- Research article
- Open Access
- Open Peer Review
Transgenic overexpression of miR-133a in skeletal muscle
© Deng et al; licensee BioMed Central Ltd. 2011
- Received: 16 February 2011
- Accepted: 26 May 2011
- Published: 26 May 2011
MicroRNAs (miRNAs) are a class of non-coding regulatory RNAs of ~22 nucleotides in length. miRNAs regulate gene expression post-transcriptionally, primarily by associating with the 3' untranslated region (UTR) of their regulatory target mRNAs. Recent work has begun to reveal roles for miRNAs in a wide range of biological processes, including cell proliferation, differentiation and apoptosis. Many miRNAs are expressed in cardiac and skeletal muscle, and dysregulated miRNA expression has been correlated with muscle-related disorders. We have previously reported that the expression of muscle-specific miR-1 and miR-133 is induced during skeletal muscle differentiation and miR-1 and miR-133 play central regulatory roles in myoblast proliferation and differentiation in vitro.
In this study, we measured the expression of miRNAs in the skeletal muscle of mdx mice, an animal model for human muscular dystrophy. We also generated transgenic mice to overexpress miR-133a in skeletal muscle.
We examined the expression of miRNAs in the skeletal muscle of mdx mice. We found that the expression of muscle miRNAs, including miR-1a, miR-133a and miR-206, was up-regulated in the skeletal muscle of mdx mice. In order to further investigate the function of miR-133a in skeletal muscle in vivo, we have created several independent transgenic founder lines. Surprisingly, skeletal muscle development and function appear to be unaffected in miR-133a transgenic mice.
Our results indicate that miR-133a is dispensable for the normal development and function of skeletal muscle.
- skeletal muscle
MicroRNAs (miRNAs) are a class of ~ 22 nt non-coding RNAs that regulate gene expression post-transcriptionally [1–3]. The involvement of miRNAs in muscle biology has recently been reported [4–11]. miRNAs regulate the expression of transcription factors and signaling mediators important for cardiac and skeletal muscle development and function [7, 12–14]. Aberrant miRNA expression has been observed in muscle diseases, including cardiac and skeletal muscle hypertrophy, heart failure and muscular dystrophy [13, 15–17].
A subset of miRNAs, miR-1, miR-133, miR-206 and miR-208, are either specifically or highly expressed in cardiac and skeletal muscle and are called myomiRs [6, 7, 13]. Among them, miR-133 was shown to promote the proliferation of myoblasts and inhibits their differentiation in cultured skeletal muscle myoblasts. miR-133 enhances myocyte proliferation, at least in part, by reducing protein levels of SRF, a crucial regulator for muscle differentiation [18, 19]. miR-133 also inhibits the translation of polypyrimidine tract-binding protein (nPTB), which controls differential transcript splicing during skeletal-muscle differentiation . Paradoxically, miR-1 and miR-133 exert opposing effects to skeletal-muscle development despite originating from the same miRNA polycistronic transcript. Interestingly, miR-1 and miR-133 also produce opposing effects on apoptosis . Additionally, embryonic stem (ES) cell differentiation towards cardiomyocytes is promoted by miR-1 and inhibited by miR-133 . Furthermore, miR-1 and miR-133 are also important regulators of cardiomyocyte differentiation and heart development [22–24].
Primary skeletal-muscle disorders involve different groups of diseases, including muscular dystrophies, inflammatory myopathies and congenital myopathies. Although the number of genes that are involved in muscle disorders increases every year and histological pathology of disease tissue is well documented, the underlying molecular pathways remain poorly understood . Recent studies have begun to link miRNAs to certain muscle-related diseases [6, 13, 15, 26, 27]. In a recent report, comprehensive miRNA expression profiling revealed that a total of 185 miRNAs were dysregulated in samples of diseased muscle tissue from 10 different muscle disorders. Five miRNAs (miR-146b, miR-221, miR-155, miR-214 and miR-222) were consistently regulated in almost all samples that were examined , suggesting a possible involvement of common miRNA-mediated regulatory mechanisms in muscle disorders. In addition to those studies of miRNA expression on muscle disorders, a direct genetic link has connected miRNA function to muscular hypertrophy . A mutation that is responsible for the exceptional muscularity of Texel sheep has been mapped to a single G-to-A mutation within the 3' UTR of the mRNA encoding myostatin, a member of the transforming growth factor-β (TGFβ) family, which functions to repress muscle growth. This mutation creates a binding site for miR-1 and miR-206, leading to the translational repression of myostatin, which phenocopies the "muscle doubling" that results from the loss of myostatin in mice, cattle, and humans [29, 30]. These findings underscore the importance of miRNA-mediated regulation in diverse muscle biological processes and disease status.
In this study, we attempted to determine the function of miR-133 in skeletal muscle. We employed a gain-of-function approach and generated transgenic mice to overexpress miR-133a-1 in skeletal muscle, using the well-characterized muscle creatine kinase (MCK) promoter. Surprisingly, we found that miR-133a-1 transgenic mice appear to be normal. Additional analyses indicated that skeletal muscle development and function were not altered in miR-133a-1 transgenic mice. Our study therefore suggests that miR-133a is dispensable for skeletal muscle development.
The mdx mice which carry a point mutation in the dystrophin gene were obtained from the Jackson Lab . Skeletal muscle was collected from the hind legs of 1 month old mdx and control mice for RNA extraction. In order to generate miR-133a-1 transgenic mice, a genomic fragment encoding the precursor and franking sequences of the miR-133a-1 gene, which is located on mouse chromosome 18, was amplified by PCR using mouse genomic DNA as a template. The primers used for amplification are: miR-133a-1F: 5' AAGCTAGCGAATTC CATGTGACCCCTCACACACA 3'; miR-133a-1R: 5' TTCTCGAG ACAAGGGGAGCCTGGATCCC 3'. Underlined nucleotide sequences are added adaptors for restriction enzyme digestion. The same primers were used for genotyping of transgenic mice.
The DNA fragment was cloned into a transgenic vector plasmid driving by a muscle-specific muscle creatine kinase (MCK) promoter . The miR-133a-1 transgenic construct was injected into the pronuclei of C57/Bl6 X C3H hybrid embryos and implanted into pseudo-pregnant recipient females by the University of North Carolina Animal Models Core. Five positive founder lines were obtained.
All animal procedures were approved by and performed in accordance with the University of North Carolina Institutional Animal Care and Use Committee under the protocol 08-227.0.
Northern Blot and RT-PCR Analysis
Taqman-based miRNA quantitative RT-PCR (Applied Biosystems) was performed as described  using total RNAs isolated from skeletal muscle of 1 month old mdx and the control mice (n = 4). MicroRNA Northern blot analyses were performed as described previously [8, 18]. Briefly, 20 μg of total RNAs isolated from skeletal muscle of 1 month old mdx and the control mice (Figure 1), or from the heart, skeletal muscle and liver tissues of miR-133a-1 transgenic and the control mice (Figure 2), were used and miRNA oligonucleotides with corresponding miRNAs (miR-1a, miR-133a and miR-206) sequences were used as probes. qRT-PCR was repeated three times and Northern blots were performed two times on skeletal muscle of four mice per genotype tested.
Histological and immunohistochemistry analyses
Histological processing and immunohistochemistry staining of skeletal muscle tissues were performed as described previously [8, 34]. Samples were stained with Hematoxylin and Eosin (H&E) for routine examination. Laminin conjugated staining was applied to identify sarcolemmal membranes so that myofiber diameter could be directly visualized. Histological and immunohistochemistry analyses were performed on least 4 animals per genotype and repeated independently. All images were acquired by a camera (UFX-DX; Nikon) mounted on an inverted (TE2000, Nikon) or an upright fluorescence microscope (Microphot-SA; Nikon). Digital fluorescent images were captured at room temperature and the images were processed using SPOT (version 3.5.4 for MacOS; Diagnostic Instruments) software and were scaled down and cropped in Photoshop (Adobe) to prepare the final figures. Gel images were quantified using the Photoshop Histogram Analysis and was plotted using Microsoft excel and is shown as relative expression level after normalized by controls.
Changed expression of muscle miRNAs in the skeletal muscle of mdx mice
Transgenic overexpression of miR-133a-1 in the skeletal muscle
Normal skeletal muscle development in miR-133 transgenic mice
In this study, we investigated the function of miR-133a in skeletal muscle. We found that the expression miR-133a, together with that of miR-206 and miR-1a, was induced in the skeletal muscle of mdx mice. However, transgenic overexpression of miR-133a-1 in skeletal muscle did not result in a noticeable change in skeletal muscle development and morphogenesis. Our results are consistent with a recent report in which miR-133 loss-of-function mice did not induce overt defects in skeletal muscle .
It is known that many miRNAs are tissue-specifically expressed. Among them, miR-1, miR-133, miR-206, miR-208 and miR-499 have been described as muscle specific miRNAs, or myomiRs [6, 13]. In addition to their muscle specific expression in normal physiological condition, the expression of some of those myomiRs was shown to be dynamically regulated in pathological and/or diseased muscles. For example, miR-206 levels are elevated in the diaphragm muscle of the mdx mouse, an animal model of muscular dystrophy . Furthermore, miR-1 and miR-206 also participate the regulation of skeletal muscle satellite cell proliferation and differentiation . Both gain- and loss-of-function studies have started to uncover the in vivo function of those muscle miRNAs [10, 23, 34, 38–41]. Interestingly, gene targeting of some of the myomiRs demonstrated that they are dispensable for the normal development and function of cardiac and skeletal muscles [10, 40]. However, many of those miRNAs are indispensable for stress-responsive muscle remodeling. Recently, we reported that overexpression of miR-208a in the heart, which is normally restricted to cardiac tissue, was sufficient to induce cardiac hypertrophy in transgenic mice . However, in the current study, we did not observe any overt muscle defect in mice expressing the MCK-miR-133a-1 transgene. This may suggest that cardiac and skeletal muscle have adapted distinct requirement for miRNAs for tissue homeostasis.
Given the vast number of miRNAs and the diverse functions in different biological processes observed in the relatively small number of miRNAs studied thus far, it is apparent that many new and unanticipated functions of miRNAs in normal muscle development, function and disorders are waiting to be discovered. Considering that many miRNAs fine-tune gene-expression programs and the intrinsic complexity of miRNA functional models [42, 43], it will important in the future to systematically analyze the potential function of miRNAs in both gain- and loss-of-function animal models.
In this study, we demonstrate that miR-133a is dispensable for the normal development and function of skeletal muscle.
We thank members of the Wang and Deng laboratories for discussion and support and Dr. John Mably for critical reading of the manuscript. This work was supported by the March of Dimes Birth Defect Foundation, National Institutes of Health and Muscular Dystrophy Association. Z Deng is supported by the Chinese National Natural Science Foundation # 30772211. DZ Wang is an Established Investigator of the American Heart Association.
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