陆军军医大学学报 (Sep 2023)
Role and mechanism of ACSS2-mediated epithelial-mesenchymal transition in ethanol-promoted migration of colorectal cancer
Abstract
Objective To explore the role and underlying mechanisms of Acyl-CoA synthetase short chain family member 2 (ACSS2) in ethanol-promoted migration of colorectal cancer (CRC) cells. Methods The ACSS2 expression level in cancer tissues and adjacent normal tissues was analyzed by online tools Gene Expression Profiling Interactive Analysis (GEPIA) and Human Protein Atlas (HPA). The transcription factor enrichment analysis tool ChIP-X Enrichment Analysis 3 (ChEA3) was used to identify ACSS2 upstream transcription factors. The ACSS2 expression level in cancer tissue and related clinical characteristic parameters of 513 CRC patients were obtained from The Cancer Genome Atlas (TCGA) database. After HCT116 and SW480 cells were treated with ethanol at a dose of 0, 22, 44 and 88 mmol/L for 24, 48 and 72 h, respectively, cell viability, migration and expression of related molecules at mRNA and protein levels were detected with CCK-8 assay, cell scratch test and transwell assay, and RT-qPCR and Western blotting respectively. Results ① After treated by ethanol for 48 and 72 h, the 3 doses of 22, 44 and 88 mmol/L showed a significant inhibition of proliferation for both HCT116 and SW480 cells (P < 0.05). ②After 48 h of ethanol treatment, RT-qPCR and Western blotting revealed that the expression levels of ACSS2 were increased significantly in HCT116 and SW480 cells, cell scratch test and Transwell assay indicated that the cell healing rate and number of perforated cells were also increased obviously (P < 0.05), and epithelial-mesenchymal transformation (EMT) related proteins, E-cadherin was down-regulated, while N-cadherin and Vimentin were up-regulated (all P < 0.05). ③ Inhibition of ACSS2 expression resulted in obviously decreased cell healing rate and number of perforated cells (P < 0.05), enhanced expression level of E-cadherin and decrased levels of N-cadherin and Vimentin (all P < 0.05). ④ HPA database analysis showed that ACSS2 has a significantly higher expression level in CRC cancer tissues than in normal tissues, and upstream regulators enrichment analysis suggested 8 transcription factors which probably regulate ACSS2 expression. Western blotting showed CEBPB, SMC1A and CTCF had notably increased expression in both SW480 and HCT116 cells after ethanol treatment, which was consistent with the increase trend of ACSS2 expression. ⑤ Of the 513 CRC patients, those with higher ACSS2 expression had a significantly higher incidence of peripheral nerve or vascular infiltration, and higher rate of existed distant metastases than those with lower expression (Chi-square=6.411, P=0.011). Conclusion Ethanol treatment may probably induce EMT by upregulating ACSS2 expression and its upstream transcription factors to promote the migration of CRC cells.
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