Prognostic Model Of Differentially Expressed Genes In Pancreatic Cancer And Clinical Correlation And Function Study Of MCM7 Expression

Pancreatic adenocarcinoma(PDAC)is a malignant gastrointestinal tumor with only a 9%five-year survival rate.Although radical resection is the major approach to cure pancreatic cancer,the number of cases having the opportunity to do surgery at the diagnosis is only 15%.Factors contributing to the post-surgery prognosis are a research hotspot,because it is common although patients at the same clinical staging,their prognoses could be largely different.Sustaining proliferative signaling and enabling replicative immortality are two hallmarks of cancer.Evaluating the tumor grade based on a proliferation index is crucial in some types of pancreatic cancer,which guides the clinical decision-making and prognostic prediction.Benefiting from the application of high throughput technology broadens the research scope by analyzing landscapes of genome/transcriptome/proteomics and characterizing molecular pathology features in common.Previous researches reported there are no specific genetic variation alterations between pancreatic cancer patients with short-or very long-term survival,and the prognosis value of molecular classification based on the DNA copy number variation pattern is limited.Thus,this project was focusing on the transcriptome of pancreatic cancer by integrating multiple high-quality microarrays or RNA-seq data to analyze consistently differentially expressed genes,combining with clinical-pathological information to establish and validate the prognosis model.Concerning the crucial role of proliferation in pancreatic cancer,we also focused on MCM7,a DNA replication related protein.We investigated its expression pattern,prognostic correlation and molecular functionsWe systematically developed a prognostic model for pancreatic cancer that was compatible across different transcriptomic platforms and patient cohorts.After performing quality control measures,we used seven microarray datasets and two RNA sequencing datasets(348 normal pancreas tissues and 404 pancreatic ductal adenocarcinoma tissues in total)to identify consistently dysregulated genes in pancreatic cancer patients.Weighted gene co-expression network analysis was performed to explore the associations between gene expression patterns and clinical features.The least absolute shrinkage and selection operator(LASSO)and Cox regression were used to construct a prognostic model.We tested the predictive power of the model by determining the area under the curve of the risk score for time-dependent survival We also investigated the correlation between the expression of MCM7 and clinical characteristics,and the molecular functions of MCM7 in pancreatic ductal adenocarcinoma.The immunohistochemistry for MCM7 was performed in a 58-case training cohort,including normal pancreas,pancreatitis,pancreatic ductal adenocarcinoma,adjacent non-tumor tissue,metastasis organ,positive-and negative-lymphoma nodes.Further,we analyzed the correlations between MCM7 expression pattern with clinical-pathological features in an additional 57 PD AC and its matched adj acent tissues.After evaluating the endogenous expression of MCM7 in normal human pancreatic ductal epithelial cell line HPDE and six PD AC cell lines,we used two knockdown strategies(siRNA and shRNA)to do phenotype assays in at least two PD AC cell lines.We performed alamarBlue cell viability assay to evaluate cell proliferation,PI staining to test cell cycle and Annexin V/PI assay to analyze cell apoptosis.Besides its knockdown effect on proliferation,we also tested the changes of several DNA damage markers and the sensitivity of MC M7 knockdown MIA PaCa-2 cells to gemcitabine.Most of the differentially expressed genes in pancreatic cancer were enriched in functions pertaining to the tumor immune microenvironment.The transcriptome profiles were found to be associated with overall survival,and four genes(DSG3,ARNTL2,NUSAP1,KR T7)were identified as independent prognostic factors.A prognostic risk score was then proposed,which displayed moderate accuracy in the training and self-validation cohorts.Furthermore,patients in two independent microarray cohorts were successfully stratified into high-and low-risk prognostic groups.PD AC cases with high expression of MCM7 tended to have shorter overall survival.Multivariate Cox regression analysis showed MCM7 was an independent factor for pancreatic cancer prognosis.Significantly increased MCM7 expression presented in cancerous primary and metastasis lesions,compared with normal pancreas tissue.The expression of MCM7 also exhibited a negative correlation with cancer differentiation.Consistently,MCM7 was overexpressed in multiple PD AC cell lines,comparing with its endogenous expression in HPDE.Knocking down MCM7 inhibited the proliferation of PDAC and affected cell cycle progress via inducing S phase arrest.MCM7 could interact with DNA damage response factor ATRIP.Silencing MCM7 activated DNA damage response signaling.The in vitro assay showed knocking down MCM7 promoted the sensitivity of MIA PaCa-2 to gemcitabine treatment.Taken together,through a combined analysis of multiple datasets,we identified 542 genes that were consistently differentially expressed between normal pancreatic tissues and PDAC tissues.Most of the DEGs were associated with the pancreatic extracellular stroma and the tumor immune microenvironment.Our prognostic model containing four DEGs(DSG3,ARNTL2,NUSAP1 and KRT7)was used to determine the risk scores of pancreatic cancer patients,and patients with high-risk scores were found to exhibit poor overall survival.The expression level of MCM7 is an independent prognostic factor.MCM7 presented increased expression in PDAC and its metastatic lesions.Silencing MCM7 suppressed PDAC proliferation,activated DNA damage response,promoted gemcitabine sensitivity.This indicates MCM7 could be a valuable prognosis prediction marker and a potential therapeutic target.