The Molecular Mechanism Of Tumor-associated Macrophages On Sorafenib Inhibited Hepatocellular Carcinoma

PART I INDUCTION AND PHENOTYPIC IDENTIFICATION OF TUMOR-ASSOCIATED MACROPHAGESObjective: To induce PBMC into M1 or M2 macrophages in vitro, and analyze their phenotypic characteristics.Methods: In this study, monocytes were isolated from PBMC and cultured in the presence of granulocyte-macrophage colony-stimulating factor(GM-CSF) for one week to generate M1 macrophages, and macrophage colony-stimulating factor(M-CSF) for one week to generate M2 macrophages. Furthermore, the expression of surface molecule CD68、HLA-DR、CD206 in M1 or M2 macrophages was analyzed by Real-time PCR. Then, the protein expression of IL-10, IL-12 and CD206 in M1 or M2 macrophages upon LPS stimulation was analyzed by ELISA.Results: In GM-CSF-induced M1 macrophages, the CD68 and HLA-DR m RNA expression were increased, and IL-12 protein expression was also increased. In M-CSF-induced M2 macrophages, the m RNA level of CD206 was increased, and the protein level of IL-10 was increased.Conclusions: We successfully induced PBMC into M1 or M2 macrophages in vitro, and analyze their phenotypic characteristics.PART II THE EFFECT AND MECHANISM OF TUMOR-ASSOCIATED MACROPHAGES ON GROWTH OF HEPATOCARCINOMAObjective: To investigate the effect and molecular mechanism of tumor-associated macrophages on growth of hepatocellular carcinoma.Methods: We investigated the proliferation, migration and invasion of Hep G2 and Huh-7 cells cultured alone or co-cultured with various treated M1 or M2 macrophages. The abilities of proliferation, migration and invasion were detected by CCK-8 kit and transwell assay respectively. The expression of EMT-related proteins such as E-cardherin、Vimentin in hepatoma cells were detected by western blot. The direct target of mi R-101, DUSP1, was identified in silico and validated using a 3￠-UTR reporter assay. To examine the effect of mi R-101 on DUSP1 expression, M1 and M2 macrophage cells were transfected with mi R-101 mimics or negative control and stimulated with LPS. Activation of MAPKs was measured by western blot.Results: Hepatoma cells proliferated faster co-cultured with M2 macrophage compared with co-cultured with M1 macrophage or cultured alone. The abilities of migration and invasion of hepatoma cells co-cultured with were increased than other co-cultured with M1 macrophage or cultured alone, respectively. Hepatoma cells co-cultured with M2 macrophage down-regulated E-cadherin expression and up-regulated vimentin, N-cadherin, and fibronectin expression. DUSP1 is a direct target of mi R-101 and mi R-101 regulates the LPS-induced activation of ERK、p38 and JNK.Conclusions: We found M2 macrophages could enhance the abilities of proliferation, migration and invasion in hepatoma cells, due to mi R-101 regulates the LPS-induced activation of ERK, p38 and JNK.PART III THE MECHANISM OF SORAFENIB REGULATES MIR-101 EXPRESSION AND INHIBITS TGF-β EXPRESSION IN HEPATOCARCINOMAObjective: To investigate the molecular mechanism of related signaling pathways in sorafenib inhibited HCC progression.Methods: To examine whether sorafenib alters mi R-101, M2 cells were treated with LPS, LPS plus sorafenib, or sorafenib only for different time periods. The m RNA expression of mi R-101 was detected by Real-time PCR, the protein level of DUSP1 and downstream related MAPKs were measured by western blot. We analyzed the expression of TGF-β and CD206 in different treated M2 macrophages by Real-time PCR and /or ELISA. Hepatoma cells co-cultured with M1 or M2 macrophages treated sorafenib, the abilities of proliferation, migration and invasion were detected by CCK-8 and transwell assay respectively. The expression of EMT-related proteins such as E-cardherin、Vimentin in hepatoma cells were detected by western blot. Mice received DEN to induce HCC and sorafenib treated. Immunohistochemistry assay was carried out to detect the expression of TGF-β 、CD68、CD206 and HLA-DR in tumor tissues. The expression of TGF-β and CD206 in human hepatoma tissues by Real-time PCR and /or immunohistochemistry, then analyze the correlation CD206 expression and other pathological characteristics.Results: After LPS stimulation, treatment sorafenib markedly increased DUSP1 expression and attenuated activation of ERK, p38 and JNK, and markedly decreased the expression of mi R-101、TGF-β and CD206. Hepatoma cells co-cultured with M2 macrophage treated sorafenib showed a significant decrease in the number of invading cells compared with the control. Hepatoma cells co-cultured with M2 macrophage treated sorafenib up-regulated E-cadherin expression and down-regulated vimentin, N-cadherin, and fibronectin expression. In vivo, treatment with sorafenib as monotherapy significantly decreased HCC burden, and the M2 marker CD206 was significantly decreased in the tumor tissue, and down-regulation of TGF-β occurred in HCC tissue treated with sorafenib. The expression of CD206 and TGF-β was increased in human hepatoma and increased CD206 expression in HCC indicates a poor prognosis.Conclusions: Sorafenib may occur through inhibition of LPS-induced activation of PI3K/AKT, reducing mi R-101 expression and subsequent induction of DUSP1 to deactivate MAPKs. Sorafenib alters the function of M2 macrophages and reduces TGF-β-driven HCC progression.