Prognostic Analysis And Mechanistic Exploration Of Autophagy-Related Exosomes Genes ITGA3,ITGB4,and PTK6 In Pancreatic Cancer

Objective: Pancreatic cancer(PC)is a highly malignant tumor with a very low 5-year survival rate,and pancreatic ductal adenocarcinoma(PDAC)accounted for 90% of PC.This study aims to explore the expression and mechanism of autophagy-related exosomal genes in the malignant biology of PC by using bioinformatics methods,and to verify the expression of autophagy-related exosome genes in PDAC pathological tissues by immunohistochemistry to identify related genes with diagnostic and prognostic value for PC.Methods:The RNA sequencing files and clinical files of PC and normal pancreatic tissues were downloaded from the UCSC xena website and GEO database for expression differential analysis.Based on the Exosome-related genes(ERGs)in the exo Rbase database and Autophagy-related genes(ARGs)in the HADb database,autophagy-related exosomal genes were extracted from the up-regulated m RNAs with significant expression differences.The autophagy-related exosomal genes were used to construct the Lasso model for prognosis assessment,and the genes most relevant to PC prognosis,named the target genes,were screened out.A risk scoring system was constructed based on the expression of the target genes of the Lasso model and their penalty coefficients,and all samples were classified into high and low risk groups using this risk scoring system.Survival analysis to assess the difference in overall survival between high and low expression groups and high and low risk groups of target genes.Univariate and multifactorial cox analyses were performed to assess whether the target gene,multiple clinical features were independent prognostic risk factors.Nomogram were performed to assess the probability of survival at 1,3 and 5years for PC.A random forest model was constructed to assess the diagnostic efficacy of the target gene,and the GEO dataset was used to validate the diagnostic efficacy of the model.Highly related genes of target genes(HRGTGs)were screened using pearson correlation analysis,GO and KEGG functional enrichment analyses were performed on HRGTGs,and PPI networks were constructed.Immune infiltration analysis was performed to assess immune cell infiltration in tumor and non-tumor,high and low risk groups of patients.Drug sensitivity analysis of target genes was performed by cell Miner database.Immunohistochemical staining was performed to detect target gene expression in cancer and paracancerous tissues of PDAC after pancreaticoduodenectomy.Results: The matrix files of 178 PC tissues(including 143 PDAC tissues)and 167 healthy human pancreatic tissues obtained from UCSC xena,and eight autophagy-related exosome genes: CDKN2 A,ITGB4,ATG9 B,PTK6,ITGA3,IL24,NRG1,and GRID1 were extracted by expression difference analysis and database information.Lasso regression analysis showed that PTK6,ITGA3,and ITGB4 were most strongly associated with prognosis,with AUCs of 0.719,0.722,and 0.685 at 1,3,and 5 years.Both univariate and multifactorial cox analyses showed that PTK6,ITGA3,ITGB4,and risk score were independent risk factors affecting the prognosis of PC patients(p<0.05).According to the scoring system,patients with high scores had a worse prognosis than those with low scores(p<0.001).Survival analysis showed that patients in the group with high expression of ITGA3 and PTK6 in PC had significantly lower OS than those with low expression(p<0.05),high and low expression of ITGB4 only had an impact on patients’ survival at 1 year after diagnosis(p<0.05).The random forest model constructed using PTK6,ITGA3,and ITGB4 had excellent diagnostic efficacy,with AUCs of 0.982,0.972,and 0.823 for the training set,test set,and external validation set,respectively.gene set enrichment analysis showed important roles of PTK6,ITGA3,and ITGB4 in cell-cell intercellular adhesion and cell-matrix adhesion.Immune infiltration analysis showed that PC samples contained higher levels of macrophages(M0,M1 and M2 types),dendritic cells,and regulatory T cells than healthy human pancreatic tissue,and lower levels of immune cells such as T-cell follicular helper cells and CD8+ T cells than normal tissue,moreover,PC samples contained higher levels of M0 and M2 macrophages in the high-risk group than in the low-risk group,and lower levels of CD8+ T cells than normal tissue.Drug sensitivity analysis showed that crizotinib and tamoxifen inhibited the expression of ITGA3,isazolomib and bortezomib inhibited the expression of ITGB4,and carboplatin and cisplatin inhibited the expression of PTK6. Immunohistochemical staining showed that the expression of PTK6,ITGA3,and ITGB4 was significantly higher in PDAC tissues than in paracancerous tissues(P<0.0001),furthermore,ITGA3 and ITGB4 were found to be highly expressed in neural tissues infiltrated by PDAC.Conclusion: Autophagy-related exosome genes PTK6,ITGA3,and ITGB4 are independent risk factors for PDAC,and potential biological marker for early diagnosis and prognosis assessment of PC,which may contribute to the development and invasive metastasis of PC by promoting cell-cell adhesion,cell-matrix adhesion,inhibiting tumor cell autophagy,promoting neural infiltration,decreasing CD8+ T cell content,and elevating M0 and M2 macrophage content.The diagnostic model constructed by PTK6,ITGA3 and ITGB4 has good diagnostic performance.