| Objective: Pancreatic carcinoma is a common digestive cancer,ranking fourth in cancerrelated mortality in the world,and the incidence rate is increasing year by year.Early diagnosis and monitoring are difficult due to the relatively obscure anatomical location of the pancreas and the lack of specific biomarkers.Most patients of pancreatic cancer are diagnosed with advanced or distant metastases at their first visit,missing the opportunity for radical surgery.In addition,pancreatic cancer is not sensitive to radiation therapy or chemotherapy,which leads to a poor prognosis.However,traditional clinical indicators are not satisfactory for the prognosis and efficacy prediction of pancreatic cancer patients.Therefore,effective predictive prognostic models and biomarkers that can guide individualized systemic therapy are crucial for prolonging the survival time and improving the quality of life of pancreatic cancer patients.In recent years,many cancer-related microarrays and sequencing platforms have emerged,so it is very important to integrate a large amount of available data and turn these molecular research results into clinical decision-making tools.Integrins(ITGs),also known as integrins,are non-covalently linked heterodimers that form a family of cell surface adhesion receptors.Integrins are abnormally expressed in a variety of diseases and play an important role in the regulation of malignant biological behaviors of many tumors,which are closely related to the prognosis of tumor patients.At present,the development of new anti-tumor drugs targeting integrins has become a research hotspot in the field of tumor therapeutic drug research and development.In this study,we first reviewed the development and research hotspots of integrin in the field of tumor prognosis in recent 20 years,and then constructed a prognosis prediction risk model of pancreatic cancer based on integrin family genes,combined with the in vitro and in vivo experiments of cell biological function,it is further indicated that the key genes in the prognostic model may participate in the development of pancreatic cancer,and the design and synthesis of antineoplastic peptides for key genes,which provides a new direction for studying new molecular approaches of pancreatic cancer pathogenesis and developing new strategies for diagnosis and treatment of pancreatic cancer.Methods:1.Bibliometric analysis: search on the core database of the web of science,screen the articles and reviews related to integrin and tumor prognosis published from 2002 to 2021,and analyze and visually display the publication time,country/institution,journal,author,and keywords by using VOSviewer software and Cite Space software;2.Bioinformatics analysis: The original microarray data and related clinical information of pancreatic cancer patients based on the TCGA(The Cancer Genome Atlas)database,and the original microarray data of normal pancreatic samples based on the GTEx(genotype-tissue Expression)database were downloaded from UCSC and normalized.Univariate Cox regression analysis was used to identify prognostic genes and construction of prognostic risk model by Least Absolute Shrinkage and Selection Operator(LASSO)and multivariate Cox regression analysis.According to the median risk score obtained from the prognostic model,the patients were divided into high-risk and low-risk groups.Log-rank test was used to analyze whether there were differences in the overall survival rate of patients in high-risk and low-risk groups.The area under the ROC curve is used to evaluate the accuracy of the prognosis risk model.Univariate and multivariate Cox regression analyses were performed to assess whether risk scores could serve as independent prognostic factors compared with other clinical characteristics,including gender,age,and tumor grade or stage.Then,a nomogram model was constructed based on multivariate regression analysis,and its consistency and prognostic stratification ability were evaluated.Gene set enrichment analysis(GSEA)was used to explore the difference of enriched pathways between high and low risk.3.Experimental validation study: 1)Based on GEO,TCGA,and The Human Protein Atlas(HPA)database,the m RNA and protein expression levels of ITGβ5 in pancreatic cancer were studied;2)Transient si RNA or plasmid transfection with jet PRIME reagent were used to silence or up-regulate the expression of target protein;3)The Ed U experiment was applied to detect the cell proliferation ability;4)The Colony formation assay was adopted to measure the cell clonal expansion ability;5)The Transwell test was performed to detect cell migration and invasion ability;6)The protein expression of ITGβ5 and 14-3-3ζ was detected by Western blot;7)Detect cell cycle changes by flow cytometry after PI staining;8)The protein mass spectrometry analyzed ITGβ5 interacting proteins;9)The formation of ITGβ5-14-3-3ζ complex in pancreatic cancer cells was verified by co-immunoprecipitation;10)Immunofluorescence confocal confirmed the colocalization of ITGβ5 and 14-3-3ζ in pancreatic cancer cells;11)Immunohistochemical staining was used to detect ITGβ5 and 14-3-3ζ in pancreatic cancer tissues;12)Design and synthesis of polypeptide B5-X that targets the binding of ITGβ5 to 14-3-3ζ;13)To establish an intraperitoneal injection model of subcutaneous tumorigenesis in nude mice,and to detect the antitumor activity and safety of polypeptide B5-X in vivo;14)Prediction of 14-3-3ζ upstream transcriptional regulatory miRNAs in miRBase and Target Scan databases;15)Quantitative real-time RT-PCR was performed to measure miRNA-490-5p expression in pancreatic cancer tissues;16)Animal experiments establish subcutaneous xenograft tumor in nude mice to confirm the inhibitory effect of miR-490-5p on pancreatic cancer subcutaneous tumor formation;17)Statistical analysis: Statistical analysis and graphing were performed using Graph Prism8 and R software(version 3.6).Each experiment was repeated three times,and the data were presented as mean ± standard deviation(SD).Comparison between groups was performed by Student’s t-test,P < 0.05 was considered as statistically significant.Results:1.Bibliometric analysis: The number of papers published has gradually increased during 2002 and 2021,and China published the most articles.The University of California and the University of Texas are institutions with more scientific research output.Articles related to integrin and tumor prognosis are mainly published in JCR district I high-quality journals such as《Cancer Research》.The most cited article was a review published on《Nat Rev Cancer》in 2010,focusing on the important regulatory effect of integrin on the occurrence,development,and metastasis of solid tumors and the therapeutic effect of integrin antagonists in clinical trials.The burst analysis of keywords shows that the focus of research has changed over time.The cluster analysis revealed that the influence of integrin on tumor prognosis was related to tumor cell invasion,cell stem,chemoresistance,overactivation of FAK,and Akt signal pathways.2.Bioinformatics analysis: Five integrin genes ITGA2,ITGA3,ITGA6,ITGB5,and ITGB6 were selected by the Lasso-Cox regression model to construct a prognostic risk model.Patients in the high-risk group consistently had poorer clinical outcomes and shorter survival times than those in the lowrisk group in both TCGA and GEO databases.Multivariate Cox regression analysis demonstrated risk scoring models can serve as independent prognostic factors for patient survival.The nomogram of risk score and clinical risk factors is constructed.The risk score of the nomogram model has good accuracy in predicting the 1-year,3-year,and 5-year overall survival rate of pancreatic cancer patients.GSEA analysis results showed that the high-risk group was enriched in the Wnt signaling pathway,P53 signaling pathway,TGF-β signaling pathway,and other signaling pathways involved in tumorigenesis.3.Experimental verification research: 1)In pancreatic cancer tissue,the m RNA and protein expressions of ITGβ5 were increased;2)ITGβ5 upregulation was associated with poor clinicopathological features and poor prognosis in pancreatic cancer patients;3)ITGβ5overexpression promotes pancreatic cancer cell growth,migration and invasion,while knockdown of ITGβ5 inhibits these malignant behavior;4)The mass spectrum analysis,immunoprecipitation results confirmed that ITGβ5 interacted with 14-3-3ζ,and immunofluorescence confocal confirmed that ITGβ5 and 14-3-3ζ colocated in pancreatic cancer cells;5)CHX protein degradation experiments,MG132-treated cell experiments,CQ-treated cell experiments verified that knockdown of 14-3-3ζ can lead to an increase in the ITGβ5 lysosomal degradation pathway and reduce the stability of ITGβ5 protein;6)Knockdown of 14-3-3ζ in ITGβ5-overexpressing pancreatic cancer cell lines reversed ITGβ5-promoted proliferation,migration and invasion effects;7)Molecular docking predicts the binding site of ITGβ5 and 14-3-3ζ,14-3-3ζ protein can bind to the serine/threonine-rich region in the intracellular segment of ITGβ5,and can target and inhibit the binding of ITGβ5 to 14-3-3ζ protein;8)Polypeptide B5-X can significantly inhibit the cell proliferation,migration and invasion;9)Subcutaneous tumorigenesis and intraperitoneal injection models in nude mice show that B5-X polypeptide has significant antitumor effect and good safety;10)The miRTar Base database found that miRNA-490-5p is directly bound to 14-3-3ζ protein,and Target Scan database also shows that miRNA-490-5p and 14-3-3ζ protein binds and displays specific binding sites;11)miRNA-490-5p is downregulated in pancreatic cancer tissues compared with adjacent normal tissues;and compared with the good prognosis group,the expression of miRNA-490-5p was downregulated in the poor prognosis group;miRNA-490-5p is lowly expressed in the tissues of pancreatic cancer patients with poor prognosis.Furthermore,patients in the lowexpression group had a shorter survival time,compared with those in the high-expression group;12)miRNA-490-5p overexpression inhibited the proliferation,migration,and invasion of pancreatic cancer cells in vitro,and significantly inhibited the growth of xenograft tumors in vivo.Conclusion:1.The number of research papers related to integrins and tumor prognosis are gradually increasing,and the research focused on the molecular mechanism of how integrins affect tumor prognosis;2.The risk prediction model composed of integrin genes ITGA2,ITGA3,ITGA6,ITGB5,ITGB6 can effectively provide prognosis prediction of pancreatic cancer;3.The high expression of ITGβ5 is related to poor prognosis and promotes proliferation,migration,and invasion of pancreatic cancer cells;4.14-3-3ζprotein interacts with ITGβ5 to enhance the stability of ITGβ5 protein by inhibiting the lysosomal degradation pathway of ITGβ5 and promotes its function;5.Polypeptide B5-X inhibits the formation of ITGβ5 and 14-3-3ζ complexes in pancreatic cancer cells and effectively exhibits antitumor function;6.Mi RNA-490-5p is an independent prognostic factor in pancreatic cancer patients,targeting down-regulating 14-3-3ζ expression,which causes cell cycle S-phase arrest,inhibits cell growth,migration,and invasion in vitro,and inhibits tumor growth in nude mice... |
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