BAG3 Knockdown Suppresses Stem Cell-like Features Of Pancreatic Ductal Adenocarcinoma Via Translational Suppression Of ISG15

Objective:Pancreatic cancer is a common malignant tumor that is difficult to diagnose and treat.In recent years,the incidence of pancreatic cancer has increased significantly,and its prognosis is very poor.Therefore,the study on the molecular mechanism of pancreatic cancer is helpful to target potential therapeutic targets.BAG3 is a member of the human BAG co-chaperone family(BAG1-6),which has an influence on cell survival,autophagy cytoskeleton arrangement and replication of some viruses.BAG3 is highly expressed in various cancers,and its expression is related to the poor prognosis of cancer such as pancreatic cancer glioblastoma and thyroid cancer.Interferon-stimulated gene ISG15 is present in both free-form and protein-bound forms in cells,and is often highly expressed in a variety of primary human cancers,including pancreatic adenocarcinoma and malignant tumors of the digestive system.It has been reported that intracellular ISG15 can promote the proliferation and migration of hepatocellular carcinoma cells.However,it is not clear whether extracellular ISG15 is a tumor promoter or a tumor suppressor.In pancreatic ductal adenocarcinoma,ISG15 protein secreted by tumor-associated macrophages(TAMs)enhances tumor stem cell phenotype.In breast cancer,extracellular free ISG15 induces an anti-tumor immune response that inhibits the growth of breast cancer.In this study,we found that the protein expression level of ISG15 was down-regulated when BAG3 was missing,while the mRNA level of ISG15 was up-regulated.We also found that BAG3 did not enrich ISG15 mRNA.Both BAG3 and ISG15 deletion inhibit stem cell-like phenotypes.In summary,we continue to explore the specific mechanism of BAG3 and ISG15 on the basis of previous experiments and the effect of BAG3 and ISG15 on the tumor stem cell-like phenotype of pancreatic cancer,so as to provide new therapeutic targets for the treatment of pancreatic cancer.Methods: 1.Pancreatic cancer cell lines with stable expression of BAG3 and ISG15 were constructed.2.mRNA expression levels of ISG15 and BAG3 were detected by RT-PCR.3.Western blot was used to detect changes in protein levels of the target gene.4.Ortho-linkage technology PLA technology detects whether BAG3 interacts with ISG15.5.The clonal formation ability of pancreatic cancer cells with BAG3 or ISG15 deletion was detected by clonal formation assay.6.Actinomycin D was used to detect the stability of the target gene mRNA by time gradient processing.7.mRNA was obtained from pancreatic cancer cells using the kit.8.Spheroid formation ability of pancreatic cancer cells with loss of BAG3 or ISG15 was detected by balloon formation assay.9.The interaction between BAG3 and ISG15 mRNA was detected by rna-binding protein immunoprecipitation.10.Transwell assay was used to detect the migration and invasion ability of pancreatic cancer cells with BAG3 or ISG15 deletion.11.ISG15 extracellular secretion was detected in BAG3-deficient pancreatic cancer cells by Dot Blot assay.12.The influence of BAG3 deletion on tumor formation was detected by tumor formation assay in nude mice.Results:1.BAG3 positively regulates ISG15 expression in pancreatic cancer cell lines.2.Knockout of BAG3 in pancreatic cancer cell lines stabilizes ISG15 mRNA and inhibits its translation.3.Ago2 is implicated in regulation of ISG15 expression by BAG3 deletion in PDACs.4.Knockdown of BAG3 inhibited the stem cell-like phenotype of pancreatic cancer cell lines.5.Knockdown of ISG15 inhibited the stem cell-like phenotype of pancreatic cancer cell lines.6.Forced ISG15 expression rescues BAG3 deletion-mediated supression of stem cell-like phenotypes in PDAC cells.Conclusion: In pancreatic cancer,BAG3 positively regulates ISG15 protein expression and stabilizes ISG15 mRNA.Forced ISG15 expression rescues BAG3 deletion-mediated supression of stem cell-like phenotypes in PDAC cells.