TIPE1 Regulates The Alternative Activation Of Tumor-associated Macrophage And Involves In HCC Development

BackgroundHepatocellular carcinoma(HCC)is one of the most common malignant tumors in human beings,and its mortality ranks third among human tumor diseases,thus it is extremely harmful to human health.The deep understanding of the pathogenic mechanism of HCC will provide a theoretical basis for the development of a comprehensive individualized treatment plan.Tumor necrosis factor a-induced protein 8(TIPE)family includes 4 members:TIPE,TIPE1,TIPE2,TIPE3,and have been reported to involve in inflammation,infection,autoimmune diseases,tumor and other diseases by regulating cell death,proliferation and migration ability.TIPE1 gene is located in human chromosome 19p13.3,encoding a cytosolic protein containing 188 amino acids,and widely expressed in a variety of adult and embryonic tissues.In 2008,TIPE1 protein was first identified as a candidate molecule that can regulate both apoptosis and necroptosis.However,so far,little is known about the specific biological function of TIPE1 protein.Studies showed that TIPE1 promoted the autophagy of dopaminergic neuron induced by oxidative stressby inhibiting TSC2 degradation through competitive binding to FBXW5,and then negatively regulating mTOR activation,which might contribute to the pathogenesis of Parkinson's disease.Previous study of our research group found that TIPE1 promoted hepatocyte death by inhibiting the activation of Rac,and functioned as a tumor suppressor gene in the progression of liver cancer.However,at present,the role of TIPE1 in immune regulation has not been reported at all.Our previous study indicated that cells with macrophage like morphology in liver cancer tissues highly expressed TIPE1.Based on these clues,the present study intends to demonstrate the expression and function of TIPE1 protein in tumor associated macrophages,and to explore its molecular mechanisms preliminarily.ObjectivesIn the present study,we aim to explore the role of TIPE1 in regulating the activation and function of tumor associated macrophages,and to uncover a novel mechanism of hepatocarcinogenesis,and to identify a novel target for the diagnosis,treatment and prognosis of liver cancer.Specific objectives are as follows:1.To detect the expression of TIPE1 in tumor associated macrophages and its regulatory mechanism;2.To clarify the biological role of macrophage TIPE1 in the development of hepatocellular carcinoma;3.To investigate the molecular mechanism of TIPE1 in regulating the activation and function of tumor associated macrophages.Methods and results1.TIPE1 expression in immune organs and macrophagesAt present,the expression of TIPE1 in immune organs and immune cells has not been reported.Therefore,we firstly detected the expression level of TIPE1 in immune organs such as thymus,spleen and lymph nodes.RT-PCR showed that there was substantial background expression level of TIPE1 in thymus,spleen and lymph nodes.Double immunofluorescence staining further ascertained the expression of TIPE1 protein in thymus,spleen and lymph nodes.Moreover,a small part of CD68+macrophages showed TIPE1 overlapping staining.In order to further clarify the expression of TIPE1 in macrophages,we performed immunofluorescence staining for TIPE1 with THP-1 cells.Results showed strong cytoplasmic TIPE1 expression.We also isolated the murine peritoneal macrophages induced by 6%starch and bone marrow-derived macrophages.RT-PCR and Western Blot analysis showed the obvious background expression of TIPE1 mRNA and protein.2.High TIPE1 expression in tumor-associated macrophages negatively correlated with patient survivalDouble immunofluorescence staining was used to detect the expression of TIPE1 in human hepatocellular carcinoma tissues and corresponding adjacent tissue.Results showed that TIPE1 was expressed by liver parenchymal cells and CD68+macrophages.Statistical analysis showed that the percentage of TIPE1+CD68+macrophages in HCC tissues was significantly higher than that in adjacent tissues.Importantly,the percentage of TIPE1+CD68+ showed significant negative correlation with patient survival.Nodular HCC tumors had higher proportion of TIPE1+CD68+macrophages than giant HCC tumors.3.Tumor microenvironment promotes TIPE1 expression in macrophagesIn order to simulate tumor microenvironment in vitro and observe its effect on TEPE1 expression in macrophages,hepatocellular carcinoma conditioned medium(HCM)was prepared from human liver cell line HepG2,Huh7 and mouse hepatoma cell line H22 cell culture supernatant.Different time course(0,12,24,48h)and doseage(0,100,200,300?l)were designed to stimulate human monocyte cell line THP1 and mouse primary macrophages PM,BMDM,and the expression of TIPE1 was detected by RT-PCR and Western Blot.The results showed that HCM significantly upregulated the expression of TIPE1 in THP1,PM and BMDM in a time-and dose-dependent manner.4.TGF-? significantly upregulates the expression of TIPE1 in macrophagesTGF-? is an important cytokine in tumor microenvironment,which plays an important role in the occurrence and development of tumor.Therefore,we further detected the effect of TGF-? on the expression of TIPE1 in macrophages by RT-PCR and Western Blot.Results showed that TGF-? significantly enhanced the expression of TIPE1 mRNA and protein in THP1,RAW264.7 and PM cells in a dose dependent manner.In order to further verify the role of TGF-p in macrophage TIPE1 upregulation by tumor microenvironment,we also used different concentrations of TGF-? receptor blocker SB431542(0,2.5,5,10?g/ml)for the pretreatment of peritoneal macrophages,then conditioned medium HCM stimulation was added,and the expression of TIPE1 in PM was detected.Results showed that SB431542 inhibited the expression of TIPE1 in PM induced by HCM in a dose-dependent manner.5.TIPE1 promotes the polarization of macrophages into M2 phenotypeIn order to further explore the mechanism of macrophage TIPE1 involved in tumorigenesis,we investigated the effect of TIPE1 on macrophage activation and differentiation,which were induced into in M2 like phenotype in tumor microenvironment.Firstly,isolatd PM were induced into M1 or M2-like phenotype by LPS or IL-4 respectively,and the expression of TIPE1 was detected by RT-PCR and Western Blot.Results showed that LPS-induced M1 macrophages showed high iNOS expression,while IL-4-induced M2 macrophages had high expression of Argl,Accordingly,the expression of TIPE1 mRNA and protein was significantly higher in IL-4-induced M2 macrophages than that in LPS induced M1 macrophages.Based on this difference,we hypothesized that TIPE1 might involve in the induction and maintenance of M2 macrophage.Thus,we knocked down the expression of TIPE1 in PM and BMDM by using small interference RNA and the M2 markers was detected,Flow cytometry analysis showed that TIPE1 interference significantly downregulated CD206 expression and IL-10 production in macrophages stimulated by IL-4.In addition,TIPE1 interference reduced the secretion of IL-10 in the supernatant of BMDM stimulated by TGF-P and HCM.6.TIPE1 regulates signaling pathways related to macrophage polarizationBased on the results metioned above,we further detected the effect of TIPE1 on the activation of the signaling pathways of macrophage polarization.Western Blot results showed that TIPE1 interference significantly downregulated the phosphorylation of STAT6 in macrophages stimulated by IL-4,while upregulated the phosphorylation of STAT1/STAT6.Related literatures reported that the transcription factor STAT1/STAT6 mediate the polarization of macrophages to M1/M2 by regulating the downstream target genes.These results suggest that TIPE1 may be involved in the regulation of macrophage polarization by modulating related signaling pathways.7.Macrophage TIPE1 enhances cell growth,migration and invasionIn order to study the biological function of TIPE1 expression in macrophages,we stimulated mouse hepatoma cell line Hepal-6 cell with the culture supernatant from mouse peritoneal macrophages PM and bone marrow-derived macrophages BMDM transfected with TIPE1-siRNA or NC-siRNA.Then,cell growth was detected by CCK-8 assay.Results showed that,compared with the control group,Hepal-6 cells,incubated with culture supernatant from TIPE1 knockdown peritoneal macrophages,growed slowly.Meanwhile,transwell assay was performed to detect the migration and invasion ability of Hepal-6 cells cocultured with TIPE1-siRNA or NC-siRNA transfected PM or BMDM.Results showed that TIPE1 knockdown in macrophage significantly inhibited the migration and invasion ability of Hepal-6 cells.8.TGF-? is responsible for enhanced protumoral activity of TIPE1 knockdown macrophagesIn order to further clarify the molecular mechanism of macrophages TIPE1 involved in tumorigenesis,we examined the effect of TIPE1 on cytokine secretion of macrophage.Results showed that TIPE1 interference significantly downregulated the secretion of TGF-?,one of important tumor-promotion mediators,both in PM and BMDM.Then,BMDM isolated from myeloid-specific TGF-? knockout mice or wild-type C57BL/6 mice were transfected with NC siRNA or TIPE1 siRNA,and were co-cultured with Hepal-6 cells in Transwell assay.Results showed that TIPE1 interference significantly inhibited the migration ability of tumor cells cocultured with wild-type BMDM,while it had no significant effect on the migration of tumor cells cocultured with TGF-? knockout mouse derived macrophages.ConclusionAccording to the above results,this project identified the molecular mechanism of TIPE1 regulation on macrophage polarization and its involvement in tumorigenesis,and made the following conclusions:1.There are TIPE1 expression in several immune organs and macrophages.2.Tumor associated macrophages have high expression of TIPE1,which negatively correlated with patient survival.TGF-? plays an important role in regulating TIPE1 expression in macrophages in tumor microenvironment.3.TIPE1 promotes M2 polarization of macrophages by regulating STAT1/STAT6 phosphorylation.4.TIPE1 upregulates the secretion of TGF-? from macrophages,which then enhances tumor growth and metastasis.Innovation and significanceIn the present study,we firstly detect the expression of TIPE1 in immune organs and macrophages,preliminarily study the effect of TIPE1 on macrophage polarization,cytokine secretion and their involvement in tumorigenesis,which would provide new data for unraveling the biological functions of TIPE1 and suggest a potential target for tumor immunotherapy.