The Mechanism Of Autophagy Promotes Immune Evasion Of P53 Mutant Hepatoma Cells By Degrading MHC-Ⅰ

Objective:Hepatocellular carcinoma(HCC)is the third leading cause of cancer death worldwide,causing great suffering and loss every year.At present,the treatment of HCC is mainly surgery combined with chemoradiotherapy,but the five-year survival rate of patients is still low under the current treatment measures.The development and prognosis of HCC are the result of the interaction between HCC cells and the body’s immune system.Most HCC patients have immune tolerance and chronic inflammation caused by underlying liver disease,further enhancing immune escape and allowing cancer cells to grow.CD8~+T lymphocytes are the main mediator of anti-tumor immunity,and regulating the response of CD8~+T cells has always been the focus of cancer immunotherapy.When CD8~+T cells specifically recognize antigenic peptides presented by major Histocompatibility complex(MHC)molecules,they can be activated and kill tumor cells.In fact,research has shown that tumors have developed various methods to limit antigen recognition and cause immune escape.Different mechanisms of tumor immune escape determine different clinical immunotherapy regimens.Therefore,it is very important to explore the molecular mechanism of tumor immune escape for the relevant strategies of tumor immunotherapy.Cancer is a collection of diseases characterized by abnormal and uncontrolled cell growth caused by genetic mutations.About 50%of tumors have p53 mutations,and about 30%of patients with HCC have p53 mutations.Recent studies have found that p53 mutations participate in the remodeling of tumor immune microenvironment and become the immune driving factor of tumor occurrence.Therefore,this study aims to deeply explore the correlation between p53 mutation and tumor immune escape,as well as the specific mechanism of p53 mutation regulation of immune escape of HCC cells,to provide a theoretical basis for the occurrence,development,and treatment of HCC.Materials and methods:1.Materials:Human HCC cell lines Hep3B and PLC/PRF/5,mouse HCC cell line Hepa1-6,C57BL/6 mice,OT-I mice,and NOD-SCID immune deficient mice.2.Methods:(1)Bioinformatics analysis:Clinical data,expression data,and mutation data of hepatocellular carcinoma were obtained from TCGA.Phenotypic information was grouped according to p53 mutation and wildness,and clinical case data of 265 liver cancer patients were extracted for subsequent statistical analysis.Based on the R environment,Limma package was used for differential gene analysis,and GSEA software was used for enrichment analysis.(2)Cell culture:Cultures human liver cancer Hep3B and PLC/PRF/5 cell lines.(3)Transfection:Lentivirus expression system technology was used to transfect PLVX in Hep3B cell line as the control group,and transfected p53 R249S as the experimental group;PLKO was transfected into the PLC cell line as the control group,while shp53,or p53 knockout,was transfected into the experimental group.Construct Hepa1-6mouse liver cancer cells with stable overexpression of p53 R249S mutations and Hepa1-6-p53R249S cells with overexpression of OVA.(4)MTT experiment:To detect the proliferation ability of liver cancer cells.(5)Clone formation experiment:To detect the colony forming ability of liver cancer cells.(6)Cell scratch test:To detect the ability of cell migration of liver cancer.(7)Mouse subcutaneous tumorigenesis experiment:Hepa1-6 mouse liver cancer cells with stable overexpression of p53 R249S mutation and Hepa1-6 p53 R249S cells with overexpression of OVA were used for subcutaneous tumorigenesis,and tumor size,weight,and volume were measured.(8)Lymphocyte extraction from mouse tumor tissue:Using tumor tissue digestion solution and density centrifugation method to extract lymphocytes from subcutaneous tumor bodies of mice.(9)Human peripheral blood CD8~+T cell extraction:Venous blood of healthy volunteers was taken to collect peripheral blood mononuclear cell with human lymphocyte isolation solution,and then CD8~+T cells were obtained by magnetic bead sorting and in vitro activator stimulation.(10)Cell co culture experiment:Coculture human liver cancer cells transfected with the virus with CD8~+T cells for subsequent flow cytometry analysis;OT-Ⅰmouse CD8~+T cells were co cultured with Hepa1-6-OVA-p53R249S mouse liver cancer cells for subsequent LDH killing experiments.(11)Flow cytometry:The infiltration of T lymphocytes in the subcutaneous tumor of mice and the levels of granzyme B and perforin in the tumor CD8~+T lymphocytes were detected;Detecting the expression of H2K in subcutaneous tumors of mice;Detect the expression of MHC-Ⅰon the surface and intracellular of liver cancer cells;The number of CD8~+T and the levels of granzyme B and perforin in CD8~+T were detected after co culture.(12)LDH killing experiment:Calculate the killing effect of CD8~+T cells on tumor cells.(13)Immunohistochemistry:Detection of protein expression levels of CD8 and H2K in subcutaneous tumor tissue of mice.(14)Immunofluorescence:Used to detect the co localization of MHC-Ⅰand LC3 in cells.(15)Real time-PCR:Detection of m RNA expression levels of TAP1,TAP2,ERAP1,ERAP2,β2m and MHC-Ⅰin HCC;Detect the m RNA levels of p53 and H2K in subcutaneous tumor tissue of mice.(16)Western blot:Detection of protein expression levels of TAP1,TAP2,ERAP1,ERAP2,β2M,MHC-I,LC3,p62,CDKN2A,BIRC5,and SPHK1 in HCC;Detect the protein levels of p53 and H2K in subcutaneous tumor tissue of mice.(17)Transmission electron microscopy:Used to observe the condition of autophagosomes.(18)Statistical analysis:All data in this experiment were analyzed using software SPSS22.0.All data were presented in the form of mean±standard deviation,and all experiments completed three independent repeated experiments.At the same time,Prism 9(Graph Pad Software)completed the histogram quantization and line chart in this experiment.When comparing two samples of normal distribution data,t-test was used,and single factor analysis of variance was used for comparison between multiple groups.When P<0.05,the data is considered to have statistical differences.3.Protocol:3.1:Clinical significance of p53 mutations in liver cancer cells and tissues(1)Based on R4.1.2 environment,gene mutation analysis was conducted on HCC sample data in the TCGA database:p53 mutation in HCC patients,overall survival of HCC patients with p53 mutation and the relationship between p53 mutation and clinicopathological characteristics were compared and analyzed;(2)The Lentivirus expression system technique was used to transfect PLVX and overexpressed p53R249S in Hep3B cell lines as the control group and experimental group respectively.PLC cell lines were transfected with PLKO as the control group and transfected with shp53(p53knockout)as the experimental group.Subsequently,the effects of p53 mutation on the malignant behavior of hepatocellular carcinoma cells were investigated by MTT proliferation assay,plate cloning formation,and cell scratch assay;(3)The effect of p53R249S mutation on tumor growth was verified in vivo;(4)A mouse model of severe immunodeficiency was used to determine whether the immune process was involved in tumor progression induced by p53R249S mutation.3.2:p53R249S mutation leads to weakened immune killing ability of CD8+T cells(1)Bioinformatics was used to analyze the correlation between p53 mutation and tumor immune-related pathways in HCC;(2)1×10~6 Hepa1-6-PLVX cells and Hepa1-6-p53R249S cells were inoculated subcutaneously into C57BL/6 mice.On the 21st day after tumor formation,lymphocytes were extracted from the tumor tissue and the infiltration of immune cells,such as CD45~+,CD3~+,and CD8~+,were analyzed by flow cytometry;(3)In vivo neutralizing antibody and immunohistochemical tests were used to determine whether CD8~+T cells participated in p53R249S mutation-mediated immune escape in mouse tumor formation experiment;(4)Flow cytometry,magnetic bead sorting,in vitro co-culture,and LDH killing test was used to verify whether p53R249S mutation regulated the function of CD8~+T cells.3.3:p53R249S mutation affects immune killing function through autophagy pathway(1)Using the OTI mouse model and constructed Hepa1-6-p53R249S cells expressing OVA,Flow cytometry,immunohistochemistry,and LDH killing experiments were used to verify whether the p53R249S mutation regulates the specific antigen processing-presentation effect and thus reduces the immune recognition and killing;(2)We analyzed the distribution of MHC-I by PCR,western blot,flow cytometry,and immunofluorescence,to explore the mechanism by which p53R249S mutations lead to an impaired immune recognition process;(3)Explore the mechanism of p53R249S mutation affecting MHC-I degradation by western blot,immunofluorescence confocal,and flow cytometry;(4)Use flow cytometry,immunohistochemistry,LDH killing experiments,and autophagy inhibitor chloroquine to determine whether autophagy is involved in p53R249S mutation-mediated immune escape;(5)The effect of targeted autophagy on tumor growth mediated by p53R249S mutation was investigated by subcutaneous tumor formation in mice.Results:Part I:p53 mutated liver cancer cells evade immune surveillance leading to poor prognosisBioinformatics analysis:1.Bioinformatics shows that the most common gene in HCC patients is p53 mutation(30%),p53R249S mutation is the most common p53mutation in HCC patients,and the overall survival and disease-free survival of HCC patients with p53 mutation are significantly shorter than those with wild-type p53(P<0.05).p53 mutation is closely related to TNM stage,differentiation degree,portal vein invasion,and lymph node metastasis;In vitro experiment:1.The activity of Hep3B cells overexpressing p53R249S mutation increased significantly,while that of PLC cells knockout p53R249S mutation decreased;2.Hep3B cells overexpressing p53R249S mutation resulted in more growth mass,while PLC cells knockout p53R249S mutation resulted in less growth mass;3.Hep3B cells overexpressing p53R249S mutation showed enhanced migration ability,while PLC cells knockout p53R249S mutation showed decreased migration ability.In vivo experiments in mice:1.The tumor volume and weight of mice transfected with p53R249S mutation increased significantly;2.The tumor growth curve and tumor volume of severely immunodeficient mice with p53R249S mutation did not change significantly.Part II:p53R249S mutation leads to weakened immune killing ability of CD8~+T cellsBioinformatics analysis:1.GSEA analysis found that p53 mutation in HCC tissue was closely related to tumor immunity;In vivo experiments in mice:1.The p53R249S mutation caused less infiltration of T lymphocytes in subcutaneous tumors,and the proportion of CD8~+T cells in all lymphocytes significantly decreased;The p53R249S mutation promotes tumor growth dependent on CD8~+T cells;3.p53R249S mutation can lead to less expression of granzyme B and perforin in CD8~+T cells in tumor;4.p53R249S weakens immune killing by affecting the specific antigen processing presentation of MHC-I molecules.In vitro experiment:1.p53R249S mutation can lead to a decrease in the killing efficiency of CD8~+T cells;2.The p53R249S mutation inhibits the immune killing function of CD8~+T cells.Part III:p53R249S mutation affects immune killing function through autophagy pathwayIn vitro experiment:1.The p53R249S mutation did not alter the m RNA expression level of MHC-I,but the protein level that can lead to MHC-I is weakened;MHC-I protein was normally expressed in the cell membrane and cytoplasm in normal hepatocytes.However,the p53R249S mutant Hep3B cells express only less MHC-I protein on the membrane surface;2.p53R249S mutation leads to increased degradation of MHC-Ⅰthrough autophagy pathway;3.p53R249S mutation promotes autophagy,leading to increased degradation of MHC-I molecules and inability to effectively present on the membrane surface.Bioinformatics analysis:CDKN2A and SPHK1 are key genes that promote autophagy through p53 R249S mutations.In vivo experiments in mice:1.Blocking autophagy could reverse p53R249S mutation and lead to less expression of granzyme and perforin in CD8~+T cells;2.Inhibition of autophagy can reduce tumor growth mediated by p53R249S mutation;3.The p53R249S mutation affects the specific antigen processing presentation of MHC-I molecules through the autophagy pathway,resulting in a weakened immune killing ability.Conclusion:1.p53R249S mutation is the most common p53 mutation in patients with HCC,and p53 mutation is closely related to the prognosis of patients with HCC;The p53R249S mutation promotes the growth of HCC,which may depend on an intact immune system.2.p53 mutation in HCC tissues is strongly correlated with tumor immune-related pathways,and p53R249S mutation results in immune escape by inhibiting the function of CD8~+T cells.3.p53R249S mutation reduces CD8~+T cell immune recognition and killing by affecting specific antigen processing and presentation of MHC-I molecules.This weakened immune response is due to the increased degradation of MHC-I molecules caused by p53R249S mutation via autophagy pathway and thus inability to effectively present on the membrane surface.In addition,inhibition of autophagy reverses p53R249S mutation-mediated immune escape.