The Effect And Mechanism Of YAP1 On The Activation And Paracrine Of Pancreatic Astrocytes

Background and objective:The incidence and mortality of pancreatic ductal adenocarcinoma(PDAC)in the population increase year by year.At present,the treatment method for this tumor is limited.Pharmacological treatment is not effective enough and need breakthrough.Severe desmoplasia is a characteristic histological manifestation of PD AC.It constitutes a microenvironmental physical barrier that prevents drugs from targeting tumor cells.Remission of PD AC desmoplasia can disrupt this physical barrier and increase the efficiency of drug treatment.Activated pancreatic stellate cells(PSCs)and a large number of extracellular matrix(ECM)secreted by them participate in the formation of PDAC desmoplasia.In addition,tumor and tumor microenvironment dependent on each other,and promote each other,and they also restrict and antagonize each other.The tumor microenvironment can influence the occurrence and development of tumors through secretion,metabolism,and immunity.PSCs,as the main component of the PDAC microenvironment,have a strong secretory function,and their effects on PCC growth cannot be ignored.Yes-associated protein 1(YAP1)plays an important role in the regulation of hepatic stellate cells and breast fibroblasts,whereas its role in PSCs is unknown.This experiment was aimed to investigate the effect of YAP 1 on PSC activation and paracrine,and its underlying mechanisms.Methods:Primary PSCs were obtained from fresh surgically resected PDAC tissues cultured in vitro.PSCs were treated with all-transretinoic acid(ATRA)to acquire a lower activation level.The phenotype of PSCs was verified by oil red O staining and alpha smooth muscle actin(a-SMA)immunofluorescence staining.The expression levels of YAP1 in control and ATRA-treated PSCs were evaluated by western blotting and immunofluorescence staining,respectively.After transfected with si-RNA or incubated with YAP1 inhibitor VP(verteporfin),the expression levels of a-SMA and collagen I,which were PSCs activation-related molecules,were detected by western blotting and real-time quantitative PCR.YAP1 expression was inhibited by si-RNA,then the morphology of the cells was observed under an inverted microscope.The CCK-8 cell proliferation assay,flow cytometry,and collagen contraction assay were used to test changes in cell proliferation,cell cycle,apoptosis,and cell contractility.The ECM genes regulated by YAP1 were screened by qRT-PCR microarray.And its expression and secretion were verified by qRT-PCR,western blotting and ELISA in three separate PSC preparations.Bisulfite sequencing(BSP),bioinformatics analysis,co-immunoprecipitation(Co-IP),chromatin immunoprecipitation(CHIP)and luciferase reporter assays were used to explore the mechanisms included in how YAP1 regulates the ECM gene.After treatment,the effect of PSCs on PCC proliferation was tested by incubation with PSCs conditioned medium or transwell co-culture system.The expressions of YAP 1 and screened cytokine proteins in PD AC stromal cells were evaluated by making PDAC tissue microarrays(TMA)and immunohistochemistry(IHC)staining.The relationship between their expression levels and clinicopathological features was analyzed.Results:After 5-day tissue culture,the PSCs began to be isolated out of PDAC.The PSCs were spindle-shaped with sharp peaks,negative for oil red O staining,and strongly positive for a-SMA immunofluorescence staining.After treatment with ATRA,PSCs became squat triangles,positive for oil red O staining,and weakly positive for a-SMA immunofluorescence staining.Western blotting and immunofluorescence staining showed that YAP1 expression in the nucleus of PSCs was significantly reduced in ATRA-treatment group,compared with control group.Inhibiting the expression of YAP1 in PSCs could down-regulate the expression of a-SMA and collagen I,change the morphology of spindle cells,inhibite cell proliferation,arrest the cell cycle in G2 phase,promote cell apoptosis,and inhibit cell contraction.PCR microarray analysis revealed that one of the most prominent ECM genes regulated by YAP1 is secreted protein acidic and cysteine rich(SPARC)(fold change of 13.3).The results of qRT-PCR,western blotting and ELISA showed that knockdown of YAP1 resulted in decreased mRNA expression,protein expression,and secretion of SPARC.Overexpression of YAP1 led to increased SPARC mRNA expression,protein expression,and secretion.BSP assay results showed YAP1 knockdown had no effect on the methylation level of SPARC promoter in PSCs.Bioinformatics analysis predicts that transcription factor RUNX1 mediates the regulation of YAP1 on SPARC.Co-IP experiments demonstrated the interaction between YAP1 protein and transcription factor RUNX1.Western blotting showed YAP1 knockdown up-regulated RUNX1 expression.CHIP,luciferase reporter assays,and western blotting demonstrated that RUNX1 inhibited SPARC expression by binding to the SPARC promoter.YAP1 knockdown in PSCs promotes the PCC proliferation.Further overexpressing SPARC in PSCs will inhibit the promotion.The overexpression of YAP1 in PSCs inhibits the PCCs proliferation.Further knocking down SPARC in PSCs will destroy the inhibition.It consistented with the results analyzed by insert co-culture system.Based on IHC analysis,the expression level of YAP1 and SPARC protein in PD AC stromal cells was significantly higher than that in para-PDAC stromal cells(P<0.05).There was a significant correlation between the expression level of YAP 1 and SPARC in stromal cells of PDAC(r = 0.433,P<0.001).The expression level of YAP1 in stromal cells of PDAC was significantly correlated with the degree of fibrosis(r = 0.543,P<0.001).High-level expression of SPARC in stromal cells was an independent poor prognostic factor for PDAC overall survival(HR = 3.216,P = 0.005).Conclusion:YAP1 is highly expressed in activated PSCs.And inhibition of YAP 1 expression can lead to PSC inactivation.In PSCs,YAP1 positively regulates SPARC expression and secretion via RUNX1.Activated PSCs inhibit PCC proliferation by SPARC paracrine.This study provide new therapeutic insights in PDAC desmoplasia remission The interaction of PSCs-PCCs is analyzed from the perspective of secretion.And the effects of changes in tumor microenvironment on tumor growth are further elucidated.