AIM To identify mi RNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine.METHODS Exponentially growing Caco-2 and HT-29 cells were harvested and prepa...AIM To identify mi RNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine.METHODS Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for m RNA, mi RNA and proteomic profiling. m RNA microarray profiling analysis was carried out using the Affymetrix Gene Chip Human Gene 1.0 ST array. mi RNA microarray profiling analysis was carried out using the Affymetrix Genechip mi RNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear iontrap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets(mi RNA, proteomics, m RNA) was conducted in the R software environment using the Euclidean distance measure and Ward's clustering algorithm. The prediction of mi RNA and oppositely correlated protein/m RNA interactions was performed using Target Scan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE mi RNA, protein and m RNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database.RESULTS Differential expression(DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 mi RNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the mi RNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology(GO) analysis of the DE m RNA identified cell adhesion, migration and ECM organization, cellular lipid and cholesterol metabolic processes, small molecule transport and a range of responses to external stimuli, while similar analysis of the DE protein list identified gene expression/transcription, epigenetic mechanisms, DNA replication, differentiation and translation ontology categories. The DE protein and gene lists were found to share 15 biological processes including for example epithelial cell differentiation [P value ≤ 1.81613 E-08(protein list); P ≤ 0.000434311(gene list)] and actin filament bundle assembly [P value ≤ 0.001582797(protein list); P ≤ 0.002733714(gene list)]. Analysis was conducted on the three data streams acquired in parallel to identify targets undergoing potential mi RNA translational repression identified 34 proteins, whose respective m RNAs were detected but no change in expression was observed. Of these 34 proteins, 27 proteins downregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 19 unique anti-correlated/upregulated micro RNAs and 7 proteins upregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 15 unique anti-correlated/downregulated micro RNAs.CONCLUSION This first study providing "tri-omics" analysis of the principal intestinal cell line models Caco-2 and HT-29 has identified 34 proteins potentially undergoing mi RNA translational repression.展开更多
基金Supported by A Strategic Alliance Programme between Alltech Ltd. and DCU and also Enterprise Ireland Innovation Partnership Grant(IP 2015 0375)
文摘AIM To identify mi RNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine.METHODS Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for m RNA, mi RNA and proteomic profiling. m RNA microarray profiling analysis was carried out using the Affymetrix Gene Chip Human Gene 1.0 ST array. mi RNA microarray profiling analysis was carried out using the Affymetrix Genechip mi RNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear iontrap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets(mi RNA, proteomics, m RNA) was conducted in the R software environment using the Euclidean distance measure and Ward's clustering algorithm. The prediction of mi RNA and oppositely correlated protein/m RNA interactions was performed using Target Scan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE mi RNA, protein and m RNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database.RESULTS Differential expression(DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 mi RNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the mi RNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology(GO) analysis of the DE m RNA identified cell adhesion, migration and ECM organization, cellular lipid and cholesterol metabolic processes, small molecule transport and a range of responses to external stimuli, while similar analysis of the DE protein list identified gene expression/transcription, epigenetic mechanisms, DNA replication, differentiation and translation ontology categories. The DE protein and gene lists were found to share 15 biological processes including for example epithelial cell differentiation [P value ≤ 1.81613 E-08(protein list); P ≤ 0.000434311(gene list)] and actin filament bundle assembly [P value ≤ 0.001582797(protein list); P ≤ 0.002733714(gene list)]. Analysis was conducted on the three data streams acquired in parallel to identify targets undergoing potential mi RNA translational repression identified 34 proteins, whose respective m RNAs were detected but no change in expression was observed. Of these 34 proteins, 27 proteins downregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 19 unique anti-correlated/upregulated micro RNAs and 7 proteins upregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 15 unique anti-correlated/downregulated micro RNAs.CONCLUSION This first study providing "tri-omics" analysis of the principal intestinal cell line models Caco-2 and HT-29 has identified 34 proteins potentially undergoing mi RNA translational repression.
