Abstract
OBJECTIVE: To investigate the expression level of aconitate decarboxylase 1 (ACOD1) in hepatocellular carcinoma (HCC) and its role in HCC cell growth, as well as the underlying mechanisms. METHODS: The expression of the ACOD1 gene in HCC and its correlation with patient survival were analyzed using the GTEx and TCGA databases. ACOD1 protein expression in four paired HCC and adjacent non-tumor tissues was detected by Western blotting. After ACOD1 knockdown in HepG2 and PLC/PRF/5 HCC cells, cell proliferation and death were assessed by MTT and LDH assays, respectively. Following ACOD1 knockdown, 4-octyl itaconate (4-OI) was administered to observe changes in cell proliferation. Potential mechanisms were predicted using the cBioPortal database through KEGG enrichment analysis, and the expression of autophagy-related proteins LC3-Ⅱ and p62 was measured by Western blotting. After ACOD1 knockdown, cells were treated with chloroquine (CQ) or supplemented with 4-OI to observe changes in autophagy marker proteins and cell proliferation. RESULTS: ACOD1 protein expression was significantly overexpressed in HCC tissues than in adjacent tissues (approximately 2-fold). Patients with ACOD1 overexpression had shorter overall survival (P < 0.05). Knocking down ACOD1 in HepG2 and PLC/PRF/5 cells, which expressed high levels of ACOD1, inhibited cell proliferation (by approximately 20% and 30%, respectively; both P < 0.05) and increased cell death (by approximately 30% in both cell lines; both P < 0.05). 4-OI promoted cell growth in a concentration-dependent manner and reversed the decrease in cell viability caused by ACOD1 knockdown, restoring it to 93% and 90% of control levels, respectively (both P < 0.05). KEGG analysis suggested a potential association between ACOD1 and the autophagy pathway. ACOD1 knockdown significantly increased LC3-Ⅱ levels (by approximately 7-fold and 10-fold, respectively; both P < 0.05) and decreased p62 levels (by 40% and 30%, respectively; both P < 0.05) in HepG2 and PLC/PRF/5 cells. CQ treatment increased the LC3-Ⅱ/LC3-Ⅰ ratio and p62 protein expression (both by approximately 2-fold; P < 0.05). Combined ACOD1 knockdown further increased the LC3-Ⅱ/LC3-Ⅰ ratio (by approximately 10%; P < 0.05), while p62 protein expression did not increase significantly (P > 0.05). Supplementation with 4-OI reversed the changes in LC3-Ⅱ and p62 protein expression induced by ACOD1 knockdown. Furthermore, CQ treatment partially rescued the decrease in viability of HepG2 and PLC/PRF/5 cells caused by ACOD1 knockdown (restoring viability to 75% and 80%, respectively; both P < 0.05). CONCLUSION: ACOD1 is overexpressed in HCC and its' overexpression is associated with poor prognosis. ACOD1 promotes HCC cell growth by synthesizing itaconate, which inhibits cellular autophagy.