Effect of EZH2 on colorectal tumor behavior and its mechanism
Objective To explore the effect and mechanism of EZH2 on the behavior of colorectal tumors.Methods ①Forty BALB/c-nu nude mice were randomly divided into four groups:EZH2-NC group,EZH2-OE group,EZH2-NC+PD1 inhibitor group,and EZH2-OE+PD1 inhibitor group.Human colorectal adenocartinoma cells SW480 were transfected with EZH2 empty vector and EZH2 overexpression plasmid,and then subcutaneously injected into BALB/c-nu nude mice to construct a graft tumor model,respectively.After successful transfection,the nude mice were injected with PD1 inhibitor(150 μg/mL)via the tail vein in EZH2-NC+PD1 inhibitor group and EZH2-OE+PD1 inhibitor group.Then the tumor volume was calculated and compared at 21 d.②The stably transfected SW480 cells were divided into EZH2-NC group,EZH2-OE group,EZH2-NC+PD1 inhibitor group(adding 1.5 ng/mL PD1 inhibitor after transfec-tion),and EZH2-OE+PD1 inhibitor group(adding 1.5 ng/mL PD1 inhibitor after transfection),and then SW480 cells and THP-1 cells were co-cultured.CCK-8 test,Scratch test and Transwell test were used to detect the proliferation,migration and invasion abilities of SW480 cells,respectively.Western blot was used to detect the expression levels of XBP-1,Cat K,Cat L,IL-6 and Arg-1 proteins in THP-1 cells and EZH2,p-STAT3 and PD-1 proteins in SW480 cells.Results ①The tumor formation results of nude mice showed that the tumor volume was increased in EZH2-OE group compared with EZH2-NC group(P＜0.01),while there was no significant difference between EZH2-OE+PD-1 inhibitor group and EZH2-NC+PD-1 group.②After co-culture of SW480 cells and THP1 cells,the proliferation,migration and invasion abilities of SW480 cells were enhanced in EZH2-OE group compared with EZH2-NC group(P＜0.01),while there was no significant difference between EZH2-OE+PD-1 inhibitor group and EZH2-NC+PD-1 group.The levels of IL-6 and Arg-1 proteins in THP-1 cells were higher in EZH2-OE group than in EZH2-NC group(P＜0.05),and IL-6 and Arg-1 protein levels were lower in EZH2-NC+PD1 inhibitor group than in EZH2-NC group(P＜0.01),and in EZH2-OE+PD1 inhibitor group than in EZH2-OE group(P＜0.01).The protein levels of XBP-1,Cat K and Cat L in THP-1 cells were significantly higher in EZH2-OE group than in EZH2-NC group(P＜0.01),and the protein levels of XBP-1,Cat K and Cat L were lower in EZH2-NC+PD-1 inhibitor group than in EZH2-NC group(P＜0.01),and in EZH2-OE+PD-1 inhibitor group than in EZH2-OE group(P＜0.01).Compared with EZH2-NC group,EZH2,p-STAT3 and PD-1 protein levels in SW480 cells in EZH2-OE group were increased(P＜0.01).Compared with EZH2-NC+PD-1 inhibitor group,EZH2 and p-STAT3 protein levels in EZH2-OE+PD-1 inhibitor group were increased(P＜0.05).Conclusion EZH2 may promote the progression of colorectal cancer by upregulating STAT3/PD-1 pathway,thereby affecting the tumor-associated macrophages.
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