Effect Of TRIM59 On Carbohydrate Metabolism And HIF-1α Expression In Macrophages

TRIM59 is a member of the three-domain superfamily with a RING domain,one or two Box boxes,a coiled domain,and a transmembrane domain,and plays a role in tumor,epigenetic modification,embryonic development and autophagy effect.The macrophages regulated by TRIM59 can significantly inhibit tumor growth and improve the inflammatory state of mice with sepsis,which is mainly through the NF-κB signaling pathway to inhibit macrophages to secret inflammatory cytokines and inhibit phagocytosis through Fcγ receptors.However,whether there are other mechanisms is unclear.The metabolism of cellular substances is closely related to cellular functions,and macrophages with an anti-inflammatory phenotype exhibit reduced glycolysis and enhanced oxidative phosphorylation.However,the glycolysis product lactate can reduce inflammation to a certain extent.Lactate inhibits LPS-induced IL-1βproduction in mouse macrophages and human PBMC in vitro,and this effect is achieved by inhibiting NF-κB and inflammasome activation.Whether TRIM59 affects macrophage glucose metabolism and lactate secretion is unclear.In addition,HIF-1αis closely related to glucose metabolism and also regulates enzymes related to lactate production,such as lactate dehydrogenase.Whether TRIM59 can regulate HIF-1α and what the mechanism is also needs to be further explored.MethodsRAW264.7 cells with high TRIM59 expression and their control,and bone marrow-derived macrophages from TRIM59 macrophage conditional knockout mice were cultured.1)Bioinformatics analysis was performed on the transcriptome of TRIM59 highly expressed RAW264.7 cells.COG database,GO database and KEGG database were used to find differentially expressed genes related to glucose metabolism,and analyze their expression differences.2)The culture supernatant of RAW264.7 cells with high expression of TRIM59 and control cells was extracted,and lactate detection kit was used for lactate detection.3)RNA was extracted from RAW264.7 cells with up-regulated expression of TRIM59 and control cells,and after reverse transcription,the expression of HIF-1α and TLRs was detected by real-time fluorescence quantitative PCR technology.4)The BMDM of TRIM59 macrophage conditional knockout mice were cultured,and after LPS stimulation,RNA and cell proteins was extracted,and HIF-1α gene expression was detected by real-time fluorescence quantitative PCR technology and the expression of HIF-1α,PKM2 and PHD proteins was detected by Western-blotting.Result1.The results of bioinformatics analysis suggest that TRIM59 can affect the glucose metabolism of macrophagesBy clustering the differentially expressed genes COG,GO,and KEGG in macrophages with high expression of TRIM59 and the control group,we found that after overexpression of TRIM59 in macrophages,the glucose metabolism-related genes of macrophages changed significantly,involving genes related to glycolysis,pentose phosphate pathway and oxidative phosphorylation.The up-regulated genes include Hk3,Acyp1,Hagh,Pck2,Sord,Acss2,Arid5 a,Adh7,and the down-regulated genes include Gmppb,L2 hgdh,Aacs,Hk2,Hmgcs1,Pfkl,Acly,Idh1,Ldha,Tpi1,Pfkfb1,Eno4.TRIM59 can regulate the glucose metabolism of macrophages,especially affecting many genes related to glycolysis.Among the inflammation-related factors,IL23 a and Il10 rb were up-regulated.The most obvious decrease genes were Il11、Il1b、Il21r and Il18 rap,and other factors such as Il6 ra,Il1rap and Il18 rap did not change significantly.Among Tnf-related molecules,Tnf,Tnfsf13 b,Tnfrsf8 and Tnfrsf11 a were all significantly decreased.Among IFN-related molecules,the gene that decreased was Ifnar2.2.TRIM59 promotes lactate secretion in RAW264.7 macrophagesWe measured lactate in the culture supernatant of RAW264.7 cells with high TRIM59 expression and control cells.We found that in the RAW264.7 cells with high TRIM59 expression,the lactate was significantly increased than the control group.3.TRIM59 can affect the expression of HIF-1α and TLR2 in RAW264.7 macrophagesWe found that after high expression of TRIM59,the genes of Hif-1α and Tlr2 in RAW264.7 macrophages were up-regulated.4.After TRIM59 deletion,the expression of HIF-1α in macrophages is down-regulatedTo clarify the effect of TRIM59 on HIF-1α expression in macrophages under inflammatory conditions,we stimulated the BMDM of TRIM59-CKO mice with LPS for 24 h,and the expression of Hif-1α gene was decreased.We also found that HIF-1αprotein expression was up-regulated 1 and 2 hours after LPS stimulation.However,in the absence of TRIM59,HIF-1α expression decreased at 1 and 2 hours after LPS stimulation,but was not significantly different from controls at 3 and 6 hours after stimulation.At 1 and 2 h after LPS stimulation of TRIM59-deficient BMDM,the expression of PKM2 was slightly lower than that of the control group,while the protein expression level of PHD2 had no changed.Conclusion1.Bioinformatics analysis showed that TRIM59 can regulate the glucose metabolism of macrophages.2.TRIM59 can promote the secretion of lactate in macrophages and up-regulate the expression of HIF-1α,which may be related to its role in inhibiting the secretion of inflammatory factors.3.TRIM59 up-regulated the expression of TLR2 in macrophages,which may be related to its regulation of lactate secretion and HIF-1α expression.