The Effect Of Knock-Down Immune Checkpoint B7-H3 On The Growth And Invasion Of Hepatocellular Carcinoma

Background and Objectives Hepatocellular carcinoma(HCC)is an important part of the global disease burden and mortality.Primary liver cancer has a poor prognosis,second only to the low survival rate of pancreatic cancer.In addition to surgery、TACE、local ablation therapy、radiation intervention and so on,anti-CTLA-4、anti-PD-1 and anti-PD-L1 monoclonal antibody treatment can significantly improve clinical results and immune checkpoint antibody therapy has gradually become an important treatment method.The discussion of immune checkpoints has become a hot spot in cancer research.The B7-H3 gene(CD276)was discovered in the c DNA library of human dendritic cells(DC)by Chapoval et al.and is associated with increased risk of recurrence、poor pathological type、and poor prognosis.Based on the above research background,we used RNA interference technology to knockdown mouse’s B7-H3 expression in hepatocellular carcinoma cell line and experiment biological behaviors of the stable cells in vitro,including cell apoptosis,proliferation,migration and invasion.Then orthotopic liver cancer model is constructed in a normal immune system mouse.We explore the effect of growth and invasion in mouse liver cancer cells through knock-down B7-H3 from vivo and vitro.The above research lays the foundation for further research on the effect of B7-H3 on the immune microenvironment of tumor-bearing mice and provides a theoretical basis for the application of B7-H3 antibody in the clinical treatment of HCC.Methods1.The expression level of mouse liver cancer cell lines was detected by RT-PCR.2.The knock-down B7-H3 plasmid with luciferase was constructed through RNA interference.The lentivirus were packaging by 3rd-generation lentiviral packaging system for B7-H3 knock-down(hepa1-6/shRNA-B7-H3-luc).3.Biological behaviors of the stable cells were proformed,including cell apoptosis,proliferation,migration and invasion.4.The tumorigenic ability、survival time、tumor size and tumor tissue morphology of mouse hepatocellular carcinoma were observed through constructing orthotopic hepatocellular carcinoma model of mice with hepa1-6/shRNA-B7-H3-luc cell.Result1.B7-H3 gene-expression profile were: hepa1-6>H22.2.B7-H3 knock-down vectors were successfully constructed.Then,successful knockdown of B7-H3 were confirmed by Western-blot and Q-PCR.3.The migration,invasion and proliferation decreased in hepa1-6/shRNA-B7-H3.The apoptosis was increased through flow cytometry in vitro.4.Orthotopic hepatocellular carcinoma tumor-bearing model was constructed in C57/BL6 mouse.It was found that tumor volume and growth are below in hepa1-6/shRNA-B7-H3-luc in vivo.5.The infiltration of inflammatory cell in HCC tissue increased significantly by HE staining.Conclusions1.Successfully construct a lentiviral vector with B7-H3 knockdown and obtain a mouse hepatocellular carcinoma cell line with low B7-H3 gene expression and luciferase label.2.It was found that knock-down B7-H3 can inhibit the invasion,migration and proliferation ability of mouse liver cancer cells and promote early apoptosis of mouse HCC in vitro.3.Established a mouse orthotopic liver cancer model in C57/BL6 mouse.Knock-down B7-H3 can slow down tumor growth and promote the infiltration of inflammatory cells around the tumor.It suggests that B7-H3 is related to tumor immune microenvironment.infiltration of inflammatory cells around the tumor increased.Prompt B7-H3 is related to tumor immune microenvironment...