| Research background%Lung cancer ranks among the top of all malignant tumors in terms of incidence and has been the first in terms of mortality.With the development of basic science,science and technology,and statistics,lung cancer has been studied more deeply and comprehensively.Among them,many genetic polymorphisms associated with the development of lung cancer have been found in GWAS studies of lung cancer.With the advent of the era of immunotherapy,immune-related gene polymorphisms have again become a hot spot for research.Major histocompatibility complex class I molecules(MHC-I)are central mediators of cellular immunity,regulating the function of cytotoxic T lymphocytes(CTL)primarily by controlling the antigen presentation process and serving as markers for natural killer(NK)cell recognition.m HC-I class I molecules expose intracellular proteins at the cell surface,allowing T cells to detect endogenous or mutated exogenous antigens.An effective CTL response relies on the ability of MHC-I-like molecules to present multiple peptide arrays,and on the other hand,the cellular immune effect of CTL on tumor cells is highly dependent on the expression of MHC-I complexes on the surface of tumor cells and antigen-presenting cells.Because of the important role of the MHC-I pathway in cellular immunity,we hypothesized that genetic variation in genes involved in the MHC-I pathway during tumor antigen presentation is associated with prognosis in patients with non-small cell lung cancer(NSCLC).Therefore,in this study,we used the MHC-I pathway as the target and the GWAS database analysis as the material to analyze the relationship between the MHC-I pathway and the prognosis of NSCLC patients,and to perform a preliminary exploration of newly identified genes associated with prognosis in lung cancer.The First Part Relationship between gene polymorphism and prognosis in MHC-I pathway Research Purpose:1.Two GWAS databases were used as discovery and validation libraries to discover and validate genetic polymorphisms in the MHC-I pathway associated with prognosis in NSCLC patients.Clinical data of lung cancer patients in the discovery database were used for independence testing to find SNPs that were independently and significantly associated with prognosis.2.Combined and stratified analysis of the screened SNPs independently associated with prognosis was performed separately to clarify the relationship between these SNPs and prognosis.The ROC modeling was then used to observe whether it could further improve the survival prediction ability of the already existing NSCLC patients.Materials and methods:Two GWAS databases were used in this study: a GWAS database containing 1185 NSCLC cases with the database name: PLCO;and a GWAS database containing 984 NSCLC cases with the database name: HLCS(Harvard Lung Cancer Susceptibility Study).The former serves as a discovery database and the latter as a validation database.SNPs were extracted and filled for 89 genes on the MHC-I pathway in the discovery database,and then the relationship between all 7811 SNPs and survival was analyzed by multivariate risk regression model.To reduce the possibility of false positives,SNPs significantly associated with survival were corrected for data using the BFDP(Bayesian false-discovery probability)with a correction threshold of 0.80.The corrected 206 SNPs loci were validated in the validation database,after which SNPs significantly associated with survival in both databases were derived.After the independence test,we finally obtained three SNPs on three genes that were significantly and independently associated with survival: ERAP1 rs469783 T > C;NCF2 rs36071574 G > A;PSMF1 rs13040574 C > A.After that,we performed the combination and stratification analysis for each genotype of the three SNPs.Finally,we developed a ROC prediction model to explore whether the newly discovered SNPs could enhance the predictive ability of the existing clinical data on patient survival.The software used in the study were SAS software,R language,and Haplo View.Result:1.Found the relationship between clinical treatment and survival of patients with NSCLC in LibraryIn this study,we found that clinical data from patients in the discovery database had a close correlation with the patient’s survival time and had a statistical significance(P＜0.05).2.Correlation analysis between SNPs in MHC-I pathway and overall survival of patients with NSCLCThe filling of 89 genes in the MHC-I pathway yielded 7811 SNPs,which were validated by BFDP,and 206 SNPs were found to be significantly associated with survival of NSCLC patients in the discovery library.Further validation in the validation library found 24 SNPs significantly associated with survival in both databases.3.Independent SNPs associated with overall survival of NSCLC patients in the PLCO databaseTo identify SNPs that were independently and significantly associated with survival in NSCLC patients in the Discovery Bank,we developed a Cox model in the Discovery Bank that incorporated information on clinical variables and selected components of the Discovery Bank in a multivariate stepwise regression analysis model.Twenty-four SNPs that were significant in both databases were added to the model.Ultimately,three SNPs within these 24 SNPs(i.e.,ERAP1 rs469783 T>C,NCF2 rs36071574 G>A,and PSMF1 rs13040574 C>A)were significantly and independently associated with patient survival in this model.4.Meta-analysis of independent correlation between SNPs and overall survival of NSCLCAfter heterogeneity analysis,the three SNPs in this study(ERAP1 rs469783 T>C,NCF2 rs36071574 G>A,PSMF1 rs13040574 C>A)were found to be independently and significantly associated with patient survival in both the discovery and validation libraries,and no significant heterogeneity was found between the two databases(P heterogeneity >0.05).5.The relationship between the three genotypes of each SNP and the prognosis of NSCLC patientsThe dominant model of SNPs was used to classify genotypes,and then the relationship between individual genotypes and patient survival was analyzed,and it was found that genotypes in three SNPs: ERAP1 rs469783 TC+CC,PSMF1rs13040574 CA+AA had better prognosis compared to being wild-type pure heterozygotes,with HRs of 0.67,95% CI = 0.55-0.81,P = <0.0001;0.78,95% CI =0.66-0.91,P = 0.002;while patients with NCF2 rs36071574 GA+AA genotype had a worse prognosis with HR of 1.29,95% CI = 1.03-1.62,P = 0.030.6.Combination effect of three independent SNPs in PLCO databaseTo further analyze the effect of the combination of the 3 SNPs,the risk genotypes of all genotypes,namely: ERAP1 rs469783 TT,PSMF1 rs13040574 CC,NCF2rs36071574 GA+AA,were analyzed in combination.We found that the risk of death in NSCLC patients gradually increased with increasing risk genotypes of the three SNPs in the multifactorial analysis of the discovery library(Ptrend<0.0001).Afterwards,these genotypes were further dichotomized into the group of genotypes with few risk genotypes(0-1)and the group of genotypes with many risk genotypes(2-3).In comparison with the group of genotypes with few risk genotypes,patients in the group of genotypes with many risk genotypes had significantly shorter both disease-specific survival(DSS)and overall survival(OS).The risk of death associated with DSS was significantly higher by 1.51-fold(HR=1.51,95% CI=1.22-1.87,P=0.0001).In contrast,the risk of death associated with OS was 1.52-fold higher(HR=1.52,95% CI=1.24-1.86,P<0.0001).7.Stratified analysis of the influence of three SNPs on survival.In this study,these 3 independently associated SNPs were stratified into the lessrisky genotype group(0-1)and the more-risky genotype group(2-3),stratified analysis was performed between clinical profiles,and tests of heterogeneity between interactions were performed.The results revealed no statistical difference in the test of heterogeneity between these three independently associated SNPs and clinical data after performing stratified analysis(P heterogeneity > 0.05).8.Establishment of prognosis prediction model for NSCLC patientsIn this study,we used some components of the joint discovery library of clinical data to build a ROC model,and then predicted whether the predictive ability of patient survival could be further improved by combining the 3 SNPs identified in this study on the basis of previous clinical data.We found that the 3 SNPs identified in this study did not significantly improve the predictive ability of patient survival at 5 years(5-year OS:P>0.05;5-year DSS: P>0.05),but significantly improved the ability of predicting patient survival at 10 years(10-year OS: P = 0.022;10-year DSS P = 0.030)when making longer predictions.Conclusion:(1)In this study,the clinical profile of the library was found to be closely related to the prognosis of NSCLC patients.(2)Three genetic polymorphism loci in the MHC-I pathway,ERAP1 rs469783T>C,NCF2 rs36071574 G>A,and PSMF1 rs13040574 C>A,were independently associated with prognosis in NSCLC patients.(3)The risk genotypes of the three SNPs identified in the study were associated with worse prognosis under multifactorial analysis,and the risk of patient death was progressively higher when the number of risk genotypes(ERAP1 rs469783 TT,PSMF1rs13040574 CC,NCF2 rs36071574 GG+GA)was increased,i.e.,patients with more risk genotypes were more likely to die compared to Patients with more risk genotypes had shorter survival compared to those with fewer risk genotypes.In the stratified analysis after the combination of risk genotypes,no significant heterogeneous differences were found between these three SNPs and clinical data.(4)The predictive models developed by the 3 SNPs in the MHC-I pathway were able to improve the prognostic prediction of NSCLC patients by existing clinical variables at the 10-year level.The Second Part The Effect of Regulating ERAP1 Expression on Proliferation,Invasion and Migration Ability of Lung Squamous Cell Carcinoma Cells And Mechanism Exploration.Research Purpose:To observe the effect of ERAP1 expression profile on the proliferation,migration and invasion ability of NSCLC cells.Preliminary exploration of the signaling pathways through which ERAP1 affects the ability of NSCLC cells to proliferate,migrate,and invade.Materials and methods:The expression of m RNA and related proteins in NCI-H226 and A549 cells was investigated by q RT-PCR and Western blot.The effect of ERAP1 expression on the proliferation of NCI-H226 and A549 cells was investigated by CCK8 method;the changes of cloning ability of NCI-H226 and A549 cells were detected by clone formation assay;the changes of apoptosis ability of NCI-H226 and A549 cells were detected by apoptosis assay;the effects of ERAP1 on the migration and invasion ability of NCI-H226 and A549 cells were detected by Transwell assay;the effects of ERAP1 on the migration and invasion ability of NCI-H226 and A549 cells were investigated by proteomics and GO,KEGG enrichment analysis to explore the signaling pathway of ERAP1 action.Statistical treatment: SAS 9.4 software was used for statistics,and the experimental results of each group were compared by t-test or ANOVA;P<0.05 was significant.Result:1.Establishment of ERAP1 knockdown cell lineQRT-PCR and Western blot results showed that the m RNA and protein expression levels of ERAP1 m RNA the ERAP1 silencing group were lower than those in the Control group(P<0.05).2.The effect of ERAP1 knockdown on cell proliferation,migration and invasion.In contrast to Control group: the results of CCK8 assay showed a significant decrease in cell proliferation ability in ERAP1 knockdown group(P<0.05);the results of cell clone formation assay showed a significant decrease in clone formation in ERAP1 knockdown group(P<0.05);the results of apoptosis assay showed a significant increase in apoptosis in ERAP1 knockdown group(P< 0.05);by Transwell migration and invasion tests revealed that the ERAP1 knockdown group had significantly fewer membrane piercing cells than the Control group(P<0.05);by proteomic studies,we identified 127 significantly elevated expressed proteins and 156 significantly reduced expressed proteins with differential expression levels of more than 1.5-fold when protein expression differences were set to 1.5-fold in the study were mainly enriched in pathways such as PPAR.Conclusion:1.Compared with normal cell lines,the ERAP1 knockdown cell lines have significantly decreased proliferation,migration and invasion capabilities.2.Proteomics show that the differential proteins related to pre-and post-ERAP1 knockdown are mainly enriched in PPAR pathways.ERAP1 may affect the proliferation and invasion of lung cancer cells by affecting the PPAR signaling pathway. |
