Regulation of DMD pathology by an ankyrin-encoded miRNA
© Alexander et al; licensee BioMed Central Ltd. 2011
Received: 8 June 2011
Accepted: 8 August 2011
Published: 8 August 2011
Duchenne muscular dystrophy (DMD) is an X-linked myopathy resulting from the production of a nonfunctional dystrophin protein. MicroRNA (miRNA) are small 21- to 24-nucleotide RNA that can regulate both individual genes and entire cell signaling pathways. Previously, we identified several mRNA, both muscle-enriched and inflammation-induced, that are dysregulated in the skeletal muscles of DMD patients. One particularly muscle-enriched miRNA, miR-486, is significantly downregulated in dystrophin-deficient mouse and human skeletal muscles. miR-486 is embedded within the ANKYRIN1(ANK1) gene locus, which is transcribed as either a long (erythroid-enriched) or a short (heart muscle- and skeletal muscle-enriched) isoform, depending on the cell and tissue types.
Inhibition of miR-486 in normal muscle myoblasts results in inhibited migration and failure to repair a wound in primary myoblast cell cultures. Conversely, overexpression of miR-486 in primary myoblast cell cultures results in increased proliferation with no changes in cellular apoptosis. Using bioinformatics and miRNA reporter assays, we have identified platelet-derived growth factor receptor β, along with several other downstream targets of the phosphatase and tensin homolog deleted on chromosome 10/AKT (PTEN/AKT) pathway, as being modulated by miR-486. The generation of muscle-specific transgenic mice that overexpress miR-486 revealed that miR-486 alters the cell cycle kinetics of regenerated myofibers in vivo, as these mice had impaired muscle regeneration.
These studies demonstrate a link for miR-486 as a regulator of the PTEN/AKT pathway in dystrophin-deficient muscle and an important factor in the regulation of DMD muscle pathology.
Duchenne muscular dystrophy (DMD) is a progressive, X-linked, muscle-wasting disease that occurs in 1 in 3,500 live male births . Males with inactivating mutations in the DMD (dystrophin) gene make a nonfunctional protein which results in degenerating skeletal muscles, severely elevated creatine kinase (CK) levels, cardiac arrhythmias and secondary infections and subsequent respiratory failure due to the degeneration of the diaphragm muscles [2, 3]. By contrast, patients with Becker muscular dystrophy (BMD) also have mutations in the DMD gene, but produce a partially functional dystrophin protein . Patients with BMD have mildly progressive muscle weakness and elevated CK serum levels as well as variable longevity and mobility . While the genetic mutations that cause DMD are now well established, the subsequent steps that lead to the disease pathogenesis are still emerging . In dystrophin-deficient muscles, it has been demonstrated that the platelet-derived growth factor (PDGF) receptor (PDGFR) signaling pathway is significantly altered [5, 6]. Activation of PDGF signaling has been shown to promote the proliferation of myoblasts in primary myoblasts and C2C12 cultures . PDGF signaling in skeletal muscles goes through a phosphatase and tensin homolog deleted on chromosome 10/AKT (PTEN)/AKT signaling pathway that is an important regulator of insulin signaling and myoblast differentiation, proliferation and cellular viability . Recent studies in the mdx (dystrophin) mutant mouse model have found that mdx muscle contains elevated PTEN/AKT mRNA levels [9, 10]. In addition, spontaneously occurring animal models of dystrophin deficiencies, such as the Golden Retriever muscular dystrophy dog, have been found to have elevated levels of PTEN within affected skeletal muscle fibers . Thus, in two animal models of dystrophin deficiency, along with isolated biopsies from human DMD patients, the upregulation of PTEN/AKT signaling has been shown to be a secondary consequence of the lack of a functional dystrophin protein resulting from an as yet unclear mechanism.
Recently, we have shown that distinctive patterns of microRNA (miRNA) expression are associated with different types of primary muscle disorders . One particular miRNA, miR-486, was significantly reduced in patients with DMD relative to control muscle biopsies. Intriguingly, this miRNA was reduced relative to control samples only in patients with DMD and not in those with the milder but allelic BMD. miRNA are known to be powerful regulators of multiple cell signaling pathways at critical time points in mammalian development and differentiation. miRNA can downregulate gene expression through either mRNA target degradation or inhibition of mRNA translation at the ribosome . In skeletal muscle development, a series of muscle-enriched miRNA control myoblast differentiation and overall muscle strength and contraction .
Ankyrin 1 belongs to a family of proteins that contain the ankyrin repeat domain, which is essential for cellular membrane integrity and modulation of extracellular signaling factors . mIR-486 is transcribed within the ANK1 (previously referred to as ankyrin-R) locus, from which both erythroid and skeletal muscle-specific isoforms are transcribed . The smaller isoform of the ANK1 gene, ANK1.5 (also referred to as sANK1) contains an alternately spliced, muscle-specific exon (exon 39A) and lacks the characteristic ankyrin repeat domain [16, 17]. ANK1.5 is a small, skeletal muscle-specific protein that associates with the sarcoplasmic reticulum of skeletal muscle myofibers [16, 18]. Molecular interaction experiments revealed that ANK1.5 interacts directly with obscurin at the Z-disks of striated muscle fibers .
In a recent study, miR-486 was identified as a negative regulator of PTEN expression levels in muscle and subsequently in the PTEN/AKT pathway in normal cardiac and skeletal muscle development . Using computational bioinformatics, we identified additional miR-486 downstream targets in adult skeletal muscle: PDGFRβ, PIK3R1 (p85α) and insulin-like growth factor 1 (IGF-1). Inhibition of miR-486 using a GFP-tagged lentivirus impaired cellular migration to a scratch wound injury, decreased myoblast fusion and increased cellular mitosis. Conversely, overexpression of miR-486 resulted in faster closure of the scratch wound, with no detrimental cellular apoptosis, compared to a scrambled miRNA control. Generation of muscle-specific miR-486-transgenic mice revealed perturbed muscle regeneration following cardiotoxin (CTX)-induced tibialis anterior (TA) muscle injury and dysregulation of the downstream target genes of miR-486 in vivo. Together these studies begin to characterize a functional role for miR-486 in normal and dystrophin-deficient skeletal muscle and further identify several components of the PTEN/AKT signaling pathway as potential targets for miR-486 regulation in muscle.
Results and Discussion
miRNA-486 expression is a significantly reduced miRNA in dystrophin-deficient skeletal muscle
In vivo skeletal muscle regeneration can be induced following CTX injury and is widely used to study factors thought to be essential for new muscle fiber formation . Wild-type and mdx 5cv (dystrophin-deficient) mice were injured in their TA muscles by injection of CTX. This resulted in the destruction of up to 90% of myofibers followed by the regeneration of new myofibers (n = 3 mice per time point for each cohort). Muscle biopsies from the injured wild-type and mdx 5cv mice were harvested, and total miRNA was extracted to determine whether the levels of miR-486 were dynamically regulated during skeletal muscle regeneration. CTX injury was validated by H & E staining of sections over the time course (representative images obtained on days 0 and 14 are shown in Additional file 1, Figure S1). In normal mouse muscle, miR-486 expression was significantly increased by day 5 after CTX injury, which is when nascent myotubes begin to appear [22, 23] (Figure 1D). Conversely, mdx 5cv mice, which have impaired skeletal muscle regeneration following CTX injury , showed overall reduced levels of miR-486 during skeletal muscle regeneration, which were statistically significant (P < 0.005) at day 5 after CTX injury, when there is maximum miR-486 expression in normal mouse muscle (Figure 1D). Thus, miR-486 appears to be an important miRNA that is dynamically regulated during normal skeletal muscle regeneration, and its expression is significantly reduced in mdx 5cv mice.
Inhibition of miR-486 in myoblasts and myotubes causes profound physical and cellular changes
miR-486 is essential for normal myoblast fusion, cellular kinetics and viability
miR-486 regulates a variety of PTEN/AKT signaling components in skeletal muscle
Transgenic mice expressing miR-486 have impaired skeletal muscle regeneration following CTX injury
Transgenic mice overexpressing miR-486 have altered levels of PTEN/AKT signaling components during skeletal muscle regeneration
miR-486 has also been shown to be dysregulated in three other pathological conditions: white blood cells during sepsis, glioblastomas and lung adenocarcinomas [35–37]. Many cancers have been shown to have dysregulated PTEN/AKT signaling resulting from a failure to regulate normal cell cycle kinetics and cellular apoptosis [38, 39]. miR-486's influence on cell cycle progression through the regulation of PTEN/AKT signaling components and their subsequent downstream regulation of cyclin-dependent kinase inhibitors reinforces the role of miR-486 as a biomarker in several cancers. In cardiomyocytes, miR-486 has been shown to be a negative regulator of PTEN/AKT signaling during heart ventricle postnatal development . That study also demonstrated that miR-486 expression can be activated by myocardin-related transcription factor A (MRTF-A, also referred to as MAL/MKL1) , a coregulator of the serum response transcription factor, which has been shown to regulate actin polymerization and Rho signaling in cardiac hypertrophy [40, 41]. Although MRTF-A has been studied predominately for its role in cardiac hypertrophy and heart failure, it is expressed in C2C12 myoblasts and may be essential for normal myogenic differentiation. Additionally, MRTF-A may have a function in skeletal muscle hypertrophy and atrophy through a striated activator of Rho signaling-dependent pathway [42, 43]. Another recent study identified miR-486 along with miR-206 (another muscle-enriched miRNA) as a regulator of myoblast cell cycle kinetics through its downregulation of Pax7 mRNA and another muscle-enriched miRNA, miR-206 . However, given that miR-486 expression is activated by myogenic factors that are downstream of Pax7 (such as MyoD1), it is likely that miR-486's regulation of PTEN/AKT signaling might explain the cell cycle defects due to the Pax7+ low expression in MyoD1+ myoblasts [45, 46].
miR-486 may also have important regulatory functions that are independent of PTEN/AKT signaling in other cell types and pathological diseases. Recently, miR-486 has been found to be significantly upregulated in lymphoblast cell lines derived from autistic patients . Intriguingly, patients with DMD have an increased incidence of autism or other neurological disorders [48, 49]. Given that two of the components that are essential for normal alternative splicing of mRNA (splicing factor, arginine/serine-rich 1 (SFRS1) and SFRS3) are likely targets of miR-486, it is possible that miR-486 could play a role in the translation of genes essential for normal cellular homeostasis in other tissues, such as the brain.
These studies offer a possible mechanism for the upregulation of PTEN/AKT signaling pathway components, which have previously been observed in dystrophin-deficient skeletal muscle. Recently, it has been shown that mice lacking PTEN had improved skeletal muscle regeneration when fed a high-fat diet . Modulation of the PTEN/AKT signaling pathway through miR-486 expression has the potential to be a novel therapy for treating DMD. Delivery of miR-486 in skeletal muscle might have a beneficial effect in ameliorating the secondary signaling defects that are observed in dystrophin-deficient muscle. Advances in the use of stable in vivo exon-skipping morpholinos for the correction of out-of-frame DMD gene mutations and deletions have led to an increased interest in the use of nonintegrative methods for the treatment of DMD [51, 52]. One can envision that a combination therapy involving the overexpression of intramuscular injection of stabilized miR-486 along with exon-skipping morpholinos might restore muscle function and prevent some of the muscle loss observed in DMD patients.
Materials and methods
Human biopsies and establishment of primary myoblast cell lines
DMD/BMD patient muscle tissue was obtained during either diagnostic skeletal muscle biopsies or other clinical surgeries. Control muscle tissue was obtained from patients undergoing clinical orthopedic surgery. Each biopsy sample was divided into two either to be frozen for sectioning or to be used to establish myoblast cultures. All patients were males between 6 and 65 years of age and were classified as normal, DMD or BMD based on a combination of DNA sequencing, dystrophin protein analysis by Western blotting and physician evaluation. The muscle groups biopsied were from human TA, back (latissimus dorsi) or hip muscles (vastus lateralis), depending on tissue availability. Mutations in the DMD gene locus were validated by the clinical DNA Diagnostic Center at Children's Hospital Boston by both Southern blot analysis and automated sequencing analysis using universal conditions for direct sequencing methods . All BMD samples contained separate deletions residing in exons 45 through 49 of the dystrophin gene (patient 1, exons 45 through 48 deleted; patient 2, exons 48 and 49 deleted; and patient 3, exons 45 through 49 deleted), which resulted in a truncated but partially functional dystrophin protein. All BMD muscle biopsies were analyzed by Western blotting and were determined to have truncated dystrophin protein (Dp427) molecular weights that ranged from 380 and 402 kDa. All patients gave their written and oral consent prior to surgery, and all protocols were approved by the Children's Hospital Boston human subjects internal review board (protocol 03-12-205R).
Myoblasts were isolated from fresh biopsies by first washing the samples in ice-cold 1× PBS and then mincing them with sterile scalpels on a 100-mm plate for 10 minutes until all clumps were removed. The minced muscles were then incubated with a collagenase IV neutral protease (Worthington Biochemical Corp. Lakewood, NJ, USA) at a ratio of 3.5× the gram weight of the minced muscle. The plate was incubated at 37°C for 45 minutes until the cells were in suspension by pipetting the mixture thrice at 15-minute intervals. Ten milliliters of human skeletal muscle growth medium (PromoCell, Heidelberg, Germany) were added to the mixture. The mixture of cells was then passed through a 70-μm filter and centrifuged at 514 × g at 4°C for 10 minutes. The supernatant was aspirated, and then the cells were resuspended in red blood cell lysis buffer (Qiagen, Valencia, CA, USA) and incubated for three minutes at room temperature. Following incubation, 22 mL of growth medium were added to the cells, followed by a second filtration using a 40-μm filter. The filtrate was then centrifuged at 514 × g at 4°C for 10 minutes. The supernatant was aspirated, and then the cells were resuspended in 10 mL of growth medium before being seeded onto uncoated 100-mm plates for one hour in a 37°C incubator. Following the one-hour preplating on uncoated plates, the supernatants containing unattached cells enriched for myoblasts were replated onto 0.1% gelatin-coated 100-mm plates (porcine; Sigma-Aldrich, St Louis, MO, USA). Cells were passaged every two to three days. Additionally, the cells were subjected to at least two additional preplating steps in which the fibroblasts adhered to the uncoated dishes and the slowly adhering myoblast fraction was then plated onto the gelatin-coated dishes, similarly to a protocol described previously . Myoblast enrichment was measured by immunofluorescent labeling of cells using a desmin antibody (1:500 dilution; Epitomics Inc., Burlingame CA, USA). Cellular preparations that contained over 90% desmin-positive myoblasts were considered "myoblast enriched" and thus were appropriate for cell culture experiments.
Lentivirus vectors that stably overexpress a pre-miR-486 precursor, anti-miR-486 (miRZipsSystem Biosciences Inc., Mountain View, CA, USA) or scrambled miRNA (nonsense small hairpin RNA) controls that contained bicistronic IRES-GFP tags were obtained commercially (System Biosciences Inc., Mountain View, CA, USA). Third-generation lentiviral packaging plasmids (MDL/RRE, Rev and VSV-G) were a generous gift from GQ Daley (Children's Hospital Boston) . HEK293T cells were grown in 100-mm plates in 10% fetal bovine serum (FBS)/DMEM (Mediatech Inc., Manassas, VA, USA) supplemented with 1× antibiotics (Invitrogen, Carlsbad, CA, USA). Lentiviral vectors along with packaging plasmids were transfected in ten 10-cm plates (at a ratio of 3 μg lentiviral vector to 1 μg of each packaging plasmid per 10-cm plate) containing 90% confluent HEK293T cells using Lipofectamine 2000 reagent (Invitrogen). Three days after transfection viral supernatants were pooled and filtered through 45-μm filters (VWR International LLC., Radnor, PA, USA), mixed with a 1/3rd volume of Lenti-X lentivirus concentrator (Clontech Laboratories Inc., Mountain View, CA, USA) and incubated overnight at 4°C. The following day the lentiviral concentrate elute was pelleted by centrifugation at 1,500 × g for 45 minutes at 4°C. Viral pellets were resuspended in DMEM at one-twentieth the starting volume. The lentiviruses were titered by calculating the percentage of GFP fluorescence to the total number of plated cells in subsequent tenfold serial dilutions following infection of 500,000 HEK293T cells . Most lentiviral infections were done at a viral titer of approximately 2 × 108 TU/mL.
C57BL6/J wild-type control mice and mdx 5cv mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) . All mouse husbandry and transgenic lines were approved by the Children's Hospital Boston Animal Facilities/Institutional Animal Care and Use Committee and maintained in pathogen-free cages following federal standard of care practices. Individual mice were weighed on a Denver Instrument DLT Series scale (Denver Instrument, Bohemia, NY, USA All mice were fed a 5P00 Prolab RMH 3000 standard chow diet (5% fat; LabDiet, PMI Nutrition International, St. Paul, MN, USA;) and had access to food ad libitum. All mouse lines and subsequent breeding were maintained on a C57BL6/J background under the Children's Hospital Boston ARCH animal protocol (10-12-1852R).
The MCK-miR-486-Tg overexpression mouse lines were generated using the pBS-MCK backbone plasmid obtained from Addgene (plasmid 12528; Addgene, Cambridge, MA, USA). The MCK plasmid contains a 6-kb enhancer region for the mouse muscle creatine kinase Ckm gene and has been previously characterized [29, 58]. A 500-bp fragment containing the stem loop precursor sequence of mouse miR-486 was amplified by PCR from C57BL6/J mouse genomic DNA and subcloned into the Pst I/Spe I restriction sites of the pBS-MCK plasmid. The 6-kb MCK fragment plus the miR-486 sequence and a simian virus 40 (SV40) polyadenylation (poly(A)) signal were excised from the plasmid using Kpn I/Sac I restriction enzymes. Following DNA purification using the GeneClean II kit (MP Biomedicals LLC, Solon, OH, USA), the linearized DNA fragment was then injected into the pronucleus of C57BL6/J fertilized eggs and transplanted into the uterine horn of pseudopregnant ICR foster mice. A total of 55 viable pups from five separate injections were genotyped by PCR using primers specific for the MCK enhancer element and the SV40 poly(A) signal transduction sequence. All F0 founder mice were maintained on a C57BL6/J background. Additional mouse genotyping was performed commercially by Transnetyx, Inc. (Cordova, TN, USA).
Additional cell cultures
HEK293T cells were a generous gift from the laboratory of GQ Daley (Children's Hospital Boston). The cells were maintained using 10% FBS (Atlanta Biologicals Inc., Lawrenceville, GA, USA)/DMEM (Invitrogen) supplemented with antibiotics (Antibiotic-Antimycotic; Invitrogen) as described previously .
miRNA isolation and real-time qPCR
miRNA and mRNA were extracted from either cell lines or animal tissue using the miRVana Isolation kit (Ambion Austin, TX, USA). Fifty nanograms of total enriched miRNA per sample were copied into cDNA (through RT) using the Multiscribe RT System (Applied Biosystems Carlsbad, CA, USA). One microliter of RT reaction was used for each specific miRNA TaqMan assay (Applied Biosystems), which was amplified using No-Amp Erase TaqMan Polymerase (Applied Biosystems) according to the manufacturer's instructions. All samples were analyzed in 96-well optical plates on an ABI 7900HT real-time PCR machine (Applied Biosystems). Cycle times were normalized to U6 small nuclear RNA as a loading control. Relative fold expression and changes were calculated using the 2-ΔΔCt method .
Total RNA from muscle biopsies and cell cultures was extracted using the mirVana miRNA/mRNA Isolation Kit (Ambion). One microgram of total RNA was copied into cDNA using the First-Strand cDNA Synthesis Kit (Invitrogen). Tenfold serial dilutions of cDNA were analyzed on 96-well optical plates using Power SYBR Green Master Mix (Applied Biosystems) and analyzed on the ABI 7900HT real-time PCR machine. Primers specific for each gene were designed using National Center for Biotechnology Information Primer-Blast http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome, and all cycle time values were normalized to an 18 s ribosome loading control. All real-time qPCR primers are listed in Additional file 6, Table S1.
The following primary antibodies were used in this study: anti-GFP (1:1,000 dilution Western blot; MBL International Corp. Woburn, MA, USA; and 1:50 dilution IF; Chemicon International Temecula, CA, USA), anti-β-tubulin horseradish peroxidase (HRP) (1:1,000 dilution Western blot; Cell Signaling Technology Danvers, MA, USA), anti-PTEN (1:1,000 dilution Western blot; Cell Signaling Technology), anti-PDGFRβ (1:1,000 dilution Western blot; Bioworld Technology Inc. St. Louis Park, MN, USA), anti-PIK3R1 (1:1,000 dilution Western blot; Abcam), anti-FOXO1 (C29H4 clone, 1:1,000 dilution Western blot; Cell Signaling Technology), anti-SFRS1 (1:1,000 dilution Western blot; Aviva Systems Biology San Diego, CA, USA), anti-SFRS3 (clone 2D2, 1:1,000 dilution Western blot; Sigma-Aldrich), anti-MF20 (1:25 dilution IF; Developmental Studies Hybridoma Bank, Iowa City, IA, USA); anti-Ki-67 (1:200 dilution IF; BD Pharmingen Inc., San Diego, CA, USA), anti-desmin (1:1,000 dilution IF Epitomics Inc. Burlingame CA, USA;), anti-p21 (1:1,000 dilution Western blot; Abcam) and anti-p27 (1:500 dilution Western blot; Abcam). Secondary antibodies used for Western blot analysis included anti-mouse and anti-rabbit HRP-conjugated antibodies (1:2,000 dilution Western blot; Cell Signaling Technology), along with anti-goat HRP-conjugated antibody (1:1,000 dilution Western blot; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies (Invitrogen) were used for immunofluorescent detection of primary antisera in mouse and rabbit with Alexa Fluor fluorochromes conjugated at 488 nm and 565 nm.
Myoblasts were sorted using FACS analysis for detection of the IRES-GFP reporter indicating a positively infected cell and were stained with propidium iodide (2 μg/mL; BD Biosciences San Jose, CA, USA) to exclude dead cells. The myoblasts were sorted on a FACSVantage flow cytometer (BD Biosciences), and all FACS plots were analyzed using FloJo version 6.4.7 software (Tree Star, Inc., Ashland, OR, USA).
Western blot analysis
All protein preparations were quantified using a bicinchoninic acid protein assay kit (Pierce Biotechnology Rockford, IL, USA). Thirty micrograms of whole cell protein lysate were resolved on 4% to 20% gradient Tris-acetate gels (Invitrogen). Following transfer to polyvinylidene fluoride (PVDF) membranes (Invitrogen), the membranes were blocked with 5% nonfat milk (Cell Signaling Technology)/Tris-buffered saline (TBS)-Tween 20 (Sigma-Aldrich) before being incubated overnight at 4°C with primary antisera diluted in 5% milk/TBS-Tween 20 (Boston BioProducts Inc., Ashland, MA, USA). The membranes were subjected to a series of three TBS-Tween 20 washes. The secondary HRP-conjugated antibodies were diluted in 5% milk/TBS-Tween 20 prior to being incubated with PVDF membranes (Invitrogen) for one hour at room temperature. Following additional washes prior to application of chemiluminescent substrate (Millipore, Billerica, MA, USA), the membranes were exposed on Kodak BioMax film (Sigma-Aldrich) using varying exposure times.
CTX injections and histology of mice
Four-month-old male C57BL6/J and miR-486-Tg littermate mice were subjected to injections into their TA muscles with 10 μM CTX (C9759; Sigma-Aldrich) that was resuspended in 1× PBS. Another cohort received 1× PBS sham (no CTX) control TA injections. All mice were monitored for signs of pain or distress following the injections. The mice were killed by CO2 asphyxiation, and their muscles were harvested at 0 (uninjured), 1, 3, 5, 7 and 14 days post-CTX injury. The harvested muscle biopsies were then embedded in Tissue-Tek O.C.T. compound (Sakura Finetek U.S.A. Inc., Torrance, CA, USA) and snap-frozen in liquid nitrogen chilled in isopentane (Sigma-Aldrich). The frozen muscles were then cryoembedded and sectioned on a cryostat in 7-μm-thick sections. A portion of each muscle was harvested for total RNA extraction at a later time point. The muscle sections were then placed on polylysine slides. Sections were methanol-fixed for 3 minutes at -20°C and then stained with H & E (Sigma-Aldrich) to observe morphology. Sections were imaged on a Nikon Eclipse E1000 microscope (Nikon Instruments, Melville, NY, USA using SPOT imaging software (SPOT Imaging Solutions, Sterling Heights, MI, USA). Some images were later modified using Adobe PhotoShop and Adobe Illustrator CS4 (Adobe Systems Inc., San Jose, CA, USA to enhance image resolution and add scale bars.
Myoblast wound migration assay
Primary human myoblasts were seeded onto 0.1% gelatin-coated 20-mm two-well chamber slides at 25,000 cells/well. Twenty-four hours later the cells were infected with lentivirus or a 1× PBS mock control at 10 multiplicities of infection. Twenty-four hours postinfection the cell wells were scratched vertically with a P100 pipette. The cells were at 85% to 90% confluence at the time of the scratch wound. Cell migration was measured at 0, 12 and 24 hours. By 24 hours, all but the anti-miR-486-infected cells had fully migrated to close the scratch wound (data not shown). All scratch wounds were performed in triplicate.
Each group of infected cells was fixed at each of the time points with 4% paraformaldehyde for 10 minutes at 4°C with gentile agitation. Following fixation the cells were washed twice in 1× PBS and three times in 0.1% Triton X-100/1× PBS, and then they were incubated in blocking buffer (10% horse serum (Gibco, Grand Island, NY, USA) and 0.1% Triton X-100 (Sigma-Aldrich) mixed in 1× PBS, Mediatech, Inc. Manassas, VA, USA) for one hour at room temperature with light shaking. Following blocking the cells were incubated with phalloidin conjugated to Alexa Fluor 546 (1:100 dilution in blocking buffer; Invitrogen) overnight at 4°C with gentle shaking. Cells were then washed three times with 0.1% Triton X-100/1× PBS before being mounted with VECTASHIELD mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Vector Labs, Burlingame, CA, USA). Cells that had been stained were photographed with a Nikon II(Nikon Instruments, Melville, NY, USA;) microscope using SPOT imaging software (SPOT Imaging Solutions, Sterling Heights, MI, USA) at all three time points.
miRNA reporter assays
Following prediction of the transcripts which were thought to be targets of miR-486, the 3'UTR regions of these genes were amplified by PCR from a human total mRNA library (Ambion) or from commercially cloned constructs (GeneCopoeia Inc., Rockville, MD, USA). The 3'UTR amplicons were then cloned into the Spe I and Not I restriction sites of a modified version of the pGL2Basic vector containing a novel multicloning site from the pCR4-TOPO vector (Invitrogen) between the luciferase open-reading frame and the SV40 poly(A) signal (gift from A Dostal, J Ho and JA Kreidberg; Children's Hospital Boston).
The miRNA 3'UTR-luc reporter assay was performed by first plating 20,000 HEK293T cells/well into 48-well plates. The following day the cells were transfected using Lipofectamine 2000 reagent with 30 ng of 3'UTR miRNA-luc reporter constructs and 100 ng of miR-486 overexpression plasmid (Origene, Rockville, MD, USA); pCMVmiR-IRES-GFP vector) or scrambled miRNA controls (Origene). Forty-eight hours after transfection the cells were lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA), and 20 μL of whole cell lysate were assayed with 50 μL of luciferase substrate using the Dual Reporter Assay (Promega, Madison, WI, USA). Luciferase levels were measured on a single-tube luminometer (Berthold Technologies USA, LLC., Oak Ridge, TN, USA). The seed sites in the 3'UTR-luc reporters were mutated from GT AC AG G to Gg Aa AtG using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA).
CASPASE 3/7 assays
Twenty-five microliters of total protein lysates from infected normal and DMD myoblasts were incubated with 100 μL of Caspase-Glo 3/7 reagent (Promega) according to the manufacturer's protocol. Luciferase measurements were taken on a single-tube luminometer (Berthold Technologies).
Unless otherwise stated, all statistical analyses were performed using a Student's t-test (two-tailed).
Becker muscular dystrophy
Duchenne muscular dystrophy
Dulbecco's modified Eagle's medium
fluorescence-activated cell sorting
forward scatter height
green fluorescent protein
- H & E:
hematoxylin and eosin
internal ribosome entry site
muscle creatine kinase
quantitative reverse transcriptase polymerase chain reaction
relative light unit
standard error of the mean
small nuclear RNA
standard unit of enzymatic activity
Funding for this work was generously provided by the Bernard F and Alva B Gimbel Foundation (to LMK) and through National Institutes of Health (NIH) grant P50 NS040828-10. RTG was funded by the Howard Hughes Medical Institute Exceptional Research Opportunities Program (EXROP). MSA was funded by a traineeship awarded by the NIH through the Harvard Stem Cell Institute. PBK is supported by the Muscular Dystrophy Association (MDA 186796 and MDA 114353) and the Genise Goldenson Fund. The authors express their gratitude toward the families and patients who donated their biopsies and time for these experiments.
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