Expression Of NAT10 In Endoplasmic Reticulum Stress Of Liver Cancer And Its Effect On Sorafenib Sensitivity

ObjectiveHepatocellular carcinoma（HCC）is a serious threat to human health due to its high degree of malignancy,morbidity and mortality.Targeted therapy is an extremely important means for patients with advanced HCC,and Sorafenib,as one of the few multi-target drugs in HCC targeted therapy,has been widely used in the first-line treatment of advanced HCC.It can affect the growth of tumor cells by inhibiting RAF/MEK/ERK signaling pathways and VEGF and PDGF receptors.However,there is a high level of drug resistance in the clinical practice of sorafenib,the cause of which remains to be determined,and it has been reported that it may be related to stress conditions such as autophagy and hypoxia.Endoplasmic reticulum stress（ERS）is a common stress response in cells.Appropriate ERS can play a protective role,and extreme ERS can independently induce apoptosis.HCC patients are mainly developed from viral hepatitis and alcoholic liver disease.Physiological environment such as viral infection and insufficient blood supply can easily induce er stress in liver cells.However,in HCC patients,the biological behavior of HCC cells is active,and energy deficiency,oxidative stress and Ca2+ homeostasis loss also exacerbate er stress.This process may be involved in the emergence of drug resistance.NAT10,full name of N-acetyltransferase,is located in p13 region on chromosome 11.It is the only protein encoding both acetylase domain and RNA binding domain,and is considered as RNA ac4 C modification enzyme.It is involved in histone acetylation,t RNA acetylation and18 S r RNA biosynthesis.Subsequent studies have found that NAT10 may play the role of oncogene by affecting m RNA ac4 C modification,but the specific mechanism of action remains unclear.The purpose of this study is to analyze the expression of NAT10 gene in ERS HCC cells,and to compare the inhibition of NAT10 gene in ERS HCC cells by Si-RNA and Remodelin.At the same time,we examine the rate of cell apoptosis between the experimental group and the control group after sorafenib treatment by flow cytometry,which means to evaluate the sensitivity of ERS HCC to sorafenib treatment.Methods1.Tissue wax blocks were collected from patients diagnosed with HCC in our h ospital to improve immunohistochemistry and understand the expression rate of N AT10;2.Tunicamycin（TM）was added into hepatoma cell line Hep G2 and Huh7,and the expression of GRP78 was observed by western blot to determine whether the cells induced ERS;3.Hepatoma cell lines Hep G2 and Huh7 were divided into 6 groups respectively.The first group was used as the control group,and TM was added into the other 5 groups,and the concentration of TM was 0.3125,0.625,1.25,2.5,5μM,respectively.Western blot was used to detect the expression of NAT10 gene;4.si-RNA was added into ERS treated hepatoma cell lines Hep G2 and Huh7 respectively,and Western blot was used to detect whether the expression of NAT10 gene was inhibited,and the optimal fragment was selected by repeated experiments;5.Through the preliminary experiment,and the western blot experiment this time,the cell experiment is divided into experimental group and Control group,the experimental group is Remodelin group and Si-NAT10 group,and the Control group is Control group and Si-NC group.The expression of NAT10 was inhibited by SI-RNA knockout or Remodelin,and then sorafenib was added.Apoptosis of HCC cells was detected by flow cytometry.;6.The above experimental results were recorded.SPSS 25.0 software was used for statistical analysis,and Graphpad and other software were used to draw graphs.Results1.Immunohistochemical analysis showed that the expression rate of NAT10 in HCC tissues was 87.1%.2.Western blot test showed that TM can effectively induce ERS in HCC cells,and the expression of NAT10 gene was significantly improved in the liver cancer cell lines Hep G2 and Huh7 after ERS treatment,and the Remodelin or Si-RNA can effectively inhibit the expression of NAT10（P<0.05）.3.Flow cytometry showed that in Hep G2 cells,the apoptosis rates of the Control group,the Remodelin group,the Si-NC group and the Si-NAT10 group were 11.79%,51.37%,12.63% and 29.32%.In Huh7 cells,the apoptosis rates of the control group,the Remodelin group,the Si-NC group and the Si-NAT10 group were 13.38%,38.71%,25.68% and 32.74%.The sensitivity of cells to sorafenib increased（P<0.05）.Conclusions1.In the selected hepatocellular carcinoma tissues,immunohistochemistry showed a high positive expression rate of NAT10（87.1%）.2.The expression of NAT10 gene was significantly up-regulated in ERS induced hepatocellular carcinoma.3.In hepatocellular carcinoma with ERS,inhibition of NAT10 gene expression can enhance the sensitivity of hepatocellular carcinoma cells to Sorafenib,resulting in an increased apoptosis rate of Sorafenib mediated HCC cells.