The Effect And Mechanism Of FAT10 Regulates Ubiquitination And Degradation Of FOXM1 On Epithelial Mesenchymal Transition (EMT) Of Pancreatic Cancer

Background and purpose:Pancreatic cancer(PC)is currently the fourteenth most common cancer in the world,and the seventh leading cause of cancer-related death,and the incidence rate of PC is increasing year by year.More susceptible to early,rapid metastasis and more aggressive invasive mortality,and the resistance of PC to chemotherapy and radiotherapy are all important factors that hinder the treatment of PC patients and improve their prognosis.Epithelial-mesenchymal transition(EMT)is considered as a potential mechanism for tumor progression,malignant invasion,metastasis,and resistance to radiotherapy and chemotherapy in PC.Forkhead box M1(FOXM1)can also promote tumor progression and chemoresistance by regulating EMT,and FOXM1 protein can be effectively degraded by ubiquitin-proteasome system in tumor cells.Ubiquitin like protein FAT10(FAT10)has a similar structure and function to ubiquitin(Ub),which can lead to proteasome degradation of its conjugate,and studies have proved that FAT10 can participate in the regulation of EMT in a variety of tumors,and it can also stabilize the expression of a substrate protein by inhibiting the ubiquitination and degradation of its substrate protein in tumors.The purpose of this study was to investigate the expression of FAT10 and FOXM1 in PC,non-tumor tissues,and PC cells,and to analyze their correlation and clinical significance.To further investigate the effect of FAT10 on the expression of FOXM1 to EMT in PC and its specific molecular mechanism.Methods:1.Bioinformatics analysis was used to analyze the expression and correlation of FAT10 and FOXM1 in PC tissues and non-tumor tissues;2.PC tissues and non-tumor tissues were collected from 89 patients with complete clinical pathology and follow-up data.Immunohistochemical staining was used to detect the expression of FAT10 and FOXM1 in tissues,and the correlation between FAT10 and FOXM1 expression was analyzed;3.Western blot was used to detect the expression of FAT10 and FOXM1 in tumor tissues of 40 PC cases,and the expressions of FAT10 and FOXM1 in various PC cell lines were detected.The correlation between FAT10 and FOXM1 expression was analyzed;4.The best sh-FAT10 interference plasmid was constructed and screened,and HA-FAT10 plasmid was constructed;sh-FAT10 and HA-FAT10 plasmids were transfected into PC cells to detect the expression of FAT10,FOXM1,and EMT marker proteins;A stable PC cell line with stable down-regulation of FAT10 was used to establish subcutaneous xenograft mouse model of PC,and the expression of FAT10,FOXM1 and EMT markers in tumor tissues were detected;5.sh-FOXM1 and Flag-FOXM1 plasmids were constructed and transfected into PC cells to detect the expression of FOXM1 and EMT marker proteins in PC cells.In addition,Flag-FOXM1 was transfected into PC cells with stable down-regulation of FAT10 to detect the expression of FAT10,FOXM1 and EMT marker proteins in PC cells.Sh-FOXM1 was transfected into PC cells with stable up-regulation of FAT10 to detect the expression of FAT10,FOXM1,and EMT marker proteins in PC cells;6.The specific molecular mechanism of FAT10 regulating FOXM1 expression was clarified by co-immunoprecipitation(CO-IP),and in vitro ubiquitination experiment.Results:1.Bioinformatics analysis showed that the expression of FAT10 and FOXM1 in PC was significantly higher than that in non-tumor tissues,and FAT10 was positively correlated with the expression of FOXM1 in PC tissues.The high-expression rate of FAT10 in 89 PC cases was 60.7%,the high-expression rate of FOXM1 was68.5%,and there was a positive correlation between them.Western blot analysis results showed that the expression of FAT10 and FOXM1 protein in cancer tissues was significantly higher than that in non-tumor tissues,and FAT10 was positively correlated with FOXM1,and the expression of FAT10 and FOXM1 in four PC cell lines was significantly higher than that in normal pancreatic ductal cells.2.The expression of FAT10 was associated with TNM stage and vascular invasion in PC patients,while the expression of FOXM1 correlated with lymph node metastasis.The results of survival analysis showed that the overall survival of PC patients in the FAT10 high expression group and FOXM1 high expression group was significantly shorter than that in low expression groups,and the overall survival of FAT10(High)FOXM1(High)group was the shortest;TNM stage,vascular invasion and the expression of FAT10 are independent prognostic factors for PC.3.Compared with the control group,the expression of FAT10 in PC cells with stable down-regulation of FAT10 decreased significantly,while the expression of FOXM1 decreased,and the expression of E-cadherin increased significantly,while the expression of N-cadherin and Vimentin decreased significantly.In the PC cells with stable up-regulation of FAT10,the expression of FAT10 increased significantly,the expression of FOXM1 increased,and the expression of E-cadherin decreased significantly,while the expression of N-cadherin and Vimentin increased significantly.4.In sh-FOXM1 transfected PC cells,the expression of FOXM1 protein decreased significantly,the expression of E-cadherin increased,and the expression of N-cadherin and Vimentin decreased.In Flag-FOXM1 transfected PC cells,the expression of FOXM1 protein increased significantly,the expression of E-cadherin decreased,and the expression of N-cadherin and Vimentin increased.5.The tumor volume of subcutaneous xenograft mice with stable down-regulation of FAT10 was significantly smaller than that of the control group,and the expression of FAT10 protein decreased significantly,while the expression of FOXM1 decreased,the expression of E-cadherin increased,and the expression of N-cadherin and FOXM1 decreased in tumor tissue.6.The expression of FAT10 protein increased in PC cells with stable up-regulation of FAT10,while the expression of FOXM1 increased.However,after transfection of sh-FOXM1 into the cells,the expression of FOXM1 decreased,the expression of E-cadherin increased,and the expression of N-cadherin,and Vimentin decreased.The expression of FAT10 protein decreased in PC cells with stable down-regulation of FAT10,the expression of FOXM1 decreased,however,after transfection of Flag-FOXM1 into the cells,the expression of FOXM1 increased,the expression of E-cadherin decreased,and the expression of N-cadherin and Vimentin increased.7.The results of CO-IP showed that FOXM1 can bind Ub,and FOXM1 was degraded by ubiquitin proteasome in PC cells.FOXM1 can bind FAT10;FAT10inhibits the ubiquitination and degradation of FOXM1 by competing with Ub to bind FOXM1.Conclusion:FAT10 inhibits ubiquitination degradation,and stabilize its expression of FOXM1 by competing with Ub to bind FOXM1,and ultimately promotes EMT of PC.