Acetylation Modification Of ALDH18A1 And Its Effect On The Proliferation And Metastasis Of Hepatocellular Carcinoma Cells

BackgroundTumor cells reprogram their cellular metabolism to accommodate their abnormal proliferation,invasion,and metastasis.Hepatocellular carcinoma（HCC）is the primary malignant tumor in liver with high mortality.Postoperative recurrence and metastasis are the main causes of death in HCC patients.Therefore,in order to improve the survival rate of HCC patients,it is necessary to better understand the abnormal metabolism of HCC cells.Recent studies have shown that proline metabolism plays an important role in metabolic reprogramming and affects the occurrence and development of cancer.ALDH18A1 is an ATP and NAD（P）H dependent enzyme and a key enzyme involved in proline synthesis,catalyzing the conversion of glutamic acid to δ-pyrrolin-5-carboxylic acid（P5C）,which is up-regulated in various tumor tissues such as liver cancer.Protein acetylation is an important post-translational modification and plays an important role in energy metabolism.However,there are few studies on the regulation of ALDH18A1 acetylation modification and its role in hepatoma cells.PurposeTo study the effects of ALDH18A1 on the proliferation and migration of hepatocellular carcinoma cells.The mechanism of acetylation modification of ALDH18A1 was investigated.To investigate the effect of ALDH18A1 acetylation on the proliferation and migration of hepatocellular carcinoma cells.Method1.The protein and mRNA expression levels of ALDH18A1 in HCC cells were detected by transfection,Western blot and QRT-PCR,and the effects of ALDH18A1 knockdown on the proliferation and migration of HCC cells were detected by CCK8,Wound Healing and Transwell tests.2.Western blot was used to detect the expression of ALDH18A1 in HCC cells treated with Trichostatin A（TSA）and Nicotinamide（NAM）at different concentrations.3.Co-Immunoprecipitation（Co-IP）and Western blot were used to detect the acetylase and deacetylase that regulated ALDH18A1.4.The main functional acetylation sites of ALDH18A1 in hepatocellular carcinoma cells were determined by mass spectrometry and site mutation.5.The effects of SIRT2 knockdown on the proliferation and migration of HCC cells were detected by transfection,CCK8,Wound Healing and Transwell tests.Result1.ALDH18A1 knockdown reduced the proliferation and migration ability of HCC cells and increased apoptosis.2.After NAM treatment,the expression level of ALDH18A1 was up-regulated in HCC cells.3.The results of Co-IP and Western blot showed that acetylase CBP and deacetylase SIRT2 interacted with ALDH18A1.Knockdown SIRT2 or overexpression of acetylase CBP could increase the acetylation level of ALDH18A1.4.The level of ALDH18A1 acetylation was decreased after KR mutation at K311 and K347 sites,and was increased after KQ mutation.After K311/347 dual mutation,the acetylation level of ALDH18A1 was obviously reducted.5.The proliferation and migration capacity of HCC cells were decreased and apoptosis was increased after SIRT2 knockdown.ConclusionKnockdown of ALDH18A1 inhibits the proliferation and migration of HCC cells and promotes the apoptosis of HCC cells.Sirt2-mediated deacetylation of ALDH18A1 at K311 and K347 sites promoted the proliferation and migration of HCC cells and inhibited apoptosis of HCC cells.