EHMT2 (G9a) activation in mantle cell lymphoma and its associated DNA methylation and gene expression

Objective:The function of euchromatic histone-lysine N-methyltransferase 2(EHMT2)has been studied in several cancers;however,little is known about its role in mantle cell lymphoma(MCL).Thus,this study aimed to characterize the significance and function of EHMT2 in MCL.Methods:EHMT2 expression in MCL and reactive hyperplasia(RH)were investigated by immunohistochemistry.Genome-wide analysis of DNA methylation was performed on EHMT2+MCL samples.The function of EHMT2 was determined by CCK&flow cytometry,and western blot assays.Gene expression profile analysis was performed before and after EHMT2 knockdown to search for EHMT2-regulated genes.Co-immunoprecipitation(Co-IP)experiments were conducted to identify the proteins interacting with EHMT2.Results:EHMT2 was expressed in 68.57%(24/35)of MCLs but not in any RHs.Genome-wide analysis of DNA methylation on EHMT2+MCLs revealed that multiple members of the HOX,FOX,PAX,SOX,and CDX families were hypermethylated or hypomethylated in EHMT2+MCLs.BIX0I294,a EHMT2 inhibitor,inhibited MCL cell growth and stalled cells in the G1 phase.Additionally,BIX01294 downregulated the expressions of cell cycle proteins,cyclin DI,CDK4,and P21,but upregulated the expressions of apoptosis-related proteins,Bax and caspase-3.Co-IP experiments revealed that EHMT2 interacted with UHRF1,HDAC1,and HDAC2 but not with HDCA3.After EHMT2 knockdown,multiple genes were regulated,including CD5 and CCND1,mostly enriched in the Tec kinase signaling pathway.In addition,several genes(e.g.,MARCH 1,CCDC50,HIP1,and WNT3)were aberrantly methylated in EHMT2+MCLs.Conclusions:For the first time,we determined the significance of EHMT2 in MCL and identified potential EHMT2-regulated genes.

Increased Tim-3 expression alleviates liver injury by regulating macrophage activation in MCD-induced NASH mice

As an immune checkpoint,Tim-3 plays roles in the regulation of both adaptive and innate immune cells including macrophages and is greatly involved in chronic liver diseases.However,the precise roles of Tim-3 in nonalcoholic steatohepatitis(NASH)remain unstated.In the current study,we analyzed Tim-3 expression on different subpopulations of liver macrophages and further investigated the potential roles of Tim-3 on hepatic macrophages in methionine and choline-deficient diet(MCD)-induced NASH mice.The results of flow cytometry demonstrated the significantly increased expression of Tim-3 on all detected liver macrophage subsets in MCD mice,including F4/80^(+)CD11b^(+),F4/80^(+)CD68^(+),and F4/80^(+)CD169^(+)macrophages.Remarkably,Tim-3 knockout(KO)significantly accelerated MCD-induced liver steatosis,displaying higher serum ALT,larger hepatic vacuolation,more liver lipid deposition,and more severe liver fibrosis.Moreover,compared with wild-type C57BL/6 mice,Tim-3 KO MCD mice demonstrated an enhanced expression of NOX2,NLRP3,and caspase-1 p20 together with increased generation of IL-1βand IL-18 in livers.In vitro studies demonstrated that Tim-3 negatively regulated the production of reactive oxygen species(ROS)and related downstream pro-inflammatory cytokine secretion of IL-1βand IL-18 in macrophages.Exogenous administration of N-Acetyl-L-cysteine(NAC),a small molecular inhibitor of ROS,remarkably suppressed caspase-1 p20 expression and IL-1βand IL-18 production in livers of Tim-3 KO mice,thus significantly reducing the severity of steatohepatitis induced by MCD.In conclusion,Tim-3 is a promising protector in MCD-induced steatohepatitis by controlling ROS and the associated pro-inflammatory cytokine production in macrophages.