Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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C2C12 Murine Myoblasts over-expressing Eda2r

ABSTRACT: C2C12 (ATCC, #CRL-1772), a myoblast cell line from the C3H mouse strain, was grown in DMEM (Dulbecco’s Modified Eagle’s Medium, Gibco), supplemented with 10% fetal bovine serum (FBS) (Euroclone, Pero, Italy) and 2 mM L-glutamine (Gibco), and maintained in culture at 37 °C with 5% CO₂, without reaching confluence to avoid fusion into myotubes. Transfection of murine myoblasts was performed using Lipofectamine. C2C12 myoblasts were grown in a 6-well plate and transfected with pCMX-GFP plasmid (gifted from Prof. Kakizuka, Japan) or EDA2R-pCMV6-AC-GFP-expressing plasmid (MG222258, Origene) using Lipofectamine 2000 (Life Technologies), according to the manufacturer’s directions. After 48 h, total RNA was isolated from cells with QIAzol Lysis Reagent (Qiagen) and extracted using miRNeasy Kit (Qiagen). RNA concentration was evaluated through Qubit RNA High Sensitivity Assay Kit (Invitrogen, Waltham, MA, USA) while RNA quality was established using 4200 Tapestation (Agilent Technologies, Santa Clara, CA, USA).

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Mus musculus

SUBMITTER: Luca Guarrera

PROVIDER: E-MTAB-13101 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Orphan-Nuclear-Receptors (ONRs) are a group of ligand dependent transcriptional factors belonging to the family of Steroid-Nuclear-Receptors, which includes estrogen, progesterone and retinoic acid receptors. The ONR denomination refers to the fact that the endogenous ligands of these 15 nuclear receptors have not yet been identified. The vast majority of ONRs are expressed not only in normal tissues but also in different types of solid tumors, including various types of breast cancer. In the present study, we define the expression levels of the ONR mRNAs in a large panel of 51 cell-lines, which recapitulate the heterogeneity of breast cancer. Ten ONRs are characterized by a relatively high expression level in the majority of the breast cancer cell-lines considered and this is consistent with the high levels of the transcripts observed in mammary tumor tissues. To get insights into the functional role played by the 10 ONRs in the growth/progression of breast cancer, we silenced each receptor with a siRNA approach. Effective silencing of the NR2F2 receptor in 5 different and representative breast cancer cell-lines with two separate siRNAs results in an increased growth of each cell-line consistent with a tumor-suppressive action of the receptor. The effect is independent of the cell phenotype, as it is observed in Estrogen-Receptor and HER2 positive as well as Triple-Negative cell-lines. By converse, silencing of the NR2F6 receptor reduces the growth of the 5 selected cell-lines characterized by high expression levels of the ONR, suggesting an oncogenic action. Once again, the anti-proliferative effects of NR2F6 silencing are evident in all the cell-lines considered and are not associated with any of the 3 breast cancer phenotypes. Stable silencing of NR2F6 following infection of the Estrogen-Receptor positive MCF-7 and the Triple-Negative MDA-MB231 cell-lines with 2 specific and different shRNAs support the oncogenic properties of the ORF. Indeed, these shRNAs reduce the growth and clonogenic ability of the 2 cell-lines. In a first set of studies, we performed whole-genome RNA-sequencing experiments in the shRNA infected MCF-7 cells to get insights into the molecular mechanisms underlying the oncogenic action of NR2F6. Stable silencing of the NR2F6 mRNA and protein results in an up-regulation of the gene-networks involved in the pathways controlling cell-adhesion and cell-cell interactions. As expected, silencing causes down-regulation of the gene-networks involved in cell-cycle and cell-proliferation. In particular, we identify 50 and 9 genes whose expression is up-regulated and down-regulated by the knock-down of the NR2F6 transcription factor. These genes are likely to be direct or indirect targets of NR2F6 transcriptional activity. In a second set of experiments, we identify the endogenous organic molecules capable of interacting with the NR2F6 receptor using a mass-spectrometry based approach. To this purpose, we used NR2F6 immune-precipitates obtained from MCF-7 cells, which we transfected with a full-length NR2F6 cDNA expression plasmid. These studies permitted us to identify palmitoylethanolamide as the only endogenous organic molecule binding NR2F6 with high affinity and reproducibility.

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C2C12 Murine Myoblasts over-expressing Eda2r

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