The Expression Of BAG3 In Pancreatic Stellate Cell Promotes Migration And Invasion Of Pancreatic Ductal Adenocarcinoma

Objective: Pancreatic cancer is a malignant tumor with a high degree of malignancy.Approximately,90 percent of the pathological type of pancreatic cancer is pancreatic ductal adenocarcinoma(PDAC)originating from ductal epithelium.With rapid clinical progress and insensitivity to radiotherapy and chemotherapy,the 5-year survival rate of PDAC is less than 5%,making it the fourth leading cause of cancer death in the United States.To study the molecular mechanism of PDAC progression can provide new targets for the treatment of PDAC.The most typical histological feature of PDAC is desmoplasia in its stroma,which mainly consists of pancreatic stellate cells(PSCs),immune cells,lymphocytes and vascular endothelial cells.There are complex interactions between stromal cells and tumor cells.On one hand,tumor cells secrete pro-inflammatory cytokines that activate PSCs.On the other hand,activated PSCs secrete abundant extracellular matrix(ECM)proteins and signaling molecules to remodel tumor microenvironment.PSCs can enhance the proliferation,invasion and drug resistance of PDAC cells,but some studies showed that PSCs have a protective effect in PDAC.Therefore,the role of PSCs in the development of PDAC is still unclear.Bcl-2 associated athanogene(BAG)is a kind of evolutionarily conserved antiapoptotic proteins,BAG3 is a member of the co-chaperones family with different domain.The intracellular expression of BAG3 can be induced by stress stimulation,while is constitutive in skeletal muscle,cardiac myocytes and several tumour types,such as breast cancer,thyroid cancer and PDAC.Its high expression related to tumor progression and poor prognosis.However,the function of BAG3 in the regulation of stromal microenvironment in PDAC remains undefined.In our previous study,we found that BAG3 has a clear regulatory effect on the proliferation of PDAC cells and promotes the interstitial fibrosis of PDAC.Previous researches mainly emphasized the oncogenic effect of BAG3 overexpression in tumor cells,while the stroma,which accounts for 50%~80% of PDAC tissue,has attracted more and more attention.PSCs in stroma are the main effector cells of fibrosis.This paper aims to study the effect of BAG3 positive PSCs in the microenvironment remodeling and tumor progression of PDAC.Methods:Ⅰ.The BAG3 and α-SMA expression of PSCs in 30 cases of PDAC patients were analyzed by immunohistochemical staining with tissue sections.Ⅱ.PSCs were cultured with the stimulation of different cytokines.Changes in BAG3 protein level were detected by Western blot,and total m RNA expression of BAG3 was detected by q RT-PCR.Ⅲ.PSCs with stable BAG3 knockdown were constructed by lentivirus infection.BAG3 and α-SMA protein levels were detected by Western blot.Ⅳ.PSCs with stable overexpression of BAG3 were constructed by lentivirus infection.1.RTCA and Ed U assay were used to detect the effect of BAG3 on the proliferation of PSCs.2.RTCA and Transwell assays were measured to detect the effect of BAG3 on the migration ability of PSCs.Ⅴ.PDAC cells Bx PC3,SW1990 and PSCs were cultured in the conditioned medium(CM)of PSCs with BAG3 overexpression.1.The effect of CM on the proliferation of PDAC cells was detected by Ed U assay.2.The effect of CM on the migration and invasion ability of PDAC cells were evaluated by Transwell.3.The effect of CM on the proliferation of PSCs was detected by Ed U assay.4.The effect of CM on the migration ability of PSCs was evaluated by Transwell.5.Cytokine antibody microarray,ELISA and DOT BLOT were used to analyze the protein secretion changes of PSCs with BAG3 overexpression.6.The m RNA levels of the cytokines in PSCs with BAG3 overexpression were detected by q RT-PCR.7.Neutralizing antibody or recombinant protein was added to the CM,Ed U assay was used to detect the proliferation ability of PSCs with BAG3 overexpression,Transwell was used to detect the migration ability of PSCs with BAG3 overexpression and the invasion ability of PDAC cells.Results: 1.The expression of BAG3 in activated PSCs was up-regulated.2.BAG3 overexpression promoted the activation,proliferation and migration of PSCs.3.CM of PSCs with BAG3 overexpression promoted the migration and invasion of PDAC cells.4.CM of PSCs with BAG3 overexpression promoted the proliferation and migration of PSCs.5.IL-6,IL-8,MCP1,TGF-β2 and IGFBP2 were significantly increased in the CM of PSCs with BAG3 overexpression,while CXCL6 was decreased.6.The neutralizing antibodies of IL-6 and IGFBP2 inhibit the proliferation of BAG3 positive PSCs.7.The neutralizing antibodies of IL-6,TGF-β2 and IGFBP2 inhibit the migration of BAG3 positive PSCs.8.The neutralizing antibodies of IL-6,MCP1,TGF-β2 and IGFBP2 inhibit the invasion of PDAC cells.Conclusion: In PDAC,BAG3 is expressed in activated PSCs,participates in the activation of PSCs and promotes the proliferation and migration of PSCs.The expression of BAG3 in PSCs maintains the activation of PSCs by promoting the autocrine of IL-6,TGF-β2 and IGFBP2,and promotes the invasion of pancreatic cancer cells by paracrine of IL-8,MCP1,TGF-β2 and IGFBP2.