SIRT4 Depletion Promotes HCC Development By Enhancing PPARδ Signaling-Mediated Alternative Activation Of Macrophages

Background:Hepatocellular carcinoma(HCC)is one of the most common cancers worldwide.In China,the mortality rate of hepatocellular carcinoma is second only to the lung cancer.Recent studies have shown that the tumor microenvironment plays an important role in tumor development,invasion and metastasis.Tumor microenvironment refers to cells composed of tumor cells and surrounding epithelial cells(paracancerous),fibroblasts,intrinsic and specific immune cells,tumor blood vessels and lymphatic vessels,tissue-specific mesenchymal cells and expression Products.Immune cells including tumor infiltrating lymphocytes(TILs)and tumor-associated macrophages(TAMs)are important components of tumor microenvironment inflammatory immunological factors.The activation of tumor-associated macrophages(TAMs)is a contributor to the progression of hepatocellular carcinoma(HCC).SIRT4 acts as a tumor suppressor gene for tumor growth via regulating cell metabolism,inflammation,and anti-tumorigenesis.Yet,its involvement of SIRT4 in the activation of TAMs is unknown.Based on previous findings,the expression of SIRT4 in distinct groups of TAMs as well as SIRT4 interference on macrophage polarization was investigated.Methods:1)The expression of SIRT4 in HCC tissues and para-carcinoma tissue were tested by RT-PCR,Western blotting and histological analysis.2)The Kaplan-Meier survival curve was based on the expression of SIRT4 in the HCC samples.3)Immunofluorescence was usedto detect the amount of macrophage infiltration in the tumor tissues of HCC patients,and the number of M1 and M2 macrophages.4)The correlation between the expression level of SIRT4 and the expression levels of CD16 and CD206 in macrophages was analyzed by flow cytometry.5)Western blot and qRT-PCR were used to detect the correlation between SIRT4 expression level and macrophage polarization markers in macrophages.6)Analysis of the effect of SIRT4 on cellular lipid energy metabolism by bioenergy analyzer.7)The effects of macrophages on proliferation,migration and invasion of hepatoma cells were detected by CCK-8 and Transwell method.8)Mouse liver orthotopic transplantation model was used to detect the effect of tumor-associated macrophages on the proliferation of HCC cells.Results: 1)SIRT4 was significantly downregulated in HCC tumor tissues.2)the low expression of SIRT4 in peritumor tissue was associated with poor survival in patients.3)We further found that low SIRT4 expression was related to the infiltration of TAMs and the ratio of M2/M1 macrophages in peritumor tissues.4)Using gene interference,we found that SIRT4 interference in TAMs significantly modulates the alternative activation of macrophages and promoted in vitro and in vivo HCC cell growth.5)Mechanistically,we revealed that HCM constrained the expression of SIRT4 in macrophages and promoted the alternative activation of macrophages via the FAO-PPARδ-STAT3 axis.6)Furthermore,we also revealed that elevated MCP-1 expression induced by low expression of SIRT4 in HCC cells is responsible for increased TAMs infiltration in para-cancer tissue.Conclusions: Overall,our results clarify that SIRT4 is significantly down-regulated in HCC tumor tissues,and the expression level of SIRT4 in peritumor tissues is positively correlated with the survival rate of patients,and negatively correlated with tumor size,pathological grade,T stage and clinical stage of HCC patients.We also elucidate that SIRT4 silencing in TAMs modulates the alternative activation of macrophages and promotes HCC development via the FAO-PPARδ-STAT3 axis.These results could provide a new therapeutic target for the treatment of HCC.