The Regulation And Mechanism Of FXR On HVEM Expression In Non-small Cell Lung Cancer

Background:Lung cancer is one of the most common malignant tumors in humans and one of the leading causes of cancer-related deaths worldwide.Non-small cell lung cancer(NSCLC)is the most common type of lung cancer(about 85%).In recent years,great progress has been made in comprehensive treatment of lung cancer,especially in immunotherapy represented by monoclonal antibodies targeting programmed Cell Death protein 1(PD-1)and its ligand PD-L1.However,the prognosis of non-small cell lung cancer(NSCLC)is still very poor(the 5-year survival rate is less than 20%),and the effect of immunotherapy only covers a part of patients with high PD-L1 expression.For the treatment of patients with PD-L1 negative effect is poor,and the course of treatment,some patients will appear a variety of reasons resistance and immune reactions,and for those of PD-1/PD-L1 immune therapy effect is poorer,or are not suitable for PD patients treated-L1,whether other immunoreplacement therapy can improve its prognosis becomes the next research direction.However,some recent studies have found that the expression level of herpes virus entering medium(HVEM)is increased in NSCLC patients with low PD-L1 expression,and HVEM is closely related to the progression and aggressiveness of various types of tumors.As a member of the tumor necrosis factor receptor superfamily,the Herpes Virus Entry Mediator(HVEM)is highly expressed in melanoma,lung cancer,and many gastrointestinal tumors.In recent years,studies have shown that upregulation of HVEM can promote tumor progression and aggressiveness in several cancers,including lung cancer and hematological malignancies.HVEM can bind to a variety of ligands in the immune system including TNF superfamily cytokines LTa and Light and immunoglobulin superfamily members BTLA and CD160 as checkpoint regulators.The combination of HVEM and the different ligands mentioned above can affect the immune microenvironment by producing co-stimulatory or co-inhibitory signals respectively.Previous studies have found that although co-stimulatory or co-inhibitory signals can exist in HVEM simultaneously,inhibitory signals dominated by HVEMBTLA dominate the HVEM signaling pathway and participate in tumor immune escape.Moreover,the above effects are likely to be realized through HVEM-BTLA inhibiting the function of CD8+T cells and thus changing the tumor microenvironment(TME).Farnesoid X receptor(FXR)is a member of the nuclear receptor superfamily,which is mainly expressed in liver,gastrointestinal tract and endocrine organs and regulates the expression of target genes related to enterohepatic circulation and lipid homeostasis.A number of recent studies have shown that FXR plays a pathogenic role in colon cells,esophageal cancer,breast cancer and pancreatic cancer,suggesting a correlation between FXR and tumors.It has been reported that the expression of FXR is significantly upregulated in NSCLC tumor tissues compared with adjacent lung tissues,and the expression of FXR is negatively correlated with that of PD-L1.Therefore,the upregulation of FXR can lead to negative/low expression of PD-L1,thus reducing the efficacy of anti-PD-1/PD-L1 immunotherapy in NSCLC.In addition,studies have shown that in human NSCLC cells,high expression of FXR can inhibit the proliferation and immune function of CD8+T cells,thus inhibiting the anti-tumor effect.Both FXR and HVEM change the tumor immune microenvironment through their effects on CD8+T cells,but whether there is a correlation between them has not been studied.Objective:To study the effect of FXR on HVEM expression level and its molecular mechanism in non-small cell lung cancer,and the effect of FXR and HVEM on tumor immune microenvironment.Methods:1.Immunohistochemical analysis was performed on tumor tissue samples from 149 patients with advanced NSCLC who received chemotherapy in Shandong Provincial Hospital.Mann-whitney test,Chi-square test and Spearman rank correlation analysis were used to analyze the correlation between FXR expression level and HVEM expression level in NSCLC cell lines by immunohistochemical results and clinical database results.2.In vitro experiments,we infected A549 cells with FXR lentivirus and the control MOCK lentivirus respectively,and constructed A549 cell groups with different levels of FXR expression:A549 FXR(stable overexpression of FXR gene),A549 MOCK(normal expression of FXR gene);H1975 cells were transfected with NC small interfering RNA and FXR small interfering RNA respectively,and H1975 cell groups with different levels of FXR expression were constructed:H1975NCsiRNA(normal expression of FXR gene),H1975FXRsiRNA-1(low expression of FXR gene),H1975FXRsirNA-2(low expression of FXR gene);Western Blot was used to check the expression level of FXR in the cells after the above treatment to confirm the above treatment.The expression levels of HVEM in A549 and H1975 cells with different levels of FXR expression were detected by immunolabelling assay.qPCRwas used to detect the mRNA expression levels of HVEM in A549 and H1975 cells with different levels of FXR expression.3.In order to explore the regulatory mechanism,HVEM CHIP primers were constructed,and the regulation of different FXR expression levels on the DNA of coprecipitated HVEM in A549 cells and H1975 cells was verified by CHIP immunoprecipitation assay,PCR assay and DNA gel electrophoresis.The HVEM promoter plasmid was constructed and the regulation of FXR expression levels on HVEM promoter activity in A549 cells and H1975 cells was detected by Luciferase.4.Add MK2206 or PD0325901 or Stattic into cells in vitro,corresponding to Akt or ERK1/2 or STAT3 signaling pathways in A549 cells after inhibiting FXR overexpression respectively.Western blotting assay was used to detect the trend of FXR expression levels in control and inhibitor groups and to verify the blocking levels of Akt,ERK1/2 and STAT3 signaling pathways.Flow Cytometry was used to detect the expression level of HVEM cells after the same treatment,and fluorescence quantitative PCR was used to detect the trend level of HVEM mRNA after the same treatment.5.With the consent of the patients,peripheral venous blood of confirmed NSCLC patients before chemotherapy was taken,PBMC was isolated and CD8+T cells were magnetic isolated and co-cultured with A549 cells and H1975 cells with different FXR expression levels,respectively.Apoptosis of CD8+T cells and changes in secretion cytokines were detected by flow cytology.Crystal violet staining and lactate dehydrogenase(LDH)toxicity were used to detect the killing level of tumor cells.6.In vivo experiment,40 mice were randomly divided into 4 groups(10 mice in each group).Intraoperatively,Z-Guggulone(Z-GS)with different concentrations was injected introperitoneally for 20 days,and the tumor model of Lewis lung carcinoma(LLC)was established.After completion of the treatment process,the tumor was removed from the mice,and the changes of HVEM expression level in the transplanted tumor tissues of different groups were detected by immunohistochemical staining(IHC),and the trend of HVEM mRNA expression level in the lung cancer tumor tissues of different groups of mice was detected by real-time fluorescence quantitative PCR.Results:1.Immunohistochemical analysis of human no-small cell lung cancer samples showed that FXR expression level was positively correlated with HVEM expression level.Clinical and database data analysis yielded similar results.Within a certain rage,PD-L1 mRNA and surface PD-L1 expression level gradually increased with the concentration of Z-GS and the prolongation of the treatment time in A549,H1975 and LLC.The activation of PD-L1 promoter in A549 and H1975 increased with the increasing concentration of Z-GS.2.Within a certain range,the expression level and mRNA level of HVEM cell surface in A549 cells after FXR overexpression were significantly higher than those in control cells without FXR overexpression.After intracellular FXR was inhibited by transfection of small interfering RNA,the expression level and mRNA level of HVEM cell surface in H1975 cells were significantly lower than those in cells without transfection of small interfering RNA.3.With FXR overexpression,the activity of HVEM promoter in A549 cells increased correspondingly,and the amount of co-precipitated DNA also increased correspondingly.On the contrary,after FXR,HVEM promoter activity in H1975 cells also decreased correspondingly,and the amount of coprecipitated DNA also decreased correspondingly.4.After transfection of A549 cells with FXR overexpressing lentivirus,the A549 cells were treated with Akt signaling pathway inhibitor MK2206 or ERK1/2 signaling pathway inhibitor PD325901 or SATA3 signaling pathway inhibitor STATtic.Both intracellular HVEM mRNA expression level and cell surface HVEM expression level were slightly lower than those of untreated FXR overexpressed cells,but higher than those of untreated FXR overexpressed lentivirus transfected cells5.When NSCLC cells were co-cultured with human CD8+T cells,the secretion of CD8+T cells in A549 cell group overexpressing FXR decreased compared with the control group,and the apoptosis of CD8+T cells increased,and the killing effect on tumor cells was also weakened.All these trends were reversed after the administration of BTLA inhibitors.On the contrary,compared with the control group,CD8+T cells secreted more cytokines,CD8+T cells had less apoptosis,and their killing effect on tumor cells was enhanced in H1975 cells after FXR gene deletion.6.In the mouse Model of Lewis lung cancer transplanted tumor,immunohistochemical staining of mouse tumor specimens showed that the expression level of HVEM on the surface of Lewis cells in mice transplanted tumor gradually decreased with the increase of Z-GS concentration inhibiting farnitone X receptor.The same trend was found in real-time quantitative PCR.Conclusion:FXR regulates HVEM expression in NSCLC and further influences the tumor immune microenvironment,in part through Akt and erKl/2 and STAT3 signaling pathways.