatocytes. Long-term, low-dose exposure experiments confirmed that even sub-micromolar DINCH concentrations promoted cell proliferation in both HCC cells and THLE-2 hepatocytes. A risk score model based on DINCH-related genes was constructed, and the high-risk group was associated with an immunosuppressive tumor microenvironment characterized by increased infiltration of Tregs and M2 macrophages. Integrative analyses identified NEDD1 as a key DINCH-associated candidate gene. Molecular docking suggested a potential binding mode between DINCH and NEDD1. Knockdown of NEDD1 markedly suppressed DINCH-induced proliferation, migration, invasion, and EMT in both LM3 and THLE-2 cells, whereas NEDD1 overexpression produced the opposite effects in Huh7 cells. Furthermore, DINCH activated TGF-β1/SMAD signaling and upregulated the expression of the immunosuppressive chemokines CCL2 and CSF1 in both HCC cells and THLE-2 hepatocytes, effects that were abrogated by NEDD1 depletion. CONCLUSION: This study provides evidence that DINCH exposure promotes HCC progression and EMT through NEDD1 upregulation and downstream TGF-β/SMAD signaling activation. These findings establish the DINCH/NEDD1 axis as a mechanistic link between environmental chemical exposure and liver cancer aggressiveness, and suggest that NEDD1 may represent a potential therapeutic target warranting further investigation.