miR‐145 Targets the 3′ UTR of Cited2 and Is Reduced by Myostatin in C2C12 cells
Authors: Graham Zachary A. , de Gasperi Rita , Bauman William A. , Cardozo Christopher P. 
Published: November 2023

IntroductionSpinal cord injury (SCI) results in atrophy of skeletal muscle, decreased metabolic activity and impaired contractile function. We have recently found that SCI lowers the levels of expression of five highly expressed microRNA (miRNA) in paralyzed muscle. miRNA are ~22 base pair ribonucleotides that bind to the 3′ UTR of target mRNA to inhibit or repress translation. miR‐145 is one of the miRNA that we have found to be decreased by SCI. It is predicted to target CBP/p300‐interacting transactivator with ED‐rich tail 2 (Cited2), a transcriptional regulator that interacts with CBP and its paralog p300 to alter transcription of genes such as HIF1α and PPARγ. Cited2 can also disrupt the signaling of NF‐κB. Additionally, overexpression of Cited2 can protect differentiated C2C12 myotubes from atrophy following dexamethasone administration. Myostatin is a potent negative regulator of skeletal muscle growth and its signaling is upregulated in many conditions associated with muscle wasting. Our objective in this work was to determine if miR‐145 targets mRNA of Cited2, and whether myostatin alters miR‐145 expression, in addition to other highly expressed miRNA affected by SCI, at varying times of myoblast differentiation.MethodsThe 3′ UTR of mouse Cited2 (nucleotides 1051–1853) was amplified from mouse genomic DNA and cloned into the pmirGLOW Dual Luciferase vector (Promega) downstream from the luciferase gene. A miR‐145 mimic or scrambled siRNA control was co‐transfected into proliferating C2C12 myoblasts with the Cited2 reporter gene. Cells were lysed 48 h post‐transfection and luciferase was measured and normalized to an internal Renilla control. To modify the miR‐145 target site in the 3′ UTR of Cited2, the plasmid was subjected to site directed mutagenesis and the seed sequence changed from AACUGGAA to ACAGAGAA. This mutation was verified by sequencing. To determine the role of myostatin in the expression of miR‐23b, miR‐145 and miR‐206, miRNA that we have noted to be decreased after SCI, C2C12 cells were grown to confluency and incubated in DMEM with 2% horse serum to promote differentiation. Cells were administered 100 ng/mL of recombinant myostatin for 24 h before they were harvested 1, 3, 5 and 7 d post‐differentiation.ResultsTargetScan confirmed that Cited2 has a seed sequence complementary to miR‐145 and found that this sequence is indeed a conserved 7mer in the 3′ UTR region in 20 out of 23 species, including those of rats, mice and humans. A miR‐145 mimic decreased the expression of a Cited2 luciferase reporter assay and this decrease was abrogated when the miR‐145 seed sequence was mutated. Further, incubation with myostatin led to reduced expression of miR‐145 at 1 D post‐differentiation, as well as lower expression of miR‐23b and miR‐206 at 1 and 3D.ConclusionThis is the first study to report that miR‐145 is expressed in murine myoblasts, can be reduced with recombinant myostatin, and can target Cited2 mRNA. Our findings reinforce literature that there is an established link between myostatin and miRNA in skeletal muscle and, importantly, that myostatin‐associated changes in the miRNA profile may occur in paralyzed muscle following SCI.Support or Funding InformationVA RR&D Service Center grant B9212C to WAB