Abstract
MicroRNA-95 (miR-95) is well known for its ability to promote the proliferation of a variety of cancer cells, but its function in skeletal muscle development has not been reported so far. Our laboratory has recently generated genetically engineered Meishan pigs containing a loss-of-function myostatin (MSTN) mutant (MSTN-/-). These MSTN-/- pigs grow and develop normally but show clear double muscle phenotype as observed in Belgian cattle. We observed that the expression of miR-95 was up-regulated in the longissimus dorsi from MSTN-/- Meishan pigs at day 65 during embryo development. In this study, we investigated the role of miR-95 in the myogenic differentiation using a murine myoblast cell line C2C12. Our results revealed that miR-95 may play a very important role in regulating the expression of myogenic differentiation marker genes myosin heavy chain (MHC) and myogenin. By use of bioinformatical analysis and luciferase reporter gene assay, aminoacyl-tRNA synthase complex-interacting multifunctional protein 2 (AIMP2) gene was identified as a miR-95 target gene involved in myogenic differentiation. Our results indicated that higher miR-95 expression level leads to lower level of AIMP2 protein expression. When the endogenous expression of AIMP2 is inhibited by siRNA, the expression levels of myogenic differentiation marker genes MHC and myogenin increased, implying that AIMP2 negatively regulates myogenic differentiation. Taken together, it is likely that miR-95 promotes myogenic differentiation in C2C12 myoblasts and may play a positive functional role in skeletal muscle development by down regulating the expression of AIMP2 at protein level.
Highlights
As early as 1997, McPherron and Lee [1] identified a new Transforming growth factor beta superfamily (TGF-β) member called myostatin (MSTN) in mice
We have identified several miRNAs www.impactjournals.com/oncotarget that are up-regulated in MSTN-/- pigs, and these miRNAs have previously been shown to be involved in myoblast development, including the well-known miR-1, miR206 [13, 15], and miR-486 [26] (Figure 1A)
It was speculated that based on the fact that the expression of miR-95 in skeletal muscle is higher in MSTN-/- than in wild type (WT) Meishan pigs at day E65
Summary
As early as 1997, McPherron and Lee [1] identified a new TGF-β member called myostatin (MSTN) in mice. A series of in vivo studies such as gene knockout have confirmed MSTN’s inhibitory roles in muscle proliferation and development. Gene editing methods such as nuclease-mediated zinc finger nucleases (ZFNs), transcriptional activator like effector nucleases (TALENs), and RNA-guided CRISPR-Cas nuclease (CRISPR/ Cas9) have been widely used to make specific genetic modifications. Our lab has recently generated ZFN-edited MSTN loss-of-function mutant pigs that have the same apparent phenotype as the double muscle Belgian cattle [2]. These MSTN-edited Meishan pigs are as healthy as normal wild type pigs, but produce improved quality pork with www.impactjournals.com/oncotarget greater lean yield and lower fat mass [2]. It has been demonstrated that skeletal muscle development is negatively impacted with incomplete hyperplasia in mice when endonuclease Dicer was conditionally knocked down [3,4,5], implying that microRNAs may play important roles in skeletal muscle development
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