Mechanism Studied Of Sorafenib Resistance In Hepatocellular Carcinoma Cells Induced By Activation Of Autophagy By Ubiquitin-like Protein FAT10

Background and Objective:Sorafenib,a target and multi kinase inhibitor,is a first-line drug approved by FDA for the treatment of patients with advanced liver cancer.However,sorafenib resistance is the main cause of poor prognosis in patients with advanced liver cancer.Autophagy is a kind of cell death programming different from apoptosis.A large number of studies have confirmed that autophagy is closely related to the drug sensitivity of tumor.Recently,a large number of studies have confirmed that post-translational modification of proteins is a key step in regulating the biological function of proteins,an important molecular basis for protein dynamic balance and interaction,and almost participates in all the physiological processes of cells.FAT10(Human HLA-F adjacent transcript 10)is considered to be the most important protein in post-translational modification.Studies have shown that FAT10 plays a key role in the occurrence and development of a variety of malignant tumors.A large number of previous studies have confirmed that FAT10 is highly expressed in liver cancer.Reducing the expression of FAT10 can significantly increase the proliferation,invasion and metastasis of liver cancer.However,the relationship between FAT10 and autophagy in the drug resistance of liver cancer and the relationship between FAT10 and autophagy is still unclear.The purpose of this study is to observe the relationship between FAT10 and sorafenib resistance by constructing sorafenib resistant hepatoma cell line;to determine whether FAT10 regulates autophagy and then affects sorafenib resistance of hepatoma through in vivo and in vitro experiments;finally to explore the specific molecular biological mechanism of FAT10 regulating autophagy.Methods:1.Firstly,we constructed a solafeni resistant hepatoma cell line,and observed the expression of FAT10 in solafeni resistant hepatoma cells.Further,we stably transfected the shfat10 interference plasmid into solafeni resistant hepatoma cells,anddetected the expression of FAT10 protein in solafeni resistant hepatoma cells by fluorescence quantitative PCR and Western blot The effect of reducing the expression of FAT10 on the sensitivity of sorafenib was observed through the tumor bearing model of nude mice and immunohistochemistry.2.Western blot,immunofluorescence and electron microscopy were used to observe the changes of autophagy level in sorafenib resistant hepatoma cells and the changes of autophagy level in sorafenib resistant hepatoma cells after changing FAT10.At the same time,immunohistochemistry was used to observe the changes of autophagy level in tumor tissues of nude mice.3.We found that the PTEN/AKT signaling pathway of autophagy related signal was significantly inhibited after reducing the expression of FAT10 in sorafenib resistant hepatoma cells by microarray.Further,we used western The changes of key proteins in PTEN/AKT signaling pathway were examined by blot.In order to further observe whether FAT10 affects autophagy through PTEN/AKT signaling pathway,leading to sorafenib resistance of hepatoma cells.4.We explored the specific molecular biological mechanism of FAT10 regulating PTEN/AKT signaling pathway by immunocoprecipitation,in vitro ubiquitination,immunofluorescence and other experiments.Results:1.Through the construction of sorafenib resistant hepatoma cells in vitro,we found that the expression level of FAT10 mRNA and protein increased significantly in sorafenib resistant hepatoma cells.Furthermore,the expression of FAT10 was reduced,and the apoptosis level of drug-resistant cell line was significantly increased.At the same time,the protein of cleved capse3 and cleved PARP in drug-resistant cell line was significantly increased after the expression of FAT10 was reduced.By constructing subcutaneous tumor bearing model in nude mice,it was also found that the tumor volume and tumor weight decreased significantly in the shFAT10-sorafenib group.In addition,TUNEL results also showed that the apoptosis of tumor tissue in shfat10-sorafenib group increased significantly.2.By immunofluorescence and electron microscopy,it was found that the autophagy level increased significantly when sorafenib was added to the cells.Secondly,we reduced the expression of FAT10 in sorafenib resistant hepatoma cells.The results showed that after reducing the expression of FAT10 in sorafenib resistant hepatoma cells,the autophagy level decreased,and the expression of cleaved capse3 and cleaved PARP protein decreased.Immunofluorescence and electron microscopy also showed that reducing the expression of FAT10 in sorafenib resistant hepatoma cells significantly inhibited autophagy and increased the sensitivity of sorafenib.Furthermore,the changes of autophagy level and apoptosis were detected by immunohistochemistry.The results showed that the expression of FAT10 decreased,autophagy expression decreased and apoptosis increased significantly in FAT10 group.FAT10 was reduced and rapamycin was added in sorafenib resistant hepatoma cells.The results showed that rapamycin could block the decrease of autophagy level and the increase of apoptosis related proteins such as cleaved capse3 and cleaved PARP.3.Compared with the parental cells HepG2 sensitive and Huh7 sensitive,the expression of PTEN protein in HepG2 resistance and Huh7 resistance cells decreased,the expression of p-AKT increased,and the total AKT protein did not change,suggesting that PTEN / AKT signaling pathway regulated autophagy involved in the resistance of sorafenib.Secondly,we reduced the expression of FAT10 in sorafenib resistant hepatoma cells.The results showed that after reducing the expression of FAT10 in sorafenib resistant hepatoma cells,PTEN protein decreased,p-AKT,cleaved capse3 and cleaved PARP protein increased,but the total AKT expression did not change and autophagy level decreased.Then,we reduced the expression of FAT10 in sorafenib resistant hepatoma cells,and added AKT signal activator sc79 and sc79 at the same time of reducing FAT10.The results showed that after reducing FAT10 in sorafenib resistant hepatoma cells,PTEN expression decreased,p-AKT expression increased,autophagy level decreased,cleaved capse3 and cleaved PARP protein expression increased.After adding AKT signal activator sc79,the autophagy level of drug-resistant cells increased significantly,and the expression of cleaved capse3 and cleaved PARP protein decreased.Adding AKT signal activator sc79 to shfat10 HepG2 resistance and shfat10 Huh7 resistance can block the decrease of autophagy level and the increase of apoptosis related protein cleaved capse3 and cleaved PARP caused by the decrease of FAT10.4.The results of immunocoprecipitation showed that there was no correlation between FAT10 protein and PTEN protein in solafeni resistant hepatoma cells,but FAT10 protein and the E3 ligase NEDD4 of PTEN protein were combined.The results showed that the expression of NEDD4 protein decreased,PTEN protein increased,p-AKT,cleaved capse3 and cleaved PARP protein decreased,the expression of AKT did not change,autophagy level decreased.The results showed that the expression of NEDD4 protein decreased,PTEN expression increased,p-AKT expression decreased,autophagy level decreased,cleaved capse3 and cleaved PARP protein increased after reducing the expression of FAT10 in the cells.After transfection of his-NEDD4 plasmid,Western blot showed that after increasing the expression of NEDD4 protein in HepG2 resistance,PTEN expression decreased,p-AKT expression increased,autophagy level decreased,cleaved capse3 and cleaved PARP protein expression decreased.Transfection of NEDD4 into shfat10 HepG2 resistance cells blocked the decrease of autophagy level and the increase of apoptosis related proteins cleaved capse3 and cleaved PARP caused by the decrease of FAT10.Finally,we confirmed that FAT10 can stabilize the expression of NEDD4 protein by inhibiting the ubiquitination level of NEDD4 through immunocoprecipitation and in vitro ubiquitination experiments.Conclusion:FAT10 can activate PTEN/AKT signaling pathway to induce autophagy of hepatoma cells by stabilizing the expression of NEDD4,which eventually leads to the resistance of hepatoma cells to sorafenib.