Effects Of Agps Gene Silencing On Expression Profiles Of LncRNA And MRNA In Glioma Cells And Bioinformatics Analysis

Objective:To observe the influence of AGPS gene silencing on the expression profiles of long non-coding RNA(lnc RNA)and messenger RNA(m RNA)in glioma cells,analyse the functions and the signaling pathways that differentially expressed genes enrich,explore the mutual relationship in differential genes or in differential signaling pathways,seek for the lnc RNAs that regulate m RNAs and signaling pathways,predict the functions of lnc RNAs and detect the possible pathogenesis of glioma.Methods:Human glioma cells cultured in vitro were divided randomly into sh R-AGPS-1,sh R-AGPS-2 and control groups.Each cell line was transfected with corresponding lentivirus carrying sh R-AGPS-1,sh R-AGPS-2 or empty vector respectively.The cell morphology was observed after monoclonal cells were screened and proliferated in turn to comment on in vitro growth of transfected glioma cell lines.AGPS protein levels in each group were determined by western blotting.The expression profiles of lnc RNA and m RNA in each group were detected by gene chip,and the differential lnc RNAs and m RNAs was screened with the standard of fold change greater than 1.2 or less than-1.2 and P values less than 0.05.Functional annotation of differential m RNAs was carried out with the Gene Ontology(GO)database and signal pathway enrichment analysis of differential m RNAs was carried out using the Kyoto Encyclopedia of Genes and Genomes(KEGG)database.KEGG pathways network was constructed according to the interrelationships among significant differentials pathways that m RNAs significantly enrich.The global signal transduction network was constructed according to the interrelationships among different m RNAs.Co-expression network that reflects the regulatory relationship of lnc RNA-m RNA was constituted according to co-expression datas of differential lnc RNAs and m RNAs.lnc RNA target pathway network was constructed based on property of differeftial lnc RNAs and significant pathways.Results:(1)In vitro culture of monoclonal cell lines after lentivirus transfection showed that the cells in sh R-AGPS-1 group and the sh R-AGPS-2 group presented shrinkage and proliferationinhibition,and the cells abnormalities were more obvious in the sh R-AGPS-2 group compared with the control group.(2)Western blot showed that the relative expression level of AGPS in the sh R-AGPS-2group was significantly lower than that in the control group(P<0.05).(3)The results of screening among genes suggest that 180 lnc RNAs and 110 m RNAs in group sh R-AGPS-1 were differentially expressed significantly compared with control group,among which 73 lnc RNAs and 46 m RNAs were up-regulated,while 107 lnc RNAs and 64 m RNAs were down-regulated(|FC|>1.2,P<0.05);and that 499 lnc RNAs and 531 m RNAs in the sh R-AGPS-2 group was significantly differentially expressed compared with the control group,among which 266 lnc RNAs and 144 m RNAs were up-regulated,while 233 lnc RNAs and 387 m RNAs were down-regulated(|FC|>1.2,P<0.05).(4)GO analysis results showed that differentially expressed m RNAs were mainly enriched to the functions of cellular response to hypoxia extracellular matrix organization,protein kinase R-like endoplasmic reticulum kinase(PERK)mediated unfolded protein response,cell adhesion,positive regulation of angiogenesis,immune response,response to mechanical stimulus,blood coagulation,leukocyte migration and embryo implantation and so on.KEGG pathway analysis showed that differentially m RNAs were mainly enriched to the pathways of advanced glycation end-product and its receptor(AGE-RAGE)signaling pathways in diabetic complications,rheumatoid arthritis,protein processing in endoplasmic reticulum,malaria,tumor necrosis factor(TNF)signaling pathway,hypoxia inducible factor 1(HIF-1)signaling pathway,Focal adhesion,biosynthesis of amino acids,nuclear factor kappa-B(NF-k B)signaling pathway and mammalian target of rapamycin(m TOR)signaling pathway and so on.(5)KEGG pathways mutual relationships analysis indicate that significant signaling pathways interacted with each other,in which PI3K-Akt signaling pathway,toll-like receptors(TLRs)signaling pathway,complement and coagulation cascades,focal adhesion,vascular endothelial growth factor(VEGF)signaling pathway,HIF-1 signaling pathway,m TOR signaling pathway,NF-k B signaling pathway,proteoglycans in cancer and central carbon metabolism in cancer and so on.(6)Global signal transduction network showed that there were a total of 75 key m RNAsinteracting with each other,among which the most core genes included EGFR,ITGA11,IGF1 R,AKT3,GYS1,PIK3 CA,ENPP1,JUN,SDC4,CD44,etc.(7)The co-expression analysis of lnc RNA-m RNA showed that there were co-expression correlations between 118 lnc RNAs and 193 m RNAs,among which the top 10 lnc RNAs were FENDRR,linc-ARHGAP11B-2,uc004 cic.2,linc-TNFRSF9,RP11-221N13.3,linc?luo?1223,linc-NBPF15-1,NR?044996?4,RP11-760H22.2 and uc001 tkz.2.(8)lnc RNA target pathway analysis showed that the key lnc RNAs,such as AK093732,LINC01384,FENDRR,uc011 bsz.1,linc-PTPRN2,linc-U2AF1-1,RP11-221N13.3,BC042023,BC043357,linc?luo?1251,at the core of the network,regulated the key signaling pathways,such as cytokine-cytokine receptor interaction,PI3K-Akt signaling pathway,cell adhesion molecules(CAMs),AGE-RAGE signaling pathway in diabetic complications,p53 signaling pathway.Conclusions:(1)The analysis of the interactions among pathways and global signal transduction analysis revealed the role that AGPS played in maintaining the morphology and proliferation of glioma cells closed related to the core pathways including PI3K-Akt signaling pathway,toll-like receptor signaling pathway,complement and coagulation cascades,and the VEGF signaling pathway and so on,as well as the core protein coding RNAs including EGFR,ITGA11,IGF1 R,AKT3,GYS1,PIK3 CA,ENPP1,JUN,SDC4 CD44,ect.(2)The co-expression analysis of lnc RNA-m RNA indicated that the role AGPS played in maintaining the morphology and proliferation of glioma cells was regulated by the core lnc RNAs including FENDRR,linc-arhgap11b-2,uc004 cic.2,linc-tnfrsf9,rp11-221n13.3,linc?luo?1223,linc-nbpf15-1,NR?044996?4,rp11-760h22.2,uc001 tkz.2,ect.