The Function And Molecular Mechanism Of ASS1 In Hepatocellular Carcinoma Metastasis

Objective:Primary hepatocellular carcinoma(HCC)is one of the most common malignant tumors in the world which seriously affects human health.However,the 5year survival rate is still low most due to its high recurrence and metastasis.To improve the prognosis of HCC,early prevention of metastasis is particularly important.The aim of this study is to investigate the function and molecular mechanism of ASS1 in regulating metastasis of HCC.Methods : Quantitative Real-time PCR(q RT-PCR),western blotting,and immunohistochemical staining(IHC)were performed to detect expression of ASS1 in HCC tissues.We examined ASS1 expression in an independent cohort with 225 cases of HCC tissues on a tissue microarray(TMA)by IHC to access the prognostic value of ASS1.After treatment with TSA and 5-Aza-Cd R to HCC cells,m RNA levels of ASS1 were detected by q RT-PCR.Pyrosequencing assay on 10 paired DNA samples extracted from HCC and corresponding nontumorous liver tissues was used to observe the effect of DNA methylation on ASS1 expression.Stable ASS1 overexpression cell lines and knockdown cell lines were established.Efficiency of overexpression and knockdown was evaluated by q RT-PCR and western blotting.Transwell arrays were conducted to evaluate migratory and invasive abilities of HCC cells.Tail-vein metastasis models were established to determine the effect of ASS1 on HCC metastasis.Human phosphokinase arrays were used to explore the related signaling pathway.Whole genome human microarray was utilized to identify effective genes which were regulated by ASS1.Results: ASS1 was significantly downregulated in HCC tissues compared to corresponding nontumorous liver tissues.Keplan-Meier analysis indicated that patients with low expression of ASS1 exhibited worse overall survival(OS)and shorter time to recurrence(TTR)than patients with high expression of ASS1.After treatment with various concentrations of TSA and 5-Aza-Cd R to HCC cells,results showed that there was no obvious change with the treatment of TSA.However,m RNA levels of ASS1 were gradually increased with the increase concentration of5-Aza-Cd R.Pyrosequencing assay exhibited methylation ratio of ASS1 was significantly increased in HCC tissues compared to corresponding nontumorous liver tissues.Overexpression of ASS1 significantly inhibited migratory abilities of HCC cell lines in vitro and lung metastasis in vivo.On the contrary,knockdown of ASS1 by sh RNA promoted migration of HCC cells in vitro and lung metastasis in vivo.Human phosphokinase array showed that serine 727 phosphorylation of STAT3 was decreased in ASS1 overexpression cell lines,but increased in ASS1 knockdown cell lines,phosphorylation of STAT3 was essential for ASS1 induced migration and invasion.Furthermore,ID1 was determined as a metastasis related gene regulated by phosphorylation of STAT3 and ASS1 using whole genome human microarray.Conclusion:ASS1 was downregulated in HCC tissues via DNA high methylation and might be served as a potential prognostic marker for HCC patients.ASS1 inhibited tumor progression by suppressing STAT3 and downregulating ID1 in hepatocellular carcinoma.