Putative MicroRNA-mRNA Networks Upon Mdfi Overexpression in C2C12 Cell Differentiation and Muscle Fiber Type Transformation
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ABSTRACT:
SUBMITTER: Huang B
PROVIDER: S-EPMC8560695 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Muscle development requires myoblast differentiation and muscle fiber formation. Myod family inhibitor (Mdfi) inhibits myogenic regulatory factors in NIH3T3 cells, but how Mdfi regulates myoblast myogenic development is still unclear. In the present study, we constructed an Mdfi-overexpression (Mdfi-OE) C2C12 cell line by the CRISPR/Cas9 system and performed RNA-seq on Mdfi-OE and wild-type (WT) C2C12 cells. The RNA-seq results showed that the calcium signaling pathway was the most significant. We also established the regulatory networks of Mdfi-OE on C2C12 cell differentiation and muscle fiber type transformation and identified hub genes. Further, both RNA-seq and experimental verification demonstrated that Mdfi promoted C2C12 cell differentiation by upregulating the expression of Myod, Myog, and Myosin. We also found that the positive regulation of Mdfi on fast-to-slow-twitch muscle fiber transformation is mediated by Myod, Camk2b, and its downstream genes, such as Pgc1a, Pdk4, Cs, Cox4, Acadm, Acox1, Cycs, and Atp5a1. In conclusion, our results demonstrated that Mdfi promotes C2C12 cell differentiation and positively modulates fast-to-slow-twitch muscle fiber transformation. These findings further our understanding of the regulatory mechanisms of Mdfi in myogenic development and muscle fiber type transformation. Our results suggest potential therapeutic targets for muscle- and metabolic-related diseases.
| S-EPMC7862576 | biostudies-literature
Project description:MicroRNAs (miRNAs) and fibroblast growth factor (FGF) signaling regulate a wide range of cellular functions, including cell specification, proliferation, migration, differentiation, and survival. In lens, both these systems control lens fiber cell differentiation; however, a possible link between these processes remains to be examined. Herein, the functional requirement for miRNAs in differentiating lens fiber cells was demonstrated via conditional inactivation of Dicer1 in mouse (Mus musculus) lens. To dissect the miRNA-dependent pathways during lens differentiation, we used a rat (Rattus norvegicus) lens epithelial explant system, induced by FGF2 to differentiate, followed by mRNA and miRNA expression profiling. Transcriptome and miRNome analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on miRNAs. We identified 131 FGF2-regulated miRNAs. Seventy-six of these miRNAs had at least two in silico predicted and inversely regulated target mRNAs. Genes modulated by the greatest number of FGF-regulated miRNAs include DNA-binding transcription factors Nfib, Nfat5/OREBP, c-Maf, Ets1, and N-Myc. Activated FGF signaling influenced bone morphogenetic factor/transforming growth factor-?, Notch, and Wnt signaling cascades implicated earlier in lens differentiation. Specific miRNA:mRNA interaction networks were predicted for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Cpsf6, Dicer1, and Tnrc6b (RNA to miRNA processing); and Ash1l, Med1/PBP, and Kdm5b/Jarid1b/Plu1 (chromatin remodeling). Three miRNAs, including miR-143, miR-155, and miR-301a, down-regulated expression of c-Maf in the 3'-UTR luciferase reporter assays. These present studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and predicted novel gene regulatory networks connected by multiple miRNAs that regulate lens differentiation.
| S-EPMC3852386 | biostudies-literature
Project description:Skeletal muscle plays an essential role in maintaining body energy homeostasis and body flexibility. Loss of muscle mass leads to slower wound healing and recovery from illness, physical disability, poor quality of life, and higher health care costs. So, it is critical for us to understand the mechanism of skeletal muscle myogenic differentiation for maintaining optimal health throughout life. miR-501-3p is a novel muscle-specific miRNA, and its regulation mechanism on myoblast myogenic differentiation is still not clear. We demonstrated that FOS was a direct target gene of miR-501-3p, and MyoD regulated miR-501-3p host gene Clcn5 through bioinformatics prediction. Our previous laboratory experiment found that MDFI overexpression promoted C2C12 myogenic differentiation and MyoD expression. The database also showed there is an FOS binding site in the MDFI promoter region. Therefore, we hypothesize that miR-501-3p formed a feedback loop with FOS, MDFI, and MyoD to regulate myoblast differentiation. To validate our hypothesis, we demonstrated miR-501-3p function in the proliferation and differentiation period of C2C12 cells by transfecting cells with miR-501-3p mimic and inhibitor. Then, we confirmed there is a direct regulatory relationship between miR-501-3p and FOS, MyoD and miR-501-3p, FOS and MDFI through QPCR, dual-luciferase reporter system, and ChIP experiments. Our results not only expand our understanding of the muscle myogenic development mechanism in which miRNA and genes participate in controlling skeletal muscle development, but also provide treatment strategies for skeletal muscle or metabolic-related diseases in the future.
| S-EPMC6627719 | biostudies-literature
Project description:Background: FGF signaling controls numerous processes during cell lineage specification, organogenesis and terminal differentiation. In lens, FGF signaling was implicated as the key pathway that controls lens fiber cell differentiation, but little is known about its full range and spectrum of regulated genes. Results: Herein, we employed rat lens epithelial explant system and performed RNA and microRNA expression profiling in cells induced to differentiate by FGF2. The primary data were collected at explants grown overnight in the presence of 5 ng/ml of FGF2, followed by a treatment with 100 ng/ml of FGF2 and collection of samples at 2, 4, 12 and 24 hours. Global analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on microRNAs (miRNAs). We identified a total number of 131 FGF2-regulated miRNAs. Forty-four of these microRNAs had at least two predicted and inversely regulated target RNA molecules. The genes regulated by the highest number of miRs include Nfib, Nfat5, c-Maf, Ets1 and N-Myc, all encoding DNA-binding transcription factors. Analysis of RNA data revealed that activated FGF signaling influenced other major signaling pathways known to regulate lens differentiation including BMP/TGF-M-NM-2, Notch, and Wnt signaling. In the M-bM-^@M-^\earlyM-bM-^@M-^] response phase (2-4 hours), miRNAs targeted expression of batteries of genes that control transcription, cell death, cell proliferation, cell junction, and protein serine/threonine kinase activity. In M-bM-^@M-^\lateM-bM-^@M-^] stages (12-24 hours), the main miRNA targets included regulators of cell cycle arrest and cellular differentiation. Specific miRNA:mRNA interaction networks were identified for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Dicer1, Fbx33 and Wdr47 (RNA processing); Ash1l, Med1/PBP and Kdm5b (chromatin remodeling); and c-Maf, Ets1 and Stc1 (FGF signaling). MicroRNAs including miR-9, -143, -155, -455 and -543 downregulated expression of c-Maf in the 3M-bM-^@M-^Y-UTR luciferase reporter asssays. The functional requirement for miRNAs in lens was further demonstrated via disrupted lens fiber cell differentiation in lenses with inactivated Dicer1. Conclusions: These studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and identified novel gene regulatory networks (GRNs) connected by multiple miRNAs. Differential gene expression of FGF2-treated lens cell cultures was analyzed at 0, 2, 4, 12 and 24 hours following the treatment. Two biological replicate experiments were performed.
2013-09-06 | E-GEOD-50604 | biostudies-arrayexpress
Project description:Background: FGF signaling controls numerous processes during cell lineage specification, organogenesis and terminal differentiation. In lens, FGF signaling was implicated as the key pathway that controls lens fiber cell differentiation, but little is known about its full range and spectrum of regulated genes. Results: Herein, we employed rat lens epithelial explant system and performed RNA and microRNA expression profiling in cells induced to differentiate by FGF2. The primary data were collected at explants grown overnight in the presence of 5 ng/ml of FGF2, followed by a treatment with 100 ng/ml of FGF2 and collection of samples at 2, 4, 12 and 24 hours. Global analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on microRNAs (miRNAs). We identified a total number of 131 FGF2-regulated miRNAs. Forty-four of these microRNAs had at least two predicted and inversely regulated target RNA molecules. The genes regulated by the highest number of miRs include Nfib, Nfat5, c-Maf, Ets1 and N-Myc, all encoding DNA-binding transcription factors. Analysis of RNA data revealed that activated FGF signaling influenced other major signaling pathways known to regulate lens differentiation including BMP/TGF-β, Notch, and Wnt signaling. In the “early” response phase (2-4 hours), miRNAs targeted expression of batteries of genes that control transcription, cell death, cell proliferation, cell junction, and protein serine/threonine kinase activity. In “late” stages (12-24 hours), the main miRNA targets included regulators of cell cycle arrest and cellular differentiation. Specific miRNA:mRNA interaction networks were identified for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Dicer1, Fbx33 and Wdr47 (RNA processing); Ash1l, Med1/PBP and Kdm5b (chromatin remodeling); and c-Maf, Ets1 and Stc1 (FGF signaling). MicroRNAs including miR-9, -143, -155, -455 and -543 downregulated expression of c-Maf in the 3’-UTR luciferase reporter asssays. The functional requirement for miRNAs in lens was further demonstrated via disrupted lens fiber cell differentiation in lenses with inactivated Dicer1. Conclusions: These studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and identified novel gene regulatory networks (GRNs) connected by multiple miRNAs. Differential gene expression of FGF2-treated lens cell cultures was analyzed at 0, 2, 4, 12 and 24 hours following the treatment.
2013-09-06 | GSE50604 | GEO
Project description:MicroRNA-34b down-regulation in acute myeloid leukemia was previously shown to induce CREB overexpression, thereby causing leukemia proliferation in vitro and in vivo. The role of microRNA-34b and CREB in patients with myeloid malignancies has never been evaluated. We examined microRNA-34b expression and the methylation status of its promoter in cells from patients diagnosed with myeloid malignancies. We used gene expression profiling to identify signatures of myeloid transformation. We established that microRNA-34b has suppressor ability and that CREB has oncogenic potential in primary bone marrow cell cultures and in vivo. MicroRNA-34b was found to be up-regulated in pediatric patients with juvenile myelomonocytic leukemia (n=17) and myelodysplastic syndromes (n=28), but was down-regulated in acute myeloid leukemia patients at diagnosis (n=112). Our results showed that hypermethylation of the microRNA-34b promoter occurred in 66% of cases of acute myeloid leukemia explaining the low microRNA-34b levels and CREB overexpression, whereas preleukemic myelodysplastic syndromes and juvenile myelomonocytic leukemia were not associated with hypermethylation or CREB overexpression. In paired samples taken from the same patients when they had myelodysplastic syndrome and again during the subsequent acute myeloid leukemia, we confirmed microRNA-34b promoter hypermethylation at leukemia onset, with 103 CREB target genes differentially expressed between the two disease stages. This subset of CREB targets was confirmed to associate with high-risk myelodysplastic syndromes in a separate cohort of patients (n=20). Seventy-eight of these 103 CREB targets were also differentially expressed between healthy samples (n=11) and de novo acute myeloid leukemia (n=72). Further, low microRNA-34b and high CREB expression levels induced aberrant myelopoiesis through CREB-dependent pathways in vitro and in vivo. In conclusion, we suggest that microRNA-34b controls CREB expression and contributes to myeloid transformation from both healthy bone marrow and myelodysplastic syndromes. We identified a subset of CREB target genes that represents a novel transcriptional network that may control myeloid transformation.
| S-EPMC3659992 | biostudies-literature
Project description:The exoribonuclease polynucleotide phosphorylase (PNPase, encoded by pnp) is a major player in bacterial RNA decay. In Escherichia coli, PNPase expression is post-transcriptionally regulated at the level of mRNA stability. The primary transcript is very efficiently processed by the endonuclease RNase III at a specific site and the processed pnp mRNA is rapidly degraded in a PNPase-dependent manner. While investigating the PNPase autoregulation mechanism we found, by UV-cross-linking experiments, that the ribosomal protein S1 in crude extracts binds to the pnp-mRNA leader region. We assayed the potential role of S1 protein in pnp gene regulation by modulating S1 expression from depletion to overexpression. We found that S1 depletion led to a sharp decrease of the amount of pnp and other tested mRNAs, as detected by Northern blotting, whereas S1 overexpression caused a strong stabilization of pnp and the other transcripts. Surprisingly, mRNA stabilization depended on PNPase, as it was not observed in a pnp deletion strain. PNPase-dependent stabilization, however, was not detected by chemical decay assay of bulk mRNA. Overall, our data suggest that PNPase exonucleolytic activity may be modulated by the translation potential of the target mRNAs and that, upon ribosomal protein S1 overexpression, PNPase protects from degradation a set of full-length mRNAs. It thus appears that a single mRNA species may be differentially targeted to either decay or PNPase-dependent stabilization, thus preventing its depletion in conditions of fast turnover.
| S-EPMC2578868 | biostudies-literature
Project description:We report differential gene expression in hematopoietic cells (lineage-Sca+) upon overexpression and transplantation of murine hematopoietic stem cells.
2021-02-11 | GSE158126 | GEO
Project description:BackgroundmicroRNAs (miRNAs) are a group of regulatory RNAs that regulate gene expression by binding to specific sequences on target mRNAs. However, functional identification of mRNA targets is usually difficult and time consuming. Here we report hybrid-PCR as a new and rapid approach to screen putative mRNA targets in vitro.ResultsFifteen putative target mRNAs for human cytomegalovirus (HCMV) miR-UL112-1, including previously confirmed HCMV IE72, were identified from mRNA-derived cDNAs using hybrid-PCR. Moreover, we randomly validated six different target candidates by luciferase reporter assays, and confirmed that their luciferase activities were down-regulated with co-transfection of HCMV miR-UL112-1.ConclusionsOur study demonstrated that hybrid-PCR is an effective and rapid approach for screening putative miRNA targets, with much more advantage of simplicity, low cost, and ease of implementation.
| S-EPMC3025964 | biostudies-literature
Project description:There is a lack of well validated prognostic biomarkers in osteosarcoma, a rare, recalcitrant disease for which treatment standards have not changed in over 20 years. We performed microRNA sequencing in 74 frozen osteosarcoma biopsy samples, constituting the largest single center translationally analyzed osteosarcoma cohort to date, and we separately analyzed a multi-omic dataset from a large NCI supported national cooperative group cohort. We validated the prognostic value of candidate microRNA signatures and contextualized them in relevant transcriptomic and epigenomic networks. Our results reveal the existence of molecularly defined phenotypes associated with outcome independent of clinicopathologic features. Through machine learning based integrative pharmacogenomic analysis, the microRNA biomarkers identify novel therapeutics for stratified application in osteosarcoma. The previously unrecognized osteosarcoma subtypes with distinct clinical courses and response to therapy could be translatable for discerning patients appropriate for more intensified, less intensified, or alternate therapeutic regimens.
| S-EPMC7064533 | biostudies-literature