| BackgroundGastric cancer is a common malignancy worldwide,characterized by highly invasiveness and aggressiveness.Besides operation,chemotherapy is usually used to perform adjuvant therapy for gastric cancer.Chemotherapy drugs can be divided into cytotoxic drugs and non cytotoxic drugs,and the curative effect of non cytotoxic drugs for the treatment of gastric cancer is superior to cytotoxic drugs with lower side effect.Non-steroidal anti-inflammatory drugs incluidng cyclooxygenase-2 inhibitors belong to non cytotoxic drugs and can promote the treatment of many tumors.Celecoxib as a kind of cyclooxygenase-2 inhibitor may inhibit proliferation and angiogenesis,as well as promote apoptosis in the tumor cells.However,the celecoxib for inhibiting the occurrence and development of gastric cancer is still unclear.Objective1.The effect of celecoxib on cell viability,cell cycle and cell apoptosis on human gastric carcinoma cell lines NCI-N87 were investigated.2.We verified the m RNA levels of COX-2 in NCI-N87 cells treated and untreated with celecoxib.3.The RNA sequencing data of NCI-N87 human gastric carcinomacells treated with or without celecoxib were prepared.Then,differentially expressed genes(DEGs)and lnc RNAs were identified for pathway enrichment analysis.Afterward,protein-protein interaction(PPI)network for DEGs was constructed and module analysis was performed.Additionally,co-expression analysis of differentially expressed genes and lnc RNAs was performed.This study is aimed to investigate the mechanisms of lnc RNAs in gastric cancer cell line treated with celecoxib.4.We verified the m RNA levels of lnc-SCD-1:13 and lnc-PTMS-1:3as well as ITGA3 and DVL1,aiming to further reveal whether lnc RNAs involved in the treatment of celecoxib in NCI-N87 cells.Methods1.MTT assay was used to detect cell viability of NCI-N87 cells.Besides,cell cycle and apoptosis were observed by flow cytometry analysis.2.The m RNA levels of COX-2 were detected by Real time-PCR in NCI-N87 cells treated and untreated with celecoxib.3.RNA libraries for NCI-N87 cells treated and untreated with celecoxib were built and the next generation sequencing was performed.Quality control(QC)of obtained next generation sequencing(NGS)data was conducted by NGS QC Toolkit.Cuffdiff was applied to screen differentially expressed genes and lnc RNAs with the cut-off criteria of q-value < 0.05.Gene Ontology(GO)functonal and Kyoto Encyclopediaof Genes and Genomes(KEGG)pathway enrichment analysis were performed using GO-function package and KEGGprofile in Bioconductor,respectively(p < 0.05 and gene counts ≥ 2).The PPIs for DEGs were predicted using version 9.1 of STRING database with the combine score > 0.7.Pearson correlation coefficients between DEGs and lnc RNAs were firstly calculated.The co-expressed genes and lnc RNAs pairs were selected with |Pearson correlation coefficient| > 0.98.4.With key genes in NCI-N87 cells treated with celecoxib were selected as experimental objects,the m RNA levels of lnc-SCD-1:13 and lnc-PTMS-1:3 as well as ITGA3 and DVL1 were detected by Real time-PCR in NCI-N87 cells.Results1.Effect of celecoxib on cell proliferation,cell cycle and cell apoptosis in NCI-N87 cellsThe results of MTT analysis showed that the cell viability was significantly inhibited in celecoxib treated cells group compared with untreated cells(p < 0.05).The results of flow cytometry suggested that compared with control group,celecoxib treated cells had a significantly increased percentage of cells in G0/G1 phase(p < 0.05)and lower percentage of cells in S and G2 phase(p < 0.05).The results of flow cytometry showed that the cell viability in early and late apoptosis were both significantly increased in celecoxib treated cells compared withuntreated cells(p < 0.05).2.Effect of celecoxib on the m RNA levels of COX-2 in NCI-N87cellsWe found that the expression of COX-2 m RNA in celecoxib with effective dose treated cells had no significant difference compared with untreated cells.3.Differentially expressed genes and lnc RNAsA total of 490 DEGs,including 302 up-regulated and 188down-regulated DEGs,were identified between the celecoxib group and the control group.Meanwhile,37 differentially expressed lnc RNAs,including 19 up-regulated and 18 down-regulated differentially expressed lnc RNAs,were screened.4.Functional enrichment analysis for DEGsGO enrichment analysis revealed that the up-regulated DEGs mainly were enriched in small molecule metabolic process,involving in ABCC3,ACAA1,B3GNT3 and CD320,and extracellular region,related to ITGA3,ITGA6,ITGB4 and ITGB5 respectively.The down-regulated DEGs were enriched in tissue development(ADAM9,ALDH1A3 and FNDC3B),extracellular region(ADAM9,CCDC80 and CLIC5)and chemokine activity(ADAM9,HSPB1,MARCKS and PKP2),respectively.5.Pathway enrichment analysis for DEGsPathway enrichment analysis indicated that the up-regulated DEGs were significantly enriched in the metabolic pathways(ATP5G1,ATP5G3,ATP6AP1,COX8 A,CYC1,NDUFS3,UQCRC1,UQCRC2 and UQCRFS1)and oxidative phosphorylation(ATP5G1,ATP5G3,ATP6AP1,COX8 A,CYC1,NDUFS3,UQCRC1,UQCRC2 and UQCRFS1).On the other hand,the down-regulated DEGs were enriched in epithelial cell signaling in helicobacter pylori infection(CXCL1,CXCL8 and MAP3K14),chemokine signaling pathway(BCAR1,CXCL1,CXCL3,CXCL5 and CXCL8)and cytokine-cytokine receptor interaction(BMPR2,CXCL1,CXCL3,CXCL5,CXCL8 and TNFRSF19).6.PPI network and module analysisAfter the PPIs of DEGs was predicted,two modules(Module 1 and Module 2)with the highest significance were selected.The DEGs in module 1(ITGB6,LAMA3,ITGA6,ITGB4,ITGB5,ITGA3 and ITGB8)were mainly related to functions about integrin-mediated signaling pathway,extracellular matrix organization and cell adhesion.In module 2,DEGs,including CYC1,COX8 A,UQCRC1,NDUFS3,UQCRC2 and UQCRFS1,were involved in the respiratory electron transport chain and mitochondrial inner membrane.The DEGs(such as LAMA3,ITGA3,ITGA6,ITGB4,ITGB5,ITGB6 and ITGB8)in the module 1 were mainly enriched in the focal adhesion pathway and ECM-receptorinteraction pathway.Meanwhile,three up-regulated DEGs(LAMA3,ITGA6 and ITGA3)were enriched in pathways in cancer.The seven up-regulated DEGs including ATP5G3,CYC1,COX8 A,UQCRC1,NDUFS3,UQCRC2 and UQCRFS1 in module 2 were enriched in oxidative phosphorylation and metabolic pathways.7.Co-expression analysis of differentially expressed genes and lnc RNAsThe DEGs co-expressed with lnc-SCD-1:13,lnc-LRR1-1:2,lnc-PTMS-1:3,lnc-S100P-3:1,lnc-AP000974.1-1:1 and lnc-RAB3IL1-2:1 were enriched in the pathways related to cancer,such as basal cell carcinoma(FZD7,DVL1 and WNT7B)),pathways in cancer(FZD7,DVL1 and ITGA3)and ECM-receptor interaction(ITGA3).The DEGs(DVL1,NFAT5,WNT11 and WNT7B)co-expressed with lnc-SCD-1:13,lnc-LRR1-1:2 and lnc-S100P-3:1were enriched in Wnt signaling pathway.The DEGs(BMP4,FZD7,WNT11 and WNT7B)co-expressed with lnc-SCD-1:13,lnc-LRR1-1:2,lnc-PTMS-1:3,lnc-S100P-3:1 and lnc-AP000974.1-1:1 were enriched in Hedgehog signaling pathway.8.Effect of celecoxib on the m RNA levels of lnc-SCD-1:13 and lnc-PTMS-1:3 as well as ITGA3 and DVL1 in NCI-N87 cellsRT-PCR analysis demonstrated increased m RNA levels of these lnc-RNAs and genes in celecoxib treated cells compared with untreated cells(p < 0.05).Conclusion1.Low concentration of celecoxib plays a important role of anti-cancer via regulating the cell cycle and apoptosis for gastric cancer by mediating the other mechanisms rather than the dependency mechanism of COX-2.2.Celecoxib may reduce the cell migration and proliferative activity to inhibit the development of gastric cancer by downregulation the CXCL1,CXCL3,CXCL5 and CXCL8,which are participated in the chemokine signaling pathway and cytokine-cytokine receptor interaction.3.Celecoxib can reduce the expression of WNT7 B,and may inhibit the progression of gastric cancer via suppressing Wnt signaling pathway.4.Celecoxib can inhibit the development of gastric cancer by ATP5G1,ATP5G3,COX8 A,CYC1,NDUFS3,UQCRC1,UQCRC2和 UQCRFS1,which are participated in the oxidative phosphorylation.5.The DEGs co-expressed with lnc-SCD-1:13,lnc-LRR1-1:2,lnc-PTMS-1:3,lnc-S100P-3:1,lnc-AP000974.1-1:1 and lnc-RAB3IL1-2:1 may play a key role on the development of gastric cancer via participating in many pathways.6.ITGA3 as co-expression gene with Lnc-SCD-1:13 and lnc-PTMS-1:3 may play a key role on the development of gastric cancer via participating in the integrin-mediated signaling pathway. |
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