N-acetyltransferase 10 Expression In Hepatocellular Carcinoma And The Role In Epithelial-mesenchymal Transformation

Background:Hepatocellular carcinoma(HCC)is one of the malignant tumors which threaten human health today.The disease incidence ranks at 6th worldwide,and in China HCC has been the second malignant tumor.Every year more than half of million patients are diagnosed with HCC,and the disease occurrence is still increasing.In recent years,with the continuous progress of liver cancer diagnosis and treatment technology,surgical resection-based treatment strategy largely improves the survival rate of patients.Especially the emergence of liver transplantation provides a thorough cure possibility.However,80%to 90%of patients diagnosed with HCC already have intrahepatic or extrahepatic metastases,and the surgical resection rate becomes very low.50%of HCC patients show metastasis after hepatectomy resection,resulting in poor life quality and prognosis,recurrence and metastasis is still the main cause of death.Tumor metastasis is a complex process involving multiple gene changes,in which epithelial-mesenchymal transition(EMT)plays a key role in this process.Present studies show that EMT can promote the invasion and metastasis of HCC and has close relationship with prognosis and treatment.Therefore,it is of great clinical significance to study the molecular regulation mechanism of EMT in hepatocellular carcinoma cells.Objectives:The aim of this study was to investigate the expression of N-acetyltransferase 10(NAT10)in HCC tissues and paired adjacent normal tissues,and to analyze the correlation of NAT10 protein expression and the clinicopathological parameters with the HCC prognosis.Moreover,by inhibiting expression of NAT 10 in HCC cell line,to study the effect of NAT10 on the proliferation and migration of HCC cells as well as molecular phenotype changes of EMT.Last but not least,to investigate the role of NAT10 in the EMT of HCC.Methods:1.Collect HCC and paired-adjacent normal tissue from 186 HCC patients,and use immunohistochemical staining(IHC)and real-time fluorescent quantitative PCR(qRT-PCR)to study the expression of NAT 10 in these tissues.2.According to the results of immunohistochemistry,to analyze the relationship between NAT10 and clinical and pathological parameters as well as the prognosis.3.By in vitro transfection of HCC cell lines with Hep3B,Huh7,SNU387,SNU449(NAT10-siRNA)and NAT 10 small molecule inhibitor Remodelin,use the Transwell cell invasion and cell scratch healing methods to test the effect of NAT 10 on the HCC invasion and migration ability.At the same time,detect changes of the E-carherin and vimentin of EMT.4.By create hypoxia situation to induce the EMT of E-cadherin marked HCC cell lines Hep3B?Huh7,and use NAT10-siRNA and Remodelin to down-regulate the NAT10 expression in hypoxia-induced cell lines,to study the effect of NAT10 on cell invasion and migration and changes of the E-carherin and vimentin of EMT.5.Use Remodelin to down-regulate the NAT10 expression in Huh7 cells,by observing tumorigenesis in nude mice to investigate the tumorigenesis effect of NAT 10 in the HCC cells,and by immunohistochemical staining to study changes of tumor cell proliferation index of Ki-67 and EMT molecular phenotypic.Results:1.Immunohistochemistry and real-time fluorescent quantitative PCR results revealed that the expression of NAT 10 protein and NAT 10 mRNA in the HCC was significantly higher than the paired-adjacent normal tissue(P<0.01).IHC results additionally showed that NAT 10 expression was significantly correlated with HCC histological differentiation(P<0.01)and TNM stages(P<0.01),but not significantly correlated with gender,age,tumor size and liver cirrhosis.2.Survival analysis showed the overall survival and disease-free survival of patient group with high NAT10 expression was significantly lower than the low NAT10 expression patient group(P =0.006 and P =0.009).Multivariate COX regression curve demonstrated that both TNM stages(HR= 1.523;95%CI=1.102-2.103,P =0.026)and NAT10 expression(HR=2.469;95%CI=1.563-3.788,P =0.005)can be the independent risk factor for evaluating the prognosis of HCC.3.Under normoxia conditions,Transwell invasion experiments and scratch experiments showed that interfering with NAT 10 expression could inhibit the invasion and migration of HCC cells.Western blot showed that the expression of E-cadherin was up-regulated and the expression of vimentin was decreased after down-regulation of NAT 10 expression.4.Under hypoxia conditions,Transwell invasion experiments and scratch experiments showed that inhibition of NAT 10 activity could reverse enhanced biological effects of the hypoxia-induced cell invasion and migration.Western blot results also revealed that hypoxia could induce EMT in HCC cells,and by inhibiting the NAT10 activity the EMT phenotype changes could be reversed.5.In vivo tumorigenesis in nude mice showed that interfering with the expression of NAT10 could attenuate the tumorigenic ability of HCC cells,Immunohistochemistry results showed that interfering with NAT 10 expression in vivo could reduce the expression of ki-67,the expression of E-cadherin was increased,and the expression of vimentin decreased.Conclusion:1.The expression of NAT 10 in the HCC tissue was significantly higher than the paired-adjacent normal tissue,suggesting that NAT10 may be correlated with HCC occurrence and progressing.2.HCC patients with high NAT10 expression had poorer prognosis compared to those with low NAT 10 expression,suggesting NAT 10 can be a promising marker for evaluating HCC prognosis.3.Inhibition of NAT10 activity could reduce the proliferation and metastasis ability of HCC cells.4.NAT 10 worked mainly by regulating EMT thereby promoting HCC progression and metastasis.