Transcriptomics Study On Dads Inhibiting The Proliferation Of Poorly Differentiated Gastric Cancer

Objective: Diallyl disulfide (DADS) has inhibitory effects on various cancer cell lines in vitro, but its mechanisms remain unclear. This study was designed to construct the differential gene expression profile , differential transcription factors profile and microRNA profile induced by DADS on human gastric cell line MGC803 and make bioinformatics analysis, to investigate molecular mechanisms on proliferative inhibition.Methods: The differential expression profile of genes in MGC803 cells exposed to DADS was identified by SuperArray Human Whole Genome Array. Real-time PCR analyzed the expression of differential expressed genes.The differential expression profile of microRNA was identified by microRNA Arrays and analyzed by Real-time PCR. The differential profile of transcription factor was identified by TranSignalTM Protein/DNA Arrays.Results:By statistically filter of data, treat and untreat groups expressed 1677 probesets including 1293 up-regulated and 1228 down-regulated in treat group. A t test analysis(P<0.001) estimated that 292 and 116 probesets measured noticeably changed expression between treat and untreat. By unsupervised hierarchical clustering and analysis, differential gene expression profiles clearly distinguished treat from untreat group. After treated with 30mg?ml-1DADS for 4h, Real-time PCR showed the TIMP3 was up-regulated while MMP9 was down-regulated.The result of bioinformatic analysis estimated the functional classification of differential expressed genes systematically.Three pathways, such as"Angiogenesis","Integrin signalling pathway"and"Oxidative stress response", at least P<0.05, were examined for enrichment in up-regulated genes in treat group among 152 up-regulated pathways by Panther software. There were no down-regulated pathway in up-regulated genes.17 GO BP categories, including"Signal transduction","Cell structure and motility","Cell proliferation and differentiation"and"Cell cycle control", were examined for enrichment which P values less than 0.05 in up-regulated genes in treat group."Lactation,mamary development"was examined in down-regulated genes in treat group.The results of GO MF analysis had shown 4 up-regulated categories, including"Actin binding cytoskeletal protein","Cytoskeletal protein","Signaling molecule"and"Extracellular matrix"in treat group.There were no categories in treat group. The results of integrating analysis by DAVID software had shown 58 GO BP up-regulated categories including"angiogenesis","positive regulation of cell differentiation","positive regulation of transcription","negative regulation of cell communication","immunity and defense"and so on in treat. Up-regulated IL-18 and KLF6 were involved with antitumor immunity and inhibition of proliferation induced by DADS. Down-regulated categories in relation to cell apoptosis and signaling cascade were enriched in treat group.4 GO MF categories, including"actin binding","cytoskeletal protein binding","growth factor activity"and"heparin binding", were examined for enrichment which P values less than 0.001 in up-regulated genes in treat group."cyclin-dependent protein kinase activity"and"calcium-dependent phospholipase A2 activity"were enriched which P values less than 0.05.Through the database of KEGG-PATHWAY and BIOCARTApathway, TSP-1 Induced Apoptosis in Microvascular Endothelial Cell, Wnt signaling pathway, Colorectal cancer, Regulation of actin cytoskeleton and ErbB signaling pathway were examined for enrichment in up-regulated genes.The transcription factor differential expression profile were attained TranSignalTM Protein/DNA Arrays between treat group with DADS and untreat group. There were 14 up-regulated transcription factors including RB, GKLF/SP1, PUR, TFE3-L, beta M-globin factor B1 and so on and 68 down-regulated transcription factors including GAS/ISRE, Pbx1 and Stat5/Stat6 and so on in DADS treated group compared with untreated one. Through target gene prediction analysis, the target genes from 22 transcription factors were in accordance with those from differential expressed profile.Comprehensive analysis of pathway enrichment and differential expressed genes, DADS reduced the expression level of MMP9 through inhibition of ERK/AP-1 and ERK/Ets-1 pathway. DADS inhibited the ability to migrate and invade by inhibiting ERK/AP-1/Fra-1/uPA and wnt pathway in relation to Frizzeld, DVL, APC, DKK, MMP and transcription factor tcf/lef.MicroRNA array detected 22 up-regulated microRNA and 20 down-regulated differentially. Real-time PCR analysis showed miR-222 was down regulated while miR-665 was up regulated compared with control. Their target genes were TGFBR1, TIMP3 and THBS1 which were involved in migration and invasion in tumor, indicating that the microRNA were target molecules which DADS influenced on.Comprehensive analysis of pathway, up-regulated genes were involved with cytoskeleton regulation and cell migration and invasion compared with control. Comprehensive analysis of GO BP and MF, up-regulated genes were involved with several biological functions including angiogenesis, cell commnication, cytoskeletal protein binding and so on. The results above indicated that DADS had effects on inhibiting migration and invasion in gastric cancer cells.Conclusion:1. Applying bioinformatic analysis for high-thoroughly microarray data, differential gene expression profiles were obtained between treated group with DADS and untreated in gastric MGC803 cells, from which 316 up-regulated and 136 down-regulated genes were selected(P<0.001).Up-regulated genes were associated with cell differentiation, angiogenesis, cell apoptosis, cell communication and immunity and defense, containing TIMP3, APC2, THBS1, RND3, MMP9, KLF6 and IL-18.2. Applying TranSignalTM Protein/DNA array, differential transcription factor expression profile was obtained including 14 up-regulated transcription factors and 68 down-regulated ones. Gklf and PPARγwere associated with cell cycle arrest and inhibition of migration and invasion induced by DADS in MGC803 cells.3. Comprehensive analysis of enriched pathway, differential genes and prediction of target genes,"ERK/AP-1/MMP9","ERK/Ets-1/MMP9", Wnt and TGFβpathway were involved with inhibition of migration and invasion induced by DADS.4. Applying microRNA array, differential microRNAs were obtained including 22 up-regulated and 20 down-regulated and miR-222 and miR-665 were associated with inhibition of migration and invasion in MGC803 cells.5. By differential gene expression profiles, differential transcription factor expression profiles, differential microRNAs profiles and bioinformatic analysis, the molecular mechanism in migration and invasion became clearer in MGC803 cells induced by DADS.