Study On The Effects Of MPGES-1 Inhibitor DMC On Immune Microenvironment Of HBV-related Hepatocellular Carcinoma

Programmed death receptor 1(PD-1)is the main immune response suppressor molecule,which is especially critical in many strategies for the recovery of exhaustion-specific T cells.Tumor immunotherapy aimed at blocking PD-1 and its ligand 1(PD-L1)have become valuable means to cure inflammation and cancer.However,in the immunotherapy of hepatocellular carcinoma(HCC),especially in hepatitis B virus(HBV)-related HCC,the immune microenvironment of HBV-HCC is more immunosuppressive than that of non-virus-related HCC,the characteristics of interaction between liver cancer cells and immune cells in the microenvironment,and the targeted drugs have not yet been found breakthrough.Single PD-1 or PD-L1blocking therapy can not achieve the ideal effect.Therefore,combination therapy or multi-target effect may be an effective strategy at present.Prostaglandin E2(PGE2)is an important inflammatory mediator produced by HBV infection,which is closely related to specific T cell dysfunction.Microsomal prostaglandin E synthase-1(m PGES-1)is the terminal rate-limiting enzyme in the process of PGE2 synthesis.Targeted inhibition of m PGES-1 has both anti-inflammatory and anti-tumor functions,and can avoid the side effects of cyclooxygenase(COX)inhibitors.Previous studies in our group showed that HBV X protein(HBx)up-regulated m PGES-1 expression at the transcriptional level and promoted PGE2 secretion.2,5-dimethylcelecoxib(DMC)could target the inhibition of m PGES-1 enzyme activity and effectively down-regulate the level of PGE2,indicating that DMC can improve the immunosuppressive state of HBV-related HCC.However,many important questions such as the overall effect of DMC on HBV-related HCC immune microenvironment and whether it can regulate the expression of immune checkpoint molecules,especially the unique intervention on PD-1/PD-L1 pathway,have not been solved.To this end,our study intends to explore the ability of DMC to inhibit tumor proliferation in HBx(+)HCC cells implanted in mice and detect its effects on immune microenvironment,and to clarify the special mechanism of DMC affecting the immune function of HBV-related HCC through cytological experiments,so as to provide a new strategy for combined or multi-target immunotherapy of HBV-related HCC.The purpose of the first part of this study is to investigate the relationship between HBV infection factors and tumor immune efficacy in clinical HCC patients.Immunohistochemical technique was used to detect the expression of CD8 and PD-L1proteins in clinical HCC tissues,and the correlation between them and clinicopathological parameters was analyzed.It was found that compared with HBV negative HCC,the CD8 expression level of HBV positive HCC tissues was significantly lower(0.6424±0.05925 vs.6.221±1.071,P<0.001),while the PD-L1expression level was higher(5.566±0.5511 vs.0.5210±0.1034,P<0.001).Correlation analysis of clinicopathological parameters showed that the expression of CD8 and PD-L1 was closely related to the factors of HBV infection,but not with other parameters.Western blot confirmed that CD163(a marker of tumor-associated macrophage M2)in HBV-positive HCC was positively correlated with PD-L1expression,suggesting that CD163 was also highly expressed in HBV-positive HCC.The second part of our study is to explore the construction of m PGES-1 reporter cells by CRISPR/Cas9 technology and its application in the screening of small molecular inhibitors.After the PX459:sg RNA2 vector with the best targeted splicing efficiency was identified by T7E1 experiment,PX459:sg RNA2 and donor vector were co-transfected to construct m PGES-1 reporter cells carrying td Tomato gene(showing red fluorescence).The results of immunofluorescence showed that m PGES-1 protein(green fluorescence)superimposed with red fluorescence and co-located successfully;Sanger sequencing confirmed that the expected td Tomato sequence was successfully inserted into the stop codon of target gene m PGES-1;flow cytometry showed that the fluorescence intensity of PE channel in m PGES-1 reporter cells was significantly stronger than that in wild-type cells,and decreased significantly after m PGES-1si RNA transfection.After treatment with different m PGES-1 inhibitors,the expression level of PGE2 and red fluorescence intensity of reporter cells decreased in different degrees,and DMC was the best.Western blot assay also confirmed that DMC inhibited the expression of PD-L1 in HBx HCC cells most obviously.The aim of the third part of this study is to analyze the effects of DMC on tumor growth and immune efficacy in mice with hepatocellular carcinoma.For this reason,we successfully established a mouse model of implanting tumor with HBx(+)HCC cells(inoculated with Hepa1-6/HBx cells)and intraperitoneally injected DMC or/and Atezolizumab for one week.The results showed that compared with the control group,DMC or Atezolizumab alone could significantly inhibit the growth of tumor,increase CD8 expression and decrease PD-L1 and CD163 expression in tumor tissue,while the effect of combination group was stronger than that of each single drug group.The results suggested that DMC can improve the tumor immune microenvironment of HBx(+)HCC by inhibiting PD-1/PD-L1 pathway and immunosuppressive cells dominated by M2 TAM cells.The purpose of the fourth part of this study is to explore the mechanism of DMC inhibiting HBx-induced PD-L1 expression in HCC cells.To this end,we constructed a co-culture system of HCC cells overexpressing HBx and peripheral blood mononuclear cells(PBMCs),and treated with DMC.The effect of DMC intervention on immune factors in the co-culture system was detected by flow cytometry.The results showed that HBx could induce the expression of PD-L1 in HCC cells and immune cells and inhibit the level of CD8~+T cells,while DMC treatment could reverse these conditions.The effect of DMC on the signal pathway of HCC cells was analyzed by microarray chip.KEGG analysis revealed that AMPK pathway played an important role after the intervention of DMC.The results of western blot showed that DMC activated AMPK?(increased phosphorylation level)and inhibited PD-L1 in a time-and concentration-dependent manner.AMPK inhibitors can rescue the inhibitory effect of DMC on PD-L1.In addition,the co-immunoprecipitation assay showed that the interaction between PD-L1 and AMPK?was more obvious after DMC treatment,indicating that DMC inhibited HBx-induced PD-L1 expression by activating AMPK?.Fluorescence confocal experiment suggested that with the extension of DMC treatment,PD-L1 protein was gradually located in the endoplasmic reticulum of HCC cells.Mass spectrometric analysis also showed that PD-L1 protein processing was abnormal.Moreover,IP-Western blot confirmed that DMC led to ubiquitin degradation of PD-L1 through the action of E3 ligase RBX1.Conclusion:1.Compared with HBV negative HCC,the level of CD8 protein in HBV positive HCC tissues was significantly lower,while the level of PD-L1 protein was higher.In the correlation analysis of clinicopathological parameters,CD8 protein and PD-L1protein were only closely related to HBV factors,while CD163 was positively correlated with PD-L1 expression.2.m PGES-1 inhibitor DMC can increase the level of infiltrating CD8~+T cells and inhibit the expression of PD-L1 and CD163 in HBx(+)liver cancer model mice,and DMC can better improve the immune efficacy in the inflammatory microenvironment of HBx(+)liver cancer,which is mainly reflected in the more effective inhibition of immunosuppressive factors mediated by PD-L1 and the immunosuppressive cells dominated by M2 TAM cells.DMC combined with Atezolizumab has more significant anti-tumor effect and stronger blocking effect on PD-1/PD-L1 pathway.3.DMC promotes ubiquitin degradation of HBx-induced PD-L1 protein in HCC cells by activating AMPK pathway,and the degradation process is mediated by E3 ligase RBX1.