| Objective: Pancreatic ductal adenocarcinoma(PDAC)is a highly aggressive malignancy of the digestive tract.Due to the high incidence of distant metastasis and recurrence,lack of effective treatment,and resistance to most treatments,it is one of the malignancies with the worst prognosis.Cancer stem cells(CSC)are a small group of cells with stem cell properties existing in tumor tissues,with self-renewal,multidirectional differentiation,infinite proliferation and strong tumorigenicity.A large number of studies have shown that cancer stem cells are involved in the occurrence and progression of pancreatic ductal adenocarcinoma,and they are resistant to chemotherapy and radiotherapy,which is an important factor for its poor clinical prognosis.Therefore,it is urgent to seek new ideas for the treatment of pancreatic ductal adenocarcinoma with cancer stem cells to improve prognosis and prolong survival.TRIM29 is one of the members of the TRIM protein family,also known as ATDC,located in the chromosome 11 q23.TRIM protein family is usually defined as E3 ubiquitin ligase because it has a RING,while TRIM29 contains the B1-B2-CC domain,lacks RING rings,so it does not belong to E3 ubiquitin ligase.Recent studies have shown that TRIM29 also has a weak E3 ubiquitin ligase function.The changes of TRIM29 are closely related to tumorigenesis,proliferation,invasion and lymph node metastasis,etc.TRIM29 can promote or inhibit cancer in cancer cells from different tissue sources.TRIM29 plays a role as a carcinogen in pancreatic ductal adenocarcinoma,but the molecular mechanism remains to be further studied.ISG15 is a ubiquitin-like protein with a molecular mass of about 15-k Da,which is produced under the stimulation of type I interferon.The target protein can be covalently modified by an enzyme cascade reaction to exert its biological function.In addition to binding to hundreds of potential target proteins,ISG15 can also exist inside cells in a free form,and can also be secreted from the cell to the outside in a free form.ISG15 and ISGylation involve a variety of key cellular processes,including protein translation,autophagy,DNA repair,and immune regulation,which are involved in the regulation of cancer and immune diseases.In view of the particularity and complexity of ISG15 structure,ISG15 plays a role in promoting or inhibiting cancer in different tumors.Current research reveals that extracellular free ISG15 may serve as a supporting factor for tumor stem cells in pancreatic ductal adenocarcinoma,but the specific mechanism of action of ISG15 and ISGylation is still to be clarified.In previous work,we found that down-regulation of TRIM29 expression could inhibit the cancer stem-cell phenotype of pancreatic ductal adenocarcinoma.Analysis of 72 cases of pancreatic ductal adenocarcinoma tissue samples found that TRIM29 and ISG15 have a significant positive correlation.In pancreatic ductal adenocarcinoma cells,down-regulation of TRIM29 expression will inhibit ISG15 protein expression by promoting ISG15 protein degradation.The specific pathway of ISG15 protein degradation has not yet been reported.Therefore,this study first explored the degradation pathway of ISG15 and found the molecular mechanism that the down-regulation of TRIM29 expression promotes the degradation of ISG15 protein by promoting the expression of CAPN3.This study also found that TRIM29 expression was down-regulated to reduce the autocrine function of ISG15,and extracellular ISG15 maintained the pancreatic ducted adenocarcinoma stem cell like phenotype through autocrine action.In addition,the ISG15,independent of its conjugation function,could restore the inhibition of the stem-cell phenotype of pancreatic ductal adenocarcinoma caused by the down-regulation of TRIM29 expression.Therefore,the purpose of this study is to analyze the specific mechanism of TRIM29 regulating the stemness of pancreatic ductal adenocarcinoma through ISG15,reveal the specific degradation pathway of ISG15 protein,and clarify the role of covalently bound and free ISG15,which is helpful to find new drug targets for the treatment of pancreatic ductal adenocarcinoma,and is expected to provide new ideas and approaches for the treatment of tumor.Methods: 1.The TRIM29 knockout cell lines of PDAC were constructed,and the effects of TRIM29 on cell proliferation,invasion,tumor stem cell formation and in vivo xenograft tumor formation were studied by Edu incorporation,plate cloning,Transwell,balloon formation and tumor formation in nude mice.2.To verify the effect of TRIM29 knockout on m RNA and protein expression levels of ISG15 in PDAC cells by RT-q PCR and Western blot.3.Use Western blot technology to verify the expression of P62,LC3 and other autophagy-related proteins and calpain family members in TRIM29 knockout PDAC cells.Add protein synthesis inhibitor cycloheximide(CHX),proteasome MG132,lysosome Inhibitor E64 D + pepstain A,autophagy inhibitors CQ and Baf A,and calpain inhibitor Z-LLY-FMK were used to detect the expression of ISG15 protein in cells,and the degradation pathway of ISG15 was clarified.3.Plasmids ISG15(WT),ISG15(D76A)and ISG15(E115A)were constructed by pc DNA3.1-FLAG vector,which were transferred to TRIM29 knockout PDAC cells and added to CHX,and the expression of ISG15 protein was detected by Western blot to identify the specific degradation pathway of ISG15.4.Restore the expression of ISG15 or down-regulate the expression of CAPN3 and HERC5 in the TRIM29 knockout cell line of PDAC,and detect the changes of tumor stem cell-like phenotype using clone formation,Transwell,balloon formation,and tumor formation experiments in nude mice..5.The expression level of ISG15 secreted into the medium by TRIM29 knockout cells of PDAC was detected by Dot blot.6.After adding ISG15 neutralizing antibody and recombinant protein,the relationship between extracellular ISG15 and tumor stem cell-like phenotype was studied by Transwell experiment and balloon formation experiment,respectively.Results: 1.TRIM29 knockdown inhibits the cancer stem cell-like phenotypes in PDACs.2.TRIM29 knockdown suppresses ISG15 expression at the posttranslational proteolytic level.3.TRIM29 knockdown promotes the protein degradation of ISG15 by promoting the expression of CAPN3.4.ISG15 rescues stem cell-like phenotypes suppressed by TRIM29 knockdown independent of its conjugation function in PDAC cells.5.CAPN3 knockdown partly rescues stem cell-like phenotypes of PDAC cells suppressed by TRIM29 knockdown.6.TRIM29 knockdown inhibits the pancreatic ductal adenocarcinoma stem cell phenotype by decreasing the autocrine function of ISG15.Conclusion: Loss of TRIM29 can inhibit the pancreatic ductal adenocarcinoma stem cell phenotype accelerates the protein degradation of ISG15 by promoting the expression of CAPN3,and can restore this phenomenon by restoring the expression of free ISG15 independent of its conjugation function,and extracellular ISG15 autocrine maintenance of pancreatic ductal adenocarcinoma stem cell-like phenotype. |
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