Effect And Mechanism Of AGR2 Epitope Peptide In Enhancing The Sensitivity Of Hepatocellular Carcinoma To NK Cells

Objective:Liver cancer is the second most common cancer in the world.Hepatocellular carcinoma(HCC)is the major histopathologic subtype of primary liver cancer which accounts for 70%to 90%.At present,several clinical treatments for liver cancer include surgical resection,chemotherapy,radiotherapy,liver transplantation,transcatheter arterial chemoembolization(TACE),radio-frequency ablation(RFA)and other treatment methods.Patients with early HCC can be effectively treated with surgical resection or liver transplantation.However,when HCC patients show obvious symptoms,most in the middle and late stage,the disease is often more serious.Patients with advanced liver cancer have poor tolerance to interventional chemotherapy and other treatment methods due to their poor liver function which have poor prognosis and short survival time.Therefore,identification of patients with poor prognosis in early stage and effective treatment for advanced HCC patients are the focus of current research.The occurrence,development,metastasis and recurrence of hepatocellular carcinoma are closely related to the immune system.Liver organ contains a large number of lymphocytes,among which NK cells account for about 30%of the total hepatocellular lymphocytes,far exceeding the proportion of NK cells in the peripheral blood,suggesting the important role of NK cells in the immunotherapy for hepatocellular carcinoma.The ability of NK cells to kill tumor cells depends on the synergistic effect of inhibitory receptors and activated receptors on the cells.When the activated receptors bind to their corresponding ligand and produce a stronger activating signal,it can activate NK cells to play an anti-tumor effect.NKG2D receptor is the major activated receptor of NK cells,which is expressed in almost all NK cells.Furthermore,NK cells could be activated without antigenic stimulation.MICA and MICB are the first discovered NKG2D ligands,which are expressed on the surface of most epithelial tumor cells.They can activate the immune response by binding with NKG2D receptor,and recruit NK cells to carry out effective immune killing effect on cells expressing MICA/B.However,MICA/B could be shed for evading attack by immune cells as tumor cells proliferate.Therefore,upregulation of MICA/B molecules on the surface of tumor cells can effectively enhance the sensitivity of tumor cells to NK cell killing activities and reverse the tumor immune escape response.The regulatory mechanisms of MICA/B are different in different tissues,so it is also important to explore the regulatory mechanisms of MICA/B molecules in HCC patients.Anterior gradient protein 2(AGR2)was highly expressed in breast cancer,colorectal cancer,pancreatic cancer and prostate cancer.It has been reported that the AGR2molecule comprises human leukocyte antigen(HLA)-A*0201-binding epitopes could be presented by DC cells and recognized by human cytotoxic T lymphocytes,which could against colorectal cancer.We found in previous studies that after the incubation of DC cells with AGR2 epitope peptide,the content of the activated ligand MICA/B on NK cells increased significantly which suggesting that AGR2 epitope peptide may regulate the expression of MICA/B.Currently,the study of T cell epitope peptide is only limited to inducing tumor targeted CTL,and there is no report on the function of epitopes in NK cells anti-tumor immunity.The main research purposes:Our study aims to analyze whether AGR2 epitope peptide can enhance the killing effect of NK cells on HCC cells.AGR2 epitope peptides were studied to enhance the sensitivity of liver cancer cells to NK cell killing activity by up-regulating the expression of MICA/B molecule on HCC cells.To explore the molecular mechanism of AGR2 epitope peptide upregulating MICA/B expression in HCC cells.To verify whether AGR2 epitope peptides can affect the expression of MICA/B molecules in HCC xenograft tumors in vivo.Methods:1.NK cells were purified and collected:Fresh peripheral blood was collected from healthy volunteers,mononuclear cells were isolated by the method of Ficoll-hypaque density gradient centrifugation,and CD3~-CD56~+NK cells were purified by magnetic cell sorting(MACS).2.The detection of NK cells function:After NK cells were treated with AGR2 epitope epitopes,the contents of IFN-?,Granzyme B and Perforin in cell supernatant were detected by ELISA.Flow cytometry was used to detect the expression of NKG2D receptor on the surface of NK cells and the expression of CD107a in NK cells.The cytotoxicity of NK cells against hepatocellular carcinoma was determined by LDH release assay.3.Detection of MICA/B expression:After incubation of Bel-7402 and Huh-7 cells with AGR2 epitope peptide for 24 hours,the mRNA expression of MICA and MICB were detected by qRT-PCR.Flow cytometry was used to detect the surface expression of MICA/B on Bel-7402 and Huh-7 cells.Western blot was used to determine the protein expression of MICA/B in the membrane of Bel-7402 and Huh-7 cells.The contents of soluble MICA and soluble MICB molecules in the cell supernatant were determined by ELISA.4.Chromatin immunoprecipitation:CHOP antibody binds to the transcription activator AP-1 in the MICA/B promoter region,Western Blot was used to detect the content of AP-1,the transcription factor CHOP and AP-1 complex regulated the transcription of MICA/B molecules,and PCR was used to detect the amount of MICA and MICB promoter region.5.Animal experiments:Four weeks of female NOD/SCID mice were used to construct the transplanted tumor model of human hepatocellular carcinoma cell line Bel-7402.The AGR2 epitope peptide and NK cell tail vein was injected into the tumor to detect the changes in tumor volume growth curve and tumor weight.6.Statistical analysis:SPSS 16.0 statistical analysis software was used for data analysis.The independent sample T test was used for the comparison between groups,and the data were expressed as mean±standard deviation.Results:1.AGR2 epitope peptide enhances the killing effect of NK cells on HCC cells.The LDH release assay showed that the cytotoxicity of NK cells on hepatocellular carcinoma cells Bel-7402 and Huh-7 after treated with AGR2 epitope peptide were increased to 45.29%±1.82%and 38.87%±1.06%respectively at E:T=2:1,increased to65.31%±1.60%and 62.20%±1.59%respectively at E:T=4:1.NKG2D antibody blocks NK cells,and AGR2 epitopes cannot effectively enhance the killing ability of NK cells.2.AGR2 epitope peptide upregulated the expression of MICA/B in HCC cells.The mRNA expression levels of MICA and MICB in Bel-7402 cells were increased by 2.45±0.34 times and 1.99±0.44 times respectively.The expression mRNA levels of MICA and MICB in Huh-7 cells increased by 2.06±0.53 times and 1.74±0.46 times respectively.Western blot was used to determine the level of MICA/B proteins in Bel-7402 and Huh-7cell membranes after treated with AGR2 epitope peptide which were increased by 2.52±0.33 times and 1.51±0.26 times respectively.Flow cytometry was used to detect the level of MICA/B on the surface of Bel-7402 and Huh-7 cells treated with AGR2 epitope peptide.The average fluorescence intensity of MICA/B on the surface of the cells of Bel-7402 and Huh-7 cell increased to 488.47±24.54 and 335.94±88.02 respectively,with statistically significant differences.3.AGR2 epitope peptide upregulates the expression of transcription factor CHOP protein by activating the p38 MAPK signaling pathway,and regulate the transcription of MICA/B in HCC by binding with the transcription activator AP-1,the CHOP/AP-1 complex regulates the MICA/B transcription.p38 specific inhibitor SB203580 could significantly weakened the ability of AGR2 epitope peptide in upregulating MICA/B in HCC cells.AGR2 epitope peptide could also upregulate the expression of CHOP protein significantly.The content of AP-1 and promoter region of MICA/B increased by AGR2 epitope peptide determined by Western blot and PCR.4.NOD/SCID mouse model verified that AGR2 epitope peptide can effectively enhance the effect of NK cells in inhibiting the growth of transplanted tumor,and SB203580 can significantly inhibit the effect of AGR2 epitope peptide in promoting the immune sensitivity of HCC cell to NK cells in vivo.The mRNA and protein expression of MICA and MICB were significantly increased by AGR2 epitope peptide in xenograft tumors which were detected by q RT-PCR,Western blot,and flow cytometry.Conclusion:1.AGR2 epitope peptide upregulates the expression of MICA/B in Bel-7402and Huh-7 cells,and enhance the sensitivity of HCC cells to the cytotoxicity of NK cells.2.AGR2 epitope peptide upregulated the expression of transcription factor CHOP by activating the p38 MAPK signaling pathway.Upregulated CHOP binds to the transcription activator AP-1 in the MICA/B promoter region and the complex of CHOP/AP-1 could upregulate the transcription of MICA/B in HCC.3.AGR2 epitope peptide significantly increased the mRNA and protein expression of MICA/B in xenograft tumors.In combination with AGR2 epitope peptide,the inhibitory effect of NK cells on the growth of transplanted tumors was significantly increased.