The Functional Effect Of AHNAK2 Protein In Pancreatic Cancer Cells And Its Correlation With Autophagy

Pancreatic cancer(PC)is still recognized as one of the deadliest cancer types.Despite advances in related chemotherapy regimens and improved surgical results,the prognosis is still poor,and the 5-year survival rate is as low as 10%.The challenge remains extremely severe.Therefore,it is necessary to explore the molecular markers and their expression patterns related to the diagnosis and treatment of pancreatic cancer.Autophagy is a biological phenomenon that uses lysosomes to degrade self-damaged organelles and macromolecular substances,and is a physiological process necessary to maintain cell homeostasis.The process of autophagy involves the growth and aging of the body,and is related to the occurrence,development and drug resistance of many diseases,especially tumors.BECN1,SQSTM1 and MAP1LC3 are known and recognized autophagy-related genes,which are involved in the formation of autophagosomes in the process of autophagy.In the early stage of the research group,chloroquine diphosphate and rapamycin,which can affect the level of autophagy,were administered to human pancreatic cancer Panc-1 cells,and gene expression profiles were detected by gene chip technology.We combined Genotype-Tissue Expression(GTEx),The Cancer Genome Atlas(TCGA),Gene Expression Omnibus(GEO)and other databases of pancreatic cancer sequencing data and chip data for differential analysis,and finally calculated the potential pancreas Differential genes related to cancer autophagy.Comprehensive analysis found that ANHAK2 was differentially expressed in pancreatic cancer tissues and pancreatic cancer cells after chloroquine treatment,and was related to the prognosis.A number of existing studies have shown that AHNAK2 protein is related to a variety of tumors,and may be used as a potential diagnostic and prognostic biomarker for pancreatic cancer.However,the relationship between AHNAK2 in the cell functions mechanism and autophagy of pancreatic cancer has not been elucidated in the literature.Therefore,this study verified the expression of ANHAK2 in pancreatic cancer through detection,explored potential biological functions and molecular mechanisms,and explored its relationship with autophagy-related proteins.ObjectiveBy detecting the expression of AHNAK2 in pancreatic cancer and its correlation with autophagy-related proteins and clinicopathological parameters,in order to understand the relationship between them,and further study the effect of AHNAK2 on the biological behavior of pancreatic cancer and its potential molecular mechanisms,and based on predictions Autophagy-related genes and prognostic data construct a network related to autophagy prognosis in pancreatic cancer,and provide some theoretical foundations for research on autophagy in pancreatic cancer.Methods1.The expression level of AHNAK2 was detected by immunohistochemistry,and its relationship with autophagy-related proteins BECN1,SQSTM1 and clinical parameters were statistically analyzed.2.Pearson correlation analysis screened out genes related to AHNAK2,and used GO,KEGG,DOSE enrichment analysis to explore the potential functions,pathways and disease associations of AHNAK2 related genes.3.RNA interference technology was used to knock down the expression of AHNAK2 in pancreatic cancer cells,and detect the expression of AHNAK2 and autophagy-related proteins by Western blot.Subsequently,CCK-8 cell proliferation experiment,scratch experiment,cell cycle and apoptosis were detected by flow cytometry.To explore the effect of knocking down ANHAK2 on the biological function of pancreatic cancer cells.4.Based on the gene expression data,survival time and survival status of pancreatic cancer cases based on TCGA,a prognostic model was established through Cox regression and Lasso analysis,and prognostic factors related to pancreatic cancer autophagy were screened based on the prognostic scores.The prognostic and bioinformatics correlation analysis of the protein-protein interaction network constructed by these genes.Results1.The expression of AHNAK2 in pancreatic cancer tissue was 0.253±0.009,and in normal pancreatic tissue it was 0.072±0.003,the difference was statistically significant.Analyzing the relationship between AHNAK2 and the clinical-pathological parameters of pancreatic cancer cases showed that the expression level of AHNAK2 was statistically different in pancreatic cancer patients less than or equal to 60 years old and greater than 60 years old,and the expression of AHNAK2 was relatively low in the group older than 60 years.Survival analysis showed that compared with the lower expression group,the prognosis of patients in the AHNAK2 high expression group was significantly worse.2.Difference analysis results showed that there are differences in AHNAK2 in pancreatic cancer tissues.Meta-analysis showed that the expression of AHNAK2 in each independent study is consistent,and the expression level of AHNAK2 is significantly higher than that of control tissues.The results of GO and KEGG enrichment analysis of related genes showed that they are involved in cell cycle regulation,extracellular matrix-receptor interaction and PI3K-Akt signaling pathway.3.Compared with the blank control group and the negative control group,the tumor cell proliferation rate of the si-AHNAK2 group was lower than that of the control group,but the difference was not statistically significant.The decline of cell migration ability in the si-AHNAK2 group was statistically significant.In the si-AHNAK2 group,cells were arrested in the G1 phase,and the proportion of late apoptotic and dead cells increased significantly.4.The prediction model analysis results suggest that AHNAK2,ITGB6,CENPF,RPH3 AL,and APOL1 are valid variables.The PPI results show that AHNAK2,CENPF,RPH3 AL,and APOL1 have the highest scores,and there is a potential interaction with epithelial-mesenchymal transition related proteins relationship.Conclusion1.The protein expression and RNA expression levels of AHNAK2 in pancreatic cancer tissues are significantly higher than those in normal tissues.The prognosis of patients with low expression group in the AHNAK2 high expression group is significantly worse,suggesting that AHNAK2 may affect the progression of pancreatic cancer as an oncogene.2.Cell experiments show that knocking down the expression of AHNAK2 can significantly inhibit the migration ability of pancreatic cancer cells,block pancreatic cancer cells in G0/G1 phase to inhibit tumor development,and promote tumor cell apoptosis.3.Constructing a predictive model to screen six pancreatic cancers with differentially expressed autophagy-related prognostic factors.Survival analysis also suggests that ANHAK2,ITGB6,COL17A1,CENPF,APOL1 are risk factors;RPH3AL is a protective factor.AHNAK2 may interact with cell cycle-related proteins and EMT-related proteins,thereby participating in the regulation of pancreatic cancer cell cycle and affecting cancer progression.