BAG3 Affects The Progression Of Pancreatic Ductal Adenocarcinoma By Reg?lating The Stability Of Expression Of Interleukin Family Member IL6 And IL8 MRNA

Objective: As a malignant gastrointestinal tumor,about 90% pancreatic cancer originates from the ductal adenocarcinoma of the ductal epithelium,its morbidity and mortality have increased significantly in recent years,and the five-year survival rate is less than 5%,which is one of the worst prognosis of malignant tumors.The poor prognosis is mainly characterized by higher metastatic tendency and high resistance to radiotherapy and chemotherapy.As a lymphokine that interacts between leukocytes and immune cells,Interleukin plays an important role in mediating T,B cell activation,proliferation and differentiation,and inflammatory responses.A growing of evidence confirms that interleukins have become the focus of research on tumor cells.At least 38 interleukins have been identified,named IL1~IL38 so far.Many articles have shown that IL6,IL8 of the interleukin and pancreatic cancer family are closely related,which provides a new idea for the early diagnosis and treatment of pancreatic cancer.Researches have shown that IL6 binds to its receptor and activates the JAK tyrosine kinase family members.Activation of JAK can stim?late the activation of various pathways,such as MAPKs,PI3 Ks,and STATs,which are the main pathways involved in cell proliferation,survival and metabolic regulation,and interstitial fibrosis.It has been found that IL6 has overexpression in a variety of tumors and plays an important role in the occurrence and development of tumors.The latest research confirms that chronic inflammation plays an important role in the process of tumor formation.The tumor-associated inflammatory factor IL6 is highly expressed in the microenvironment of malignant tumors,which can increase the invasive ability of tumors,promote distant metastasis of tumors and angiogenesis and participate in the formation of tumor resistance,etc.Therefore,in-depth study of the mechanism of IL6 acting on malignant tumors can help reveal the mocular basis of the interconnection between microenvironment and solid tumors,and lay a foundation for targeting the cancer-associated inflammatory factor IL-6 in the tumor microenvironment.IL8 is the first chemokine to be discovered and plays a key role in the inflammatory process.With the in-depth study of mechanism,it was found that IL8 also has mitogenic and angiogenic effects,and IL8 regulates cell proliferation,migration and invasion in cancer by activating CXC motif chemokine receptor 1(CXCR1)and CXCR2(two cell surface G protein-coupled receptors).Clinical studies have shown that IL8 is consistently highly expressed in patients with PDAC,and its up-regulation in pancreatic cancer is associated with increased cancer metastatic potential and overall prognosis.The latest research also confirms that IL8 also plays a role in reg?lating the tumor microenvironment.The IL8-CXCR1 signaling pathway activates the self-renewal and regeneration of cancer stem cells.BAG3 is an important member of the Bcl-2 related anti-apoptotic protein family.Initially,the origin of name of the BAG family is they were found to bind to the anti-apoptotic protein Bcl-2 and promote cell survival.The BAG3 protein is mainly distributed in the cytoplasm,which contains the WW domain,a BAG domain that interacts with Bcl-2 and chaperone heat shock protein(HSP)family members,And a proline-rich region with m?ltiple PXXP motifs(P stands for proline and X stands for any amino acid residue).Because of the conserved BAG domain at the C-terminus of the BAG protein family,it interacts with the heat shock protein HSP70/HCP70,positively and negatively regulates the function of such chaperones,and produces a wide range of cell?lar biological effects.Therefore,BAG family proteins are also recognized as co-chaperones.Although with high expression levels in heart and skeletal muscle but also in the brain and the peripheral nervous system,BAG3 expression is weak or not expressed in other normal tissues;In some tumor cells,such as leukemia,solid tumor cells,BAG3 expression level is higher,maintaining the survival of tumor cells.The preliminary work of this experiment showed that BAG3 can regulate the progression of pancreatic cancer,and found that the expression of IL6 and IL8 of the interleukin family was down-regulated with the down-regulation of BAG3.BAG3 is an RNA-binding protein related to post-transcriptional regulation closely.In view of the importance of tumor microenvironment the treatment of cancer and post-transcriptional regulation in gene expression,this study aims to clarify the regulation of BAG3 on the progression of pancreatic cancer and further explore the specific mechanism of BAG3 reg?lating IL6 and IL8 expression at post-transcriptional level.Methods: Pancreatic cancer cells and pancreatic stellate cells were c?ltured,and SW1990 and BxPC3 cell lines stably knocking out BAG3 and empty vector were constructed by lentivirus infection.1.The expression level of the target mRNA was detected by qRT-PCR.2.Westernblot detected the expression level of the target protein.3.Elisa detected the release of relevant factors in the supernatant.4.EdU incorporation detected cell proliferation;Transwell detected cell migration and invasion.5.Tissue microarray for BAG3-specific immunohistochemical staining,the correlation between BAG3 expression and cell fibrosis was analyzed.6.The expression of the nascent mRNA is detected by the nascent RNA capture kit;the half-life of the target mRNA is detected by qRT-PCR using a synthetic inhibitor of RNA.7.Dual luciferase reporter system detected the activity of a promoter or other element.8.The interaction between RNA and protein was detected by RNA binding protein immunoprecipitation and biotin pull down assay.9.Immunnoprecipitation detected the interaction between the related proteins.Results: 1.BAG3 knockdown reduced the production of IL6 in PDAC and decreased the activation of HPanSteC by PDAC.2.BAG3 knockdown reduced the activation of HPanSteC by inhibiting the production of IL6.3.BAG3 knockdown in PDAC relies on Ago2 to regulate the destabilization of IL6 mRNA through 3'UTR.4.BAG3 knockdown promotes recruitment to IL6 mRNA by increasing phosphorylation of Ago2 Ser387.5.BAG3 knockdown inhibits the migration and invasion of PDAC by reducing the production of IL8.6.BAG3 knockdown regulates the stability of IL8 mRNA through its 3'UTR.7.BAG3 knockdown regulates the stability of IL8 mRNA by increasing the phosphorylation of HuR Ser202.8.The miRISC is involved in the de-stabilization of IL8 mRNA by BAG3 knockdown in PDAC.9.The miRISC and HuR are involved in the process by which BAG3 knockdown regulates the stability of IL8 mRNA together.10.In PDAC,miR-4312 competes with HuR for IL8 mRNA interaction and inhibits IL8 expression by BAG3 knockdown.11.There was a positive correlation between BAG3 and IL8 expression in pancreatic cancer tissue samples.Conclusion: 1.In PDAC,BAG3 inhibits the release of IL6 mRNA by increasing phosphorylation at S387 of Ago2,thereby promoting the activation of PSC and affecting the development of pancreatic ductal adenocarcinoma.2.In PDAC,BAG3 knockdown inhibits its binding to IL8 mRNA by promoting phosphorylation of HuR Ser202,and up-regulates Ago2 phosphorylation,promotes binding of IL8 to miRISC containing miRNA-4312,and promotes degradation of IL8,thereby reducing the migration and invasion of PDAC.