The Expression And Related Mechanisms Of Costimulatory Molecule B7-H3 In Hepatocellular Carcinoma

Objective:To investigate costimulatory molecule B7-H3 expression profile and its clinicopathological significance in HCC and explore the mechanisms of how B7-H3 involves in the biological behaviors of HCC cells.Methods:1.116 HCC tumor tissues obtained from patients undergoing liver curative resection were used to examine B7-H3 expression by immunohistochemical staining. Kaplan-Meier survival analysis and COX’s proportional hazard model were used to analyze the outcome of patients and the dangerous factors in each groups divided by B7-H3 expression level.2. HCC cell lines, HepG2 and SMMC7721, with B7-H3 depletion established by RNA interference were used to investigate the effect of B7-H3 on cell proliferation, apoptosis, movement ability and invasive ability by MTT assay, ELISA apoptosis detection kit, scratch wound healing assay and transwell invasion model, respectively.3. Western blot was used to detect the expression of invasion related molecules MMP-2, MMP-9, EMT-related molecules E-cadherin, vimentin, N-cadherin, JAK/Stat signal molecules p-stat3, p-JAK2, and transcription factors slug, snail.4.116 HCC tumor tissues were used to examine CD68 positive macrophages by immunohistochemical staining. The relationship of TAMs’ number and B7-H3 expression level was analyzed.5. RT-PCR and western blot were used to examine the expression of M1 and M2 subtype markers in THP-1-induced macrophages after 24 and 48 hours of coculturing with HepG2 cells in cell-cell contact and cell noncontact manners, respectively.6. RT-PCR and western blot were used to examine the expression of M1 and M2 markers in THP-1-induced macrophages after coculturing with HepG2 cells in which B7-H3 was depleted. B7-H3 recombinant protein was used to reverse the effect of B7-H3 depletion in HepG2 cells on macrophage polarization.Results:1. Among the 116 tissues,109 showed positive B7-H3 expression. High expression of B7-H3 correlated with late TNM stage and metastasis. Survival analysis showed that high B7-H3 expression predicts poor prognosis for HCC patients2. B7-H3 depletion had no obvious effect on HCC cell proliferation and apoptosis, whereas suppressed scratch wound healing ability and invasive ability significantly.3. B7-H3 depletion upregulated E-cadherin’s expression and downregulated MMP-2, MMP-9, N-cadherin and vimentin’s expression in HCC cells. The expression levels of p-stat3, p-JAK2 and Slug were also decreased.5. Of the 116 cases,94.8% showed positive staining of TAMs. The cell number were positively correlated with TNM stage, metastasis and B7-H3 expression.6. HepG2 cells could promote M2 phenotype macrophage polarization, which showed decreased HLA-DR, iNOS and TNF-α expression, and increased Argl, VEGF and CCL22 expression.7. Depletion of B7-H3 interfered with HepG2 cells mediated M2 macrophage polarization. B7-H3 recombinant protein could partially reverse the effect.Conclusions:1. B7-H3 was highly expressed in HCC tissues, and correlated with TNM stage, tumor metastasis and prognosis of patients.2. The expression of B7-H3 promoted invasion and metastasis of HCC cells, the mechanism of which might involve in EMT via JAK2/Stat3/Slug signal pathway.3. The infiltration of TAMs in HCC tissues correlated with the progression and prognosis of HCC patients.4. B7-H3 expressed on HCC cells could polarize macrophages in the tumor microenvironment to an M2 phenotype.