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
Background:Hypoxia is a hallmark of cancer and is associated with poor prognosis.However,the molecular mechanism by which hypoxia promotes tumor progression remains unclear.MicroRNAs dysregulation has been shown to pl...Background:Hypoxia is a hallmark of cancer and is associated with poor prognosis.However,the molecular mechanism by which hypoxia promotes tumor progression remains unclear.MicroRNAs dysregulation has been shown to play a critical role in the tumor and tumor microenvironment.Here,we investigated the roles ofmiR-495 and miR-5688 in human non-small cell lung cancer(NSCLC)and their underlying mechanism.Methods:The expression levels of miR-495 and miR-5688 in human NSCLC tissue specimens were measured by quantitative real-time polymerase chain reaction(qRT-PCR).Deferoxamine(DFO)was used to determine whether the regulation of miR-495 and miR-5688 under hypoxia was dependent on hypoxia-inducible factor 1-alpha(HIF-1α).Furthermore,the functions of miR-495 and miR-5688 in tumor progression were evaluated using colony formation,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium(MTS),wound healing,transwell assays,and xenograft model.Two algorithms,PicTAR and Targetscan,were used to predict the target gene of these two miRNAs,and dual-luciferase reporter assay was conducted to confirm the target.The unpaired two-tailed t test,Pearson correlation analysis,and Fisher’s exact probability test were performed for statistical analyses.Results:Two miRNAs,miR-495 and miR-5688,were found to participate in NSCLC progression under hypoxia.They were down-regulated in NSCLC tissues compared with normal tissues.We determined that hypoxia led to the down-regulation of miR-495 and miR-5688 in NSCLC cells,which was independent of HIF-1αand cellular metabolic energy.In addition,miR-495 and miR-5688 suppressed cell proliferation,migration,and invasion in vitro.The NSCLC xenograft model showed that miR-495 and miR-5688 inhibited tumor formation in vivo.Interestingly,we found that miR-495 and miR-5688 had the same target,interleukin-11(IL-11).Recombinant human IL-11 counteracted the effects of miR-495 and miR-5688 on NSCLC cells,suggesting that miR-495 and miR-5688 executed their tumor suppressive role by repressing IL-11 expression.Conclusion:We found that hypoxia down-regulated the expression levels of miR-495 and miR-5688 in NSCLCto enhance IL-11 expression and tumor progression,indicating that the miR-495/miR-5688/IL-11 axismay serve as a therapeutic target and potential biomarker for NSCLC.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.
基金supported by the National Natural Science Foundation of China(No.81602026)the Natural Science Foundation of Tianjin(No.18JCQNJC81600 and 18JCZDJC32600).
文摘Background:Hypoxia is a hallmark of cancer and is associated with poor prognosis.However,the molecular mechanism by which hypoxia promotes tumor progression remains unclear.MicroRNAs dysregulation has been shown to play a critical role in the tumor and tumor microenvironment.Here,we investigated the roles ofmiR-495 and miR-5688 in human non-small cell lung cancer(NSCLC)and their underlying mechanism.Methods:The expression levels of miR-495 and miR-5688 in human NSCLC tissue specimens were measured by quantitative real-time polymerase chain reaction(qRT-PCR).Deferoxamine(DFO)was used to determine whether the regulation of miR-495 and miR-5688 under hypoxia was dependent on hypoxia-inducible factor 1-alpha(HIF-1α).Furthermore,the functions of miR-495 and miR-5688 in tumor progression were evaluated using colony formation,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium(MTS),wound healing,transwell assays,and xenograft model.Two algorithms,PicTAR and Targetscan,were used to predict the target gene of these two miRNAs,and dual-luciferase reporter assay was conducted to confirm the target.The unpaired two-tailed t test,Pearson correlation analysis,and Fisher’s exact probability test were performed for statistical analyses.Results:Two miRNAs,miR-495 and miR-5688,were found to participate in NSCLC progression under hypoxia.They were down-regulated in NSCLC tissues compared with normal tissues.We determined that hypoxia led to the down-regulation of miR-495 and miR-5688 in NSCLC cells,which was independent of HIF-1αand cellular metabolic energy.In addition,miR-495 and miR-5688 suppressed cell proliferation,migration,and invasion in vitro.The NSCLC xenograft model showed that miR-495 and miR-5688 inhibited tumor formation in vivo.Interestingly,we found that miR-495 and miR-5688 had the same target,interleukin-11(IL-11).Recombinant human IL-11 counteracted the effects of miR-495 and miR-5688 on NSCLC cells,suggesting that miR-495 and miR-5688 executed their tumor suppressive role by repressing IL-11 expression.Conclusion:We found that hypoxia down-regulated the expression levels of miR-495 and miR-5688 in NSCLCto enhance IL-11 expression and tumor progression,indicating that the miR-495/miR-5688/IL-11 axismay serve as a therapeutic target and potential biomarker for NSCLC.
