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Background and purpose: Metabolic reprogramming occurs during tumor progression, and 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT), as a key enzyme in the de novo synthesis of triacylglycerol (TAG), is closely associated with tumor progression. However, one of the isoforms, AGPAT5, has been studied in cancer in a very limited way, and this study aimed to provide a new perspective on the role of AGPAT5 in hepatocellular carcinoma and its potential molecular mechanisms, providing novel ideas for the diagnosis and treatment strategies of liver cancer. Methods: AGPAT5 was knocked down in a variety of hepatocellular carcinoma cell lines using lentiviral infection, and the effects of AGPAT5 on the functions of hepatocellular carcinoma cell proliferation, migration and resistance to anoikis were detected in vitro by experiments such as Taipan blue counting, scratching, transwell and plate cloning. The wild-type or enzyme activity-deficient form of AGPAT5 was rescued to investigate whether AGPAT5, as a metabolic enzyme, plays a classical role in regulating the migration of hepatocellular carcinoma cells. We constructed a tail vein metastasis model in nude mice to validate the cellular phenotype in vitro from the in vivo level. Immunoprecipitation mass spectrum (IP-MS) identified proteins interacting with AGPAT5 and verified by co-immunoprecipitation (coIP). Protein post-translational modification identification was performed to analyze the potential modification sites of AGPAT5, and in vitro experiments were performed to explore the effects of the point mutation before and after the point mutation on the migration of hepatocellular carcinoma cells. CoIP was performed to explore the binding of AGPAT5 to the interacting protein before and after the mutation of the site. We determined its role in cell phenotype by knocking down interacting proteins. Rescue experiments were used to verify whether AGPAT5 exerts its effects through the interacting protein. We detected the expression levels of AGPAT5 and the interacting protein in wild-type hepatocellular carcinoma cell lines to examine their correlation. Results: Knockdown of AGPAT5 increased the tolerance to serum-free starvation and promoted hepatocellular carcinoma cell migration, but did not affect proliferation and anoikis. However, deletion of enzyme activity did not affect the inhibition of hepatocellular carcinoma cell migration by AGPAT5. Knockdown of AGPAT5 promoted lung and liver metastasis of hepatocellular carcinoma cells in nude mice. AGPAT5 could interact with fibrillarin (FBL), and the interaction was strengthened under serum starvation conditions. Curbing FBL expression inhibited hepatocellular carcinoma cell migration, and the effect was similar to that of overexpression of AGPAT5. Inhibition of FBL expression weakened the promoting effect of AGPAT5 knockdown on hepatocellular carcinoma cell migration; In the hepatocellular carcinoma cell lines examined, AGPAT5 and FBL did not show any correlation at the protein level. The inhibitory effect of AGPAT5 on hepatocellular carcinoma cell migration was attenuated by the K201 site mutation, and the K201 site mutation attenuated the binding of AGPAT5 to FBL. Conclusion: Knockdown of AGPAT5 can significantly enhance the migratory ability of hepatocellular carcinoma cells. AGPAT5 can interact with FBL, and in the absence of serum starvation stimulation, AGPAT5 strengthen its binding to FBL through acetylation of the K201 site, thereby more effectively inhibiting FBL, consequently inhibiting the migration of hepatocellular carcinoma cells. But this inhibitory effect is not derived from the metabolic enzyme activity of AGPAT5, but driven by non-metabolic function. |
