Project description:We used a multi-omics approach combining transcriptomics, proteomics and metabolomics to study the impact of over-expression and inhibition of the microRNA miR-223, a pleiotropic regulator of metabolic-related disease, in the RAW monocyte-macrophage cell line. We analyzed the levels of proteins, mRNAs, and metabolites in order to identify genes involved in miR-223 regulation, to determine candidate disease biomarkers and potential therapeutic targets. We observed that both up- and down-regulation of miR-223 induced profound changes in the mRNA, protein and metabolite profiles in RAW cells. Microarray-based transcriptomics evidenced a change in 120 genes that were linked predominantly to histone acetylation, bone remodeling and RNA regulation. In addition, 30 out the 120 genes encoded long noncoding RNAs. The nanoLC-MS/MS revealed that 52 proteins were significantly altered when comparing scramble, pre- and anti-miR-223 treatments. Sixteen out of the mRNAs coding these proteins genes are predicted to have binding sites for miR-223. CARM-1, Ube2g2, Cactin and Ndufaf4 were confirmed to be miR-223 targets by western blotting. Analyses using Gene Ontology annotations evidenced association with cell death, splicing and stability of mRNAs, bone remodeling and cell metabolism. miR-223 alteration changed the expression of CARM-1, Ube2g2, Cactin and Ndufaf4 during osteoclastogenesis and macrophage, indicating that these genes are potential biomarkers of these processes. The most important discriminant metabolites found in the metabolomics study were found to be hydrophilic amino acids, carboxylic acids linked to metabolism and pyrimidine nucleotides, indicating that changes in miR-223 expression alter the metabolic profile of cells, and may affect their apoptotic and proliferative state.
Project description:Total bone marrow (BM) from miR-223 knockout (mir-223-/-) and wildtype (miR-223+/+) mice 21 was extracted, prestimulated for 2 days. Then, the BM cells were simultaneously cotransduced with MSCV-Hoxa9-pgk-neomycin and a MSCV-Meis1-IRES-YFP by co-cultivation with irradiated (4,000 cGy) viral producers. HoxA9-Meis1 transduced cells were sorted for YFP expression and continuously selected with neomycin (1.4 mg/ml). Processing of the pre-miRNA through Dicer1 generates a miRNA duplex, consisting of a miRNA and miRNA* strand. Despite the general view that miRNA*s have no functional role, we further investigated miRNA* species in 10 deep sequencing libraries from mouse and human tissue. Comparing miRNA/miRNA* ratios across the miRNA sequence libraries revealed that 50% of the investigated miRNA duplexes exhibit a highly dominant strand. Conversely, 10% of miRNA duplexes show a comparable expression of both strands, while the remaining 40% exhibit variable ratios across the examined libraries as exemplified by miR-223/miR-223* in murine and human cell lines. Functional analyses revealed a regulatory role for miR-223* in myeloid progenitor cells, implying an active role for both arms of the miR-223 duplex. This was further underscored by the demonstration that miR-223 and miR-223* target the IGF1R/PIK3 axis and that high miR-223* levels associate with increased overall survival in acute myeloid leukemia (AML) patients. Thus, we found a supporting role for miR-223* in differentiating myeloid cells in normal as well as the leukemic cell state. The fact that the miR-223 duplex acts through both arms extends the complexity of miRNA-directed gene regulation of this myeloid key miRNA.
Project description:miR-223 is step-wise increasingly up-regulated in the normal esophagus - Barrett's esophagus -esophageal adenocarcinoma carcinoma sequence. In this study, we aimed to determine the function of miR-223 in esophageal adenocarcinoma carcinogenesis.
Project description:Total bone marrow (BM) from miR-223 knockout (mir-223-/-) and wildtype (miR-223+/+) mice 21 was extracted, prestimulated for 2 days. Then, the BM cells were simultaneously cotransduced with MSCV-Hoxa9-pgk-neomycin and a MSCV-Meis1-IRES-YFP by co-cultivation with irradiated (4,000 cGy) viral producers. HoxA9-Meis1 transduced cells were sorted for YFP expression and continuously selected with neomycin (1.4 mg/ml). Processing of the pre-miRNA through Dicer1 generates a miRNA duplex, consisting of a miRNA and miRNA* strand. Despite the general view that miRNA*s have no functional role, we further investigated miRNA* species in 10 deep sequencing libraries from mouse and human tissue. Comparing miRNA/miRNA* ratios across the miRNA sequence libraries revealed that 50% of the investigated miRNA duplexes exhibit a highly dominant strand. Conversely, 10% of miRNA duplexes show a comparable expression of both strands, while the remaining 40% exhibit variable ratios across the examined libraries as exemplified by miR-223/miR-223* in murine and human cell lines. Functional analyses revealed a regulatory role for miR-223* in myeloid progenitor cells, implying an active role for both arms of the miR-223 duplex. This was further underscored by the demonstration that miR-223 and miR-223* target the IGF1R/PIK3 axis and that high miR-223* levels associate with increased overall survival in acute myeloid leukemia (AML) patients. Thus, we found a supporting role for miR-223* in differentiating myeloid cells in normal as well as the leukemic cell state. The fact that the miR-223 duplex acts through both arms extends the complexity of miRNA-directed gene regulation of this myeloid key miRNA. 2 biological replicates
Project description:miR-223 is step-wise increasingly up-regulated in the normal esophagus - Barrett's esophagus -esophageal adenocarcinoma carcinoma sequence. In this study, we aimed to determine the function of miR-223 in esophageal adenocarcinoma carcinogenesis. miR-223 was transfected in OE33 cells using 10nM pre-miR hsa-miR-223 miRNA precursor (Ambion, Life Technologies, Grand Island, NY) and lipofectamin 2000 (OE33_223_1 and OE33_223_2). Mock control OE33 cells were transfected with a negative control pre-miR miRNA (OE33_NEG_1 and OE33_NEG_2). HumanHT-12 v4 Expression BeadChip arrays (Illumina, San Diego, CA) were used for microarray hybridizations to examine the global gene expression of two biological replicated experiments (four samples in total). The array targets more than 25,000 annotated genes with 47,323 unique probes derived from the National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) Release 38 and UniGene (Build 199) databases.