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Effect of Mirn378 overexpression on gene expression during C2C12 myogenic and BMP2-induced osteogenic differentiation

ABSTRACT: Background: MicroRNAs (miRNAs) are a family of small, non-coding single-stranded RNA molecules involved in post-transcriptional regulation of gene expression. As such, they are believed to play a role in regulating the step-wise changes in gene expression patterns that occur during cell fate specification of multipotent stem cells. Here, we have studied whether terminal differentiation of C2C12 myoblasts is indeed controlled by lineage-specific changes in miRNA expression. Results: Using a previously generated RNA polymerase II (Pol-II) ChIP-on-chip dataset, we show differential Pol-II occupancy at the promoter regions of six miRNAs during C2C12 myogenic versus BMP2-induced osteogenic differentiation. Overexpression of one of these miRNAs, miR-378, enhances Alp activity, calcium deposition and mRNA expression of osteogenic marker genes in the presence of BMP2. Conclusions: Our results demonstrate a previously unknown role for miR-378 in promoting BMP2-induced osteogenic differentiation.

ORGANISM(S): Mus musculus

PROVIDER: GSE51883 | GEO | 2014/01/28

SECONDARY ACCESSION(S): PRJNA225670

REPOSITORIES: GEO

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Effect of Mirn378 overexpression on gene expression during C2C12 myogenic and BMP2-induced osteogenic differentiation

Project description:Background: MicroRNAs (miRNAs) are a family of small, non-coding single-stranded RNA molecules involved in post-transcriptional regulation of gene expression. As such, they are believed to play a role in regulating the step-wise changes in gene expression patterns that occur during cell fate specification of multipotent stem cells. Here, we have studied whether terminal differentiation of C2C12 myoblasts is indeed controlled by lineage-specific changes in miRNA expression. Results: Using a previously generated RNA polymerase II (Pol-II) ChIP-on-chip dataset, we show differential Pol-II occupancy at the promoter regions of six miRNAs during C2C12 myogenic versus BMP2-induced osteogenic differentiation. Overexpression of one of these miRNAs, miR-378, enhances Alp activity, calcium deposition and mRNA expression of osteogenic marker genes in the presence of BMP2. Conclusions: Our results demonstrate a previously unknown role for miR-378 in promoting BMP2-induced osteogenic differentiation. Stable C2C12 cell lines C2C12-pMirn0 and C2C12-pMirn378 were generated by lentiviral transduction of C2C12 myoblasts with a Mirn378-overexpression construct and its parent vector, respectively. C2C12-pMirn0 and C2C12-pMirn378 cells were plated at 2.5 x 10^4 cells/cm2 (day -1), cultured for 1 day in DMEM 10%NCS, then (d0) treated with or without 300 ng/ml bone morphogenetic protein 2 (BMP2) for 6 days. RNA was extracted on d0, d3 and d6 and hybridized to GeneChip Mouse Genome 430 2.0 array (Affymetrix).

2014-01-28 | E-GEOD-51883 | biostudies-arrayexpress

Evaluation of BMP2/miRNA expression systems (hybrid vectors) for potent therapeutic efficacy in bone tissue regeneration

Project description:Reconstruction of bone defects and compensation of deficient repair mechanisms represent important goals within the field of regenerative medicine and require novel safe strategies for translation into the clinic. Therefore, we generated a nonviral osteogenic gene therapeutic (hybrid) vector system, combining an improved bone morphogenetic protein 2 (BMP2) gene cassette and pro-osteogenic microRNAs. The hybrid vectors were tested for their functionality and osteogenic differentiation potential in C2C12 and C3H/10T1/2 via alkaline phosphatase enzyme activity assay, enzyme-linked immunosorbent assay and reverse transcription quantitative polymerase chain reaction analysis of miRNA and osteogenic marker expression. In a mouse ectopic bone formation model, osteoinduction in transfected muscle tissue was analysed via micro computed tomography scans and histological staining. All tested hybrid vector systems led to an increase in BMP2 protein overexpression in C2C12 and C3H/10T1/2, when compared with miRNA-lacking constructs. Incorporated miRNAs were found to be efficiently upregulated. Distinct intra- and extracellular localization preferences were observed. The hybrid vector consisting of BMP2 and miR-590 revealed highest potential of inducing osteogenic differentiation. The favourable effect of combining overexpression of BMP2 and miRNAs has been shown in vitro and in vivo. Induction of mineralization was observed in an ectopic bone mouse model, although high variability in terms of size and shape indicates potential for improvement regarding plasmid administration and retention. We believe this study contributes in a valuable way to the preclinical knowledge of nonviral gene therapy for tissue regeneration. Also, we hope to encourage additional research on multi-target gene therapeutic approaches.

2021-12-31 | GSE166110 | GEO

MeDIP-on-chip and Pol-II ChIP-on-chip during C2C12 myogenic and BMP2-induced osteogenic differentiation

Project description:Differentiation of pluripotent embryonic stem cells, via more restricted multipotent adult stem cells, towards the multitude of terminally differentiated, specialized cell types that make up the adult body is a multi-step process characterized by a progressive restriction of differentiation potential. Previous studies have demonstrated tissue-specific differences in DNA methylation patterns that might play a role in lineage restriction and tissue-specific gene expression. However, these studies have mainly focused on either the differentiation of pluripotent ES cells or on comparing somatic cells from different tissues, so that it remains unclear at which stages during development these differences in DNA methylation profiles are established. Since data on the role of DNA methylation during late-stage development is limited, we have examined the role of this epigenetic mark during the terminal differentiation of a multipotent adult stem cell, using C2C12 as a model system. Using parallel MeDIP- and Pol-II ChIP-on-chip approaches, we demonstrate a mutually exclusive relationship between high Pol-II occupancy and high DNA methylation levels. In addition, we observe that in undifferentiated progenitor cells, osteoblast-related genes have in general higher methylation levels than myoblast-related genes, while myoblast-related genes have higher Pol-II occupancy. Induction of osteogenesis with BMP2 leads to a rearrangement of Pol-II towards osteoblast-related genes. However, methylation patterns remain unchanged. These results indicate that DNA methylation primes C2C12 multipotent stem cells for myogenic differentiation and permits induction of the osteogenic gene expression program upon growth factor stimulation. We propose that cell type-specific DNA methylation patterns are established prior to terminal differentiation of adult stem cells.

2011-02-07 | GSE22077 | GEO

The microRNA expression profile of C2C12 cell upon 72 hours of BMP2 treatment

Project description:To identify osteoblast specific miRNAs that can contribute to osteoblastogenesis by post-transcriptionally regulates their targets, BMP2 are treated to C2C12 for 72 hours and performed miRNA microarray. C2C12 cells were treated with vehicles or BMP2 (300 ng/mL) supplemented alpha-MEM media for 72 days and total RNA was harvested and performed exiqon miRNA microarray. Duplicates were used for each condition. Dye swaps were done.

2012-05-02 | E-GEOD-37036 | biostudies-arrayexpress

The microRNA expression profile of C2C12 cell upon 72 hours of BMP2 treatment

2012-05-03 | GSE37036 | GEO

Mus musculus

Project description:MeDIP-on-chip and Pol-II ChIP-on-chip during C2C12 myogenic and BMP2-induced osteogenic differentiation

| PRJNA128959 | ENA

Mouse primary osteoblasts treated with/without BMP2

2012-03-30 | GSE36925 | GEO

Mouse primary osteoblasts treated with/without BMP2

Project description:Bone morphogenetic proteins (BMPs) regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation. We used cDNA microarrays to elucidate regulators of BMP-2-induced osteoblast differentiation.

2012-03-29 | E-GEOD-36925 | biostudies-arrayexpress

Mouse C2C12 cells treated with/without hBMP2

Project description:Bone morphogenetic proteins (BMPs) regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. We used cDNA microarrays to elucidate regulators of BMP-2-induced osteoblast differentiation.

2011-11-11 | E-GEOD-33567 | biostudies-arrayexpress

Mouse C2C12 cells treated with/without hBMP2

Project description:Bone morphogenetic proteins (BMPs) regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells.

2011-11-11 | GSE33567 | GEO

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