The Use Of SFRP4 As A Biomarker In The Pancreatic Ductal Adenocarcinoma

OBJECTIVES: To investigate the aberrant expression of SFRP4 and the correlation between SFRP4 expression and the clinicopathological features and prognosis of PDAC patients.To explore the effect of SFRP4 on the recruitment of Treg cells and its possible molecular mechanism or pathway.METHODS: We utilized gene microarray to analyze the genes differently expressed in PDAC tissue and adjacent non-tumor tissue in Ren Ji cohort of 50 PDAC cases.The screening results were validated by quantitative real-time PCR(qRT-PCR)and immunohistochemistry(IHC).Furthermore,we screened for the extracellular matrix protein differently expressed in PDAC tissue and chose SFRP4 for further study.We confirmed the expression pattern of SFRP4 by analyzing the data from Gene Expression Omnibus(GEO)and testing the expression of SFRP4 at mRNA and protein level by qRT-PCR and IHC.Then we analyzed the relationship between SFRP4 expression and clinicopathological features and prognosis using the data from tissue microarray and blood sample.We also investigated the biological process influenced by Gene Set Enrichment Analysis(GSEA)and then used tissue microarray to confirm the relationship between SFRP4 and FOXP3 positive regulatory T cells(Treg).We performed chemotaxis experiment to make sure that SFRP4 influenced the recruitment of T cells.At last,we used the dual-luciferase reporter system to find the down stream pathway of SFRP4.RESULT: We found 276 genes highly expressed in PDAC tissue(FDR<0.00001,Fold change >3)and 88 genes low expressed in PDAC tissue(FDR<0.00001,Fold change <0.3)from the gene microarray data.We found that 30.8% of the genes highly expressed in PDAC tissue were extracellular matrix proteins including WISP1,CTHRC1,SFRP4 and WNT7 B,which were associated with Wnt signaling.The expression of WISP1 and CTHRC1 were significantly evaluated in PDAC at both mRNA and protein levels by qRT-PCR and IHC.Similarly,SFRP4 was highly expressed in PDAC tissue in both public microarray datasets and Ren Ji cohort.The expression level of SFRP4 in PDAC tissues was prominently correlated with T classification(p=0.017)and SFRP4 expression acted as an independent predictor of PDAC patients' overall survival(HR=1.422,P=0.039).We also found that serum level of SFRP4 was elevated in PDAC patients and Kaplan-Meier survival analysis demonstrated that concentration of SFRP4 at serum level was associated with PDAC patients' overall survival(p=0.046).GSEA showed that the expression of SFRP4 was correlated with migration of lymphocytes,we found the infiltration of Treg cells was increased in the cases with high expression of SFRP4 by the analysis of tissue microarray.SFRP4-siRNA was successfully transfected into SW1990 cell line and the conditional culture median were used to performed the chemotaxis of peripheral blood mononuclear cells.The results suggested that the recruitment of CD4 positive T cells was impaired in the group transfected with SFRP4-siRNA compared to the negative control group.We demonstrated that SFRP4 activate the Wnt/?-catenin pathway by the dual-luciferase reporter system which may explain how SFRP4 influenced the recruitment and differentiation of T cells.CONCLUSION: The high expression of extracellular matrix proteins is a character of PDAC.The expression of SFRP4 was significantly elevated in PDAC and the expression of SFRP4 in PDAC tissues was prominently correlated with T classification and the prognosis of patients.High expression of SFRP4 was associated with advanced progression and poor prognosis.SFRP4 expression status could act as an independent prognostic biomarker among PDAC patients even in the serum sample.SFRP4 is associated with infiltration of Treg cells and it can promote the recruitment of CD4 positive T cell.Wnt signaling promotes the recruitment and differentiation of T cells and SFRP4 may promote the infiltration of Treg cells by activating Wnt/?-catenin pathway.