B7-H3 promotes aggression and invasion of hepatocellular carcinoma by targeting epithelial-to-mesenchymal transition via JAK2/STAT3/Slug signaling pathway.

BACKGROUND: B7-homologue 3 (B7-H3), a recently identified immunoregulatory protein, has been shown to be overexpressed in human hepatocellular carcinoma (HCC). However, whether the dynamic expression pattern of B7-H3 contributes to early invasion of HCC is largely unknown. In addition, the biological roles of B7-H3 in HCC are still unclear. Herein, we are going to examine B7-H3 expression profile and its clinicopathological significance in primary and metastatic HCC, and further determine whether B7-H3 knockdown simulates different pathological states of HCC progression and metastasis. METHODS: Using immunohistochemistry, B7-H3 expression was studied on 116 HCC containing primary and metastatic HCCs. Survival curves and log-rank tests were used to test the association of B7-H3 expression with survival. HCC cells with B7-H3 depletion were established by RNA interference to investigate the effect of B7-H3 on cell proliferation, apoptosis, migration and invasion in vitro. RESULTS: Statistical analysis of clinical cases revealed that B7-H3 high expression group had inclinations towards late TNM stage, the presence of vascular invasion, lymph metastasis, and the formation of microsatellite tumors. Increased intensity of tumor B7-H3 staining was detected more significantly in metastatic HCC tumors. Consistently in experiments performed in vitro, B7-H3 was able to stimulate the wound healing, metastasis and invasion of hepatoma cells by targeting epithelial-to-mesenchymal transition (EMT) via JAK2/Stat3/Slug signaling pathway, while no obvious influence on cell growth and apoptosis. CONCLUSION: B7-H3 in the regulation of the metastatic capacity of HCC cells makes itself a promising therapeutic target for anti-metastasis therapy.

Construction of a replication-competent hepatitis B virus vector carrying secreted luciferase transgene and establishment of new hepatitis B virus replication and expression cell lines.

BACKGROUND: Previously, we have successfully constructed replication-competent hepatitis B virus (HBV) vectors by uncoupling the P open reading frame (ORF) from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence. Consequently, the replication-competent HBV vectors carrying foreign genes, including pCH-BsdR, carrying blasticidin resistance gene (399 bp), and pCH-hrGFP, carrying humanized renilla green fluorescent protein gene (720 bp), were successfully obtained. However, the replication efficiency of the former is higher but it is tedious to use, while that of the latter is poor and cannot be quantified. Hence, we need to search for a new reporter gene that is convenient and quantifiable for further research. AIM: To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies. METHODS: We utilized the replication-competent HBV viral vectors constructed by our laboratory, combined with the secreted luciferase reporter gene, to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase (SecNluc). HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene. RESULTS: The replication-competent HBV vector carrying the SecNluc reporter gene pCH-sNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression. HBV replication intermediates could be produced from this vector. Via transfection with pTRE-sNLuc and selection by hygromycin, we obtained isolated cell clones, named HBV-NLuc-35 cells, which could secrete secNLuc recombinant viruses, and were sensitive to existing anti-HBV drugs. Using differentiated HepaRG cells, it was verified that recombinant HBV possessed infectivity. CONCLUSION: Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability, and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene. More importantly, the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.

Hepatocellular carcinomas promote tumor-associated macrophage M2-polarization via increased B7-H3 expression.

B7 family members are aberrantly expressed on the human hepatocellular carcinoma (HCC) cell surface, and induce local and systemic immunosuppression. Tumor-associated macrophages (TAMs) are a significant immune cell subpopulation in HCC and may be induced to express co-inhibitory molecules including B7 homologue 3 (B7-H3). In the present study, 79.3% of the HCC tissue samples showed high expression of B7-H3 which was positively correlated with the number of TAMs in the evaluated cancer tissues. Furthermore, high levels of TAMs or B7-H3 were associated with a shorter survival time of the HCC patients. In vitro, B7-H3 expression was upregulated at both the mRNA and protein levels in phorbol 12-myristate 13-acetate (PMA)-induced THP-1 cells cocultured with HepG2 cells in a Transwell system. In addition, B7-H3 promoted PMA-induced THP-1 cells to differentiate into the M2 phenotype, with evidence of increases in arginase 1 (Arg1), vascular endothelial cell growth factor (VEGF) and macrophage-derived chemokine (CCL22) mRNA following coculture with HepG2 cells. However, this phenomenon was abrogated through knockdown of B7-H3 by RNA interference or by blocking the signal transducer and activator of trans-cription 3 (STAT3) signaling pathway. Overall, these results suggest that the B7-H3-mediated STAT3 signaling pathway is an important mechanism for inducing M2-type polarization of TAMs, which accelerates HCC development. Our findings may support the development of novel therapeutic strategies for HCC patients through the skewing of the TAM phenotype by targeting the B7-H3 signaling pathway.

Interleukin-21 inhibits HBV replication in vitro.

BACKGROUND: Cytokines are crucial factors in the non-cytolytic antiviral process to inhibit HBV gene expression and replication. Interleukin (IL)-21 has been suggested to play an important role in HBV infection, but it remains unknown whether IL-21 can inhibit HBV replication or how it inhibits HBV replication. METHODS: In this study, we investigated the influence of IL-21 on HBV replication based on human hepatoma Huh7.93 cells co-cultured with human peripheral blood mononuclear cells (PBMCs) and the possible correlation among IL-21, interferon-γ, tumour necrosis factor-α and IL-10. RESULTS: We demonstrated that the decrease of IL-21 expression and the increase of IL-10 expression in PBMCs could promote HBV replication in vitro. We further revealed that IL-21 is not only able to effectively suppress HBV replication directly but also reduce HBV replication by inhibition of IL-10 secretion. CONCLUSIONS: Our results provide important evidence for the non-cytolytic antiviral mechanism mediated by cytokines and their interactions in chronic hepatitis B.