Functional Analysis And Identification Of Nucleoprotein AHNAK,and Detection Of Methylation Level Of Pancreatic Stellate Cells In Pancreatic Ductal Adenocarcinoma

BackgroundPancreatic cancer(PC)is one of the highly malignant tumors in digestive system and threatening people’s health and quality of lives nowadays.The most frequent histological type of PC is pancreatic ductal adenocarcinoma(PD AC).In recent decades,the morbidity and mortality of PC remains worldwide,and latest epidemiological evidence showed maintained morbidity and upraised mortality in China.It suggests that the cognitive profundity of genetic alterations of PC remains shallow and effective therapeutic targets translated from molecular research in PC lacks.The Big Data on cancer appears and becomes popular as the improvement of molecular biological technology and diversification of bioinformatic analysis,which brings huge facilities for doctors and scholars to explore the biological nature of PC.Alterations in different omics of PC could be acquired to enhance the cognition of molecular genetic alterations in PC via analysis of big data.Nucleoprotein AHNAK is a macromolecular protein(629kDa)and plays diverse roles,including cell-cell transduction,modulation of calcium ion channel and cell adhesion,in human body.Some studies showed it upregulated in several tumors and promoted or mediated biological procedure in tumors.AHNAK was a prognostic predictor in some tumors in some reports.The most typical morphological characteristics of PC is desmoplasia,and the majority of cellular components is pancreatic stellate cells(PSCs).PSCs remain quiescent status in normal condition,however,PSCs are activated once stimulated by factors in vitro or in vivo,including PC.Nonetheless,the function of PSCs remains in veil and whether inhibition of activation of PSCs could minimize the malignant potential of PC is still equivocal.Methylation is the most frequent form of epigenetics.Subsequent studies showed DNA methylation was involved in initiation and development of multiple tumors.However,in PC,the methylation condition was not totally clearly,especially in PSCs,the most important stromal component in PC.Based on those,the study analyzed the PC high-throughput data from online database and verified the prediction via classic molecular biological experiments to understand the alteration in PC and to search for genetic alterations that might by translated into diagnostic or therapeutic targets.Then,according to the morphological feature of PC,activated PSCs were acquired from fresh tumor tissues and cultivated.All-trans retinoic acid(ATRA)was used for deactivation of PSCs.Both activated and deactivated PSCs were detected by whole genome bisulfite sequence,to explicit the epigenetic alterations in activation of PSCs and to further understand the function of PSCs in PC.Methods一、PC high-throughput data was downloaded from online database,Gene Expression Omnibus(GEO)and The Cancer Genome Atlas(TCGA),including gene expression and clinical data.R language was use for reorganization and normalization.Differential expression genes(DEGs)was acquired by differential analysis and clustering analysis(logFC>2,P<0.05),and RobustRankAggreg(RRA)analysis was used for integration of DEGs.Gene ontology(GO),KEGG(Kyoto Encyclopedia of Genes and Genomes)and Gene set enrichment analysis(GSEA)was carried for searching for relative biological functions and signaling pathways.STRING database was used for finding the protein-protein interaction(PPI)and Kaplan-Meier analysis was carried for seeking for DEGs with prognostic prediction.二、IntoGen database have predicted several driver genes in PC and we enlarged our screen condition of DEGs(logFC>0.5,P<0.05).We acquired some driver genes with differentially expressed level in PC for further examinations.三、Combining the text mining and prediction of bioinformatics,we proved that AHNAK was a driver gene with differential expression and prognostic prediction in PC.Then,the functions of AHNAK in PC were verified by molecular biological experiments subsequently.Sixty-six included PDAC FFPE specimens was detected by immunohistochemistry(IHC)and analyzed by Chi-square and t test.Prognostic value was analyzed by Kaplan-Meier and cox Regression analysis.The expression of AHNAK in pancreatic cancer cell lines was detected by qRT-PCR and Western blot and the function in cell lines was examined by cellular functional assays.Bioinformatics,including GO,KEGG and GSEA were carried to proof our results in the meantime.四、As the histological specialty of PC,PSCs was considered as the most important component in the stroma of PC.IHC showed a diverse expression of AHNAK in PSCs,with significant prognostic evaluation.Considering activated PSCs could promote the malignant potential of PC,we ran the WGBS to test the epigenetic alteration in the activation procedure of PSCs and further analyzed the diversity by combining previous RNA-seq data to understand the functions of PSCs in PC.Results一、Twelve upregulated(POSTN,ITGA2,FN1,CEACAM5,LAMC2,SLC6A14,TSPAN1,CEACAM6,GALNT5.CTSE,C1S and LAMB3)and 6 downregulated DEGs(IAPP,RN7SL186P,ALB,SERPINI2,RNU6-570P and CTRL)was discovered in One hundred and sixteen paired PC samples.GO and KEGG showed that those DEGs was involved in extracellular matrix(ECM)morphology and secretion,cell-cell interaction.PPI analysis showed that close interaction in those DEGs and prognostic analysis showed LAMB3,LAMC2,SLC6A14,ITGA2 and CIS was associated with prognosis of PC.二、Twenty driver genes were predicted in PC and 5 genes(ARID,AHNAK,ATM,PCDH18 and EPC1)was discovered after intersection with enlarged DEGs.After text mining,AHNAK was selected for further exploration.IHC showed AHNAK expression in PD AC was higher than adjacent tissues and associated with poor prognosis of PDAC,and AHNAK was an independent prognostic predictor of PD AC.三、AHNAK can mediated several signaling pathways(IL2/STAT5 signaling pathway and TP53 signaling pathway)to influence the epithelial mesenchymal transition(EMT)of PCCs to promote proliferative and migrative ability of PCCs by molecular biological experiments,but AHNAK showed no effect on cellular apoptosis.四、The IHC result showed that AHNAK high expression in PSCs was also associated with poor prognosis of PDAC.TCGA data also suggested that low methylation of AHNAK predicted worse outcome of PC.Considering previous research,we ran WGBS in both activated and deactivated PSCs to detect the methylation alteration.The result showed that mCpG in deactivated PSCs was more than activated PSCs(59.61%vs.48.8%)in the genome level,and mCpG was relatively low in deactivated PSCs(79.39%vs.92.40%).Methylation mode in motif was also different.Some diversities of mCG,mCHG and mCHH were also discovered in different function region between activated and deactivated PSCs.Deactivated PSCs also showed a higher methylation in chromosome level.五、It showed that amount of differential methylation region(DMR)between activated and deactivated PSCs and 174 DMRs contain three methylation mode(CG,CHG and CHH)in the genomic level,while 44 DMRs with the three mode was identified in the promoter region.GO showed genomic DMRs was mainly included in biological development while promoter DMRs was enriched at organelle and intracellular function and KEGG showed DMRs was involved in several tumor-related signaling pathways,including Hippo signaling pathway(hsa04390),Pathways in cancer(hsa05200),Rap1 signaling pathway(hsa04015),Hedgehog signaling pathway(hsa04340)and Wnt signaling pathway(hsa04310).六、Twenty genes with both expression and methylation differences was discovered by conjoint analysis with RNA-seq and the most significant two genes,CARMIL3 and MED12L was selected for identification.The results of methylation-specific PCR(MSP)and qRT-PCR proved the differences in WGBS.GO and KEGG showed those genes was involved in polymorphism and sequence variant etc.Conclusion一、A large amount of DEGs in PC was identified and mainly involved in extracellular matrix(ECM)mor:phology and secretion,cell-cell interaction,some of DEGs,including LAMB3,LAMC2,SLC6A14,ITGA2 and C1S,was associated with prognosis of PC.二、Driver gene AHNAK can mediated several signaling pathways(IL2/STAT5 signaling pathway and TP53 signaling pathway)to influence the epithelial mesenchymal transition(EMT)of PCCs to promote proliferative and migrative ability of PCCs,thus AHNAK was associated with outcome of PDAC and was an independent prognostic predictor in PD AC.三、The genomic methylation level altered during the activation procedure of PSCs,which might be involved in multiple biological processes and signaling pathways in PC.