Screening,Verification And Bioinformatic Analysis Of Differentially Expressed Genes Of Pancreatic Stellate Cells

Part Ⅰ Screening and bioinformatics analysis of differentially expressed genes between quiescent and activated pancreatic stellate cellsPurpose: Pancreatic cancer,which is considered as "the King of Cancer",is related to fast progress,surgery-difficulty,drug-resistance and high mortality.PSCs act as the core of the desmoplasia of tumor tissue which is mainly involved in ECM.We are entering into the age of "Big Data",with the rapid development of gene and information technology,bioinformatics,a multidiscipline subject plays a significant role in analyzing complicated data.To screen differentially expressed genes between quiescent and activated pancreatic stellate cells and make bioinformatic analysis.Method: We selected 8 tumor tissues of 8 patients from Peking Union Medical College Hospital who had been diagnosed with pancreatic ductal adenocarcinoma by pathologists and then cultured PSCs.Using ATRA to transform activated PSCs to quiescent state and sequencing the genome of two types of PSCs in order to screen DEG.Then we used GO analysis and KEGG pathway enrichment analysis for further exploration.Results: We got 192 DEG from 33289 genes which include 109 up-regulated genes and 83 down-regulated genes.The top three down-regulated genes were HR,RP11-624L4.1,and FGF9.GO enrichment analysis involves three parts—BP,CC and MF.The top 2 functional annotations of BP part were extracellular structure organization and extracellular matrix organization.The top 3 functional annotations of CC part were extracellular matrix,proteinaceous extracellular matrix and interstitial matrix.The top 3 functional annotations of MF part were sulfur compound bind,heparin binding and growth factor.TGF-β pathway rank first in KEGG analysis.We ranked those functional annotations according to P value of all the BP,CC and MF parts and found that ECM and growth factor were in the core of the activation of PSCs.Then ranked genes involved in ECM and growth factor by frequency and got 18 genes which appeared more four times in those functionalannotations.But FGF9 was the only gene which was both found in top 3 DEG and core functional annotations.Conclusion: The transformation between quiescent and activated PSCs are regulated by multi-genes.Genes related to ECM and interstitial matrix account for major part of down-regulated genes which suggest that matrix play a significant role in activation of PSCs.Part Ⅱ Verification of FGF9 and related receptors through RT-q PCRPurpose: The thousands of hundreds of data would become "trash" if we do not dig the sophisticated value.So we tried to analyze the data from part one and choose the candidate gene by combining other research.Then we selected FGF9 as the candidate gene and other receptors related to FGF9 and PSC for further verification.Method: We divided cells into 2 groups,Group activated and Group ATRA are extracted RNA respectively from which.Verified genes of FGFR2c、FGFR3b and FGFR3 c through RT-q PCR to confirm the results of sequencing.Results: Expression of FGF9、FGFR3b and FGFR3 c was significantlyConclusion: FGF9 promotes the activation of PSCs and FGFR3 b together with FGFR3 c may play the synergetic effect to FGF9.Now we do not have a specific conclusion about the function of FGFR2 c in PSCs.