MBD3 Inhibits Stemness Of Pancreatic Cancer Cells Via Hippo Signaling

Objective:In this study,we explored the effects of Methyl-CpG-binding domain 3(MBD3)on the abilities of proliferation and stemness in pancreatic cancer cells.Moreover,we clarified the underlying molecular mechanism of the effects of MBD3 on the stemness of pancreatic cancer cells.This study might offer a theoretical basis for diagnostics and prognosis of pancreatic cancer.Methods:irstly,the expression of MBD3 was detected by fluorescence quantitative PCR and Western blot in pancreatic cancer cells,and the downregulation and overexpression of sh-mbd3 and flag-mbd3 plasmids were verified.Then,the clone formation experiment,sphere formation assay and animal experiments were done to detect the effects of MBD3 on the proliferation and stemness in pancreatic cancer cells.Western blot assay was used to examine the effects of MBD3 on the stemness markers and the Hippo/YAP signal relative protein expression in pancreatic cancer cells.The relationship was analyzed between the expression levels of MBD3 and YAP in TCGA database,and the effect of MBD3 on YAP was analyzed by immunoprecipitation(IP)experiment,immunofluorescence assay and nuclear and cytoplasmic extraction.Results:In pancreatic cancer cells,Real-time PCR and Western blot was used to profile the MBD3 expression.At both mRNA and protein levels,the expression of MBD3 from high to low were: PANC1,PaTu8988 and SW1990 cells.Validation results showed that sh-MBD3 and Flag-MBD3 plasmids were effective.In PANC1 and PaTu8988 cells,MBD3 downregulation enhanced the ability of colony formation,proliferation and stemness maintenance.Meanwhile,MBD3 upregulation weakened the abilities of colony formation,proliferation and stemness maintenance.After down-regulating MBD3,the levels of NANOG,SOX2 and OCT4 in PaTu8988 and PANC1 cells were increased.In contrast,MBD3 up-regulation resulted in the decrease of NANOG,SOX2 and OCT4 expression in SW1990 and PaTu8988 cells.In PaTu8988 and PANC1 cells,the knockdown of MBD3 increased the expression levels of YAP,MOB1 and LATS1,while the expression levels of p-YAP,p-MOB1,p-LAT1,SAV1,MST1 and MST2 decreased.To the contrary,MBD3 upregulation resulted in the decrease of YAP,MOB1 and LATS1 in SW1990 and PaTu8988 cells,while the expression of p-YAP,p-MOB1,p-LAT1,SAV1,MST1 and MST2 were increased.The analysis of TCGA database showed that the expression of MBD3 was negatively correlated with that of YAP.We confirmed the binding of MBD3 to YAP by Immunoprecipitation assays in HEK293 T cells.In pancreatic cancer cells,MBD3 suppressed YAP nuclear translocation,which was examined by Luciferase reporter assay,Immunofluorescence analysis and Nuclear and Cytoplasmic Extraction.MBD3 suppresses YAP nuclear translocation.At both mRNA and protein levels,after down-regulating YAP,the MBD3 expression in SW1990 and PaTu8988 cells were increased,while the expression of MBD3 were decreased after overexpressing YAP in PaTu8988 and PANC1 cells.Conclusion:In this study,we demonstrated that MBD3 weakened the abilities of proliferation and sphere formation in pancreatic cancer cells,and inhibited tumorigenesis.Furthermore,it was found that MBD3 bound to YAP to inhibit stemness of pancreatic cancer cells via the Hippo-YAP signaling pathway.