1. Volume 20, Issue 6, Pages 1222-1233 (June 2012)
Loss of miR-29 in Myoblasts Contributes to Dystrophic Muscle Pathogenesis 
Lijun Wang, Liang Zhou, Peiyong Jiang, Leina Lu, Xiaona Chen, Huiyao Lan, Denis C Guttridge, Hao Sun, Huating Wang 
Molecular Therapy 
Volume 20, Issue 6, Pages (June 2012)
DOI: /mt
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
miR-29 is downregulated in mdx muscles. (a,b) The expressions of miR-29a, -29b, -29c in RNAs isolated from tibialis anterior (TA), gastrocnemius (Gas), quadriceps (Quad), diaphragm or heart muscles of wild-type (WT) or mdx mice. (c) The expressions of the above miRs in TA of WT, mdx, double knock-out (DKO) or heterozygous (Het) mice. Data are plotted as mean ± SD. *P < 0.05, **P < 0.01.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Loss of miR-29 in mdx myoblasts caused a defect on myogenic differentiation. (a) H&E and IHC staining for E-MyHC were performed on cryosections of mdx muscles injected with negative control (NC) or miR-29 mimics. n = 5 mice for each group. (b,c) Quantification of E-MyHC–positive fibers and fibers with centrally localized nuclei (CLN). (d) Left: immunoblotting for Pax7, MyoD, myogenin, and YY1 in the above muscles with GAPDH as a loading control. Results from three representative mice are shown. Right: quantification of western blots was done by densitometry. (e) IF staining for Pax7 was performed on the above NC or miR-29 injected muscles. (f) Positively stained cells were counted from at least 15 sections. E-MyHC, embryonic-MyHC; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H&E, hematoxylin and eosin; IF, immunofluorescence; IHC, immunohistochemistry; YY1, Yin Yang 1. **P < 0.01, ***P <
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
miR-29 is downregulated in mdx myoblasts. (a) Expression of miR-29 in primary myoblasts from WT or mdx muscles. (b) Left: primary myoblasts from mdx muscles were kept growing (DM 0 hour) or differentiated (DM) for 7, 24, or 48 hours at which times cells were immunostained for MyHC. Right: positively stained cells were quantified. Numbers indicate the average number of MyHC positive cells counted from a minimum of 10 randomly chosen fields. Graphs are plotted as mean ± SD. Images were taken at 24 hours. (c) Expressions of MyHC or troponin RNAs in WT or mdx myoblasts differentiated for the indicated times. (d) Expression of miR-29 in mdx primary myoblasts transfected with NC or miR-29 oligos. (e) Left: the above transfected cells were differentiated for 48 hours at which time the cells were photographed under phase contrasts or immunostained for MyHC. Right: positively stained cells were quantified as in b. (f) Expressions of MyHC and troponin RNAs in NC or miR-29 transfected cells at different time points of differentiation. (g) mdx myoblasts were transfected with MyHC-Luc or TnI-Luc reporter plasmids and NC or miR-29 oligos. Cells were then differentiated for 48 hours at which time luciferase activities were determined. The data represent the average of three independent experiments ± SD. *P < 0.05, **P < DM, differentiation medium; NC, negative control; WT, wild-type.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Overexpression of miR-29 in mdx muscles downregulates fibrotic genes. (a) Differentially expressed genes in mdx muscles injected with NC and miR-29 oligos as determined by mRNA-seq. X- and Y-axis represent the log2 based FPKM values for expressed genes in NC and miR-29 samples, respectively. (b) Over-represented GO terms by GO analysis of downregulated list of genes. BP, biological process; CC, cellular component; KEGG, Kyoto Encyclopedia of Genes and Genomes; SP_PIR, a database of protein super-family names. (c) Coverage plot showing a 56-kb region encompassing the ACTA2 (α-SMA) gene on chromosome (Chr) 19; the gene structure is shown in blue below the graph. (d) Expressions of collagen 1A1 (Col 1A1), collagen 1A2 (Col 1A2), collagen 3A1 (Col 3A1), α-smooth muscle actin (α-SMA) or vimentin (VIM) in NC or miR-29 injected muscles. (e) Expressions of the above genes in TA muscles injected with negative control (Anti-NC) or miR-29 inhibitor oligos (Anti-miR-29). FPKM, fragments per kilobase of transcript per million fragments mapped; GO, gene ontology; mRNA, messenger RNA; NC, negative control; TA, tibialis anterior.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Loss of miR-29 promotes myoblasts transdifferentiation into myofibroblasts. (a) Expressions of Col 1A1, Col 1A2, and Col 3A1 in primary myoblasts freshly isolated from WT or mdx muscles. (b) Primary myoblasts from mdx muscles were transfected with NC or miR-29 oligos and examined for Col 1A1, Col 1A2, and Col 3A1 expressions. (c) WT or mutant Col 1A1, Col 1A2, or Col 3A1-3′UTR luciferase reporter constructs were transfected into mdx primary myoblasts with NC or miR-29 oligos. Luciferase activities were determined at 48 hours post-transfection. Relative luciferase unit (RLU) is shown with respect to NC cells where normalized luciferase values were set to 1. The data represents the average of three independent experiments ± SD. (d) Predicted binding between mmu-miR-29c and mouse 3′UTR of Mfap5. (e) WT or mutant Mfap5-3′UTR luciferase reporter constructs were transfected into C2C12 cells with NC or miR-29 oligos. Luciferase activities were determined as above. (f) Expressions of Mfap5 in primary myoblasts from WT or mdx muscles. (g) Primary myoblasts from mdx muscles were transfected with NC or miR-29 oligos and examined for Mfap5 expression. (h) Expressions of the Mfap5 mRNAs in mdx TA muscles injected with negative control (Anti-NC) or miR-29 inhibitor oligos (Anti-miR-29). (i) IF staining for MyoD (green) and α-SMA (red) were performed on cryosections of mdx TA muscles. DAPI (blue) staining was also performed to visualize the nuclei. A field with three types of cells is shown on the left: arrows, MyoD‐/α-SMA+ cells; arrowhead, MyoD+/α-SMA‐ cells; asterisk, MyoD+/α-SMA+ cells. Higher magnification of one of the MyoD+/α-SMA+ cells is presented on the right. (j) TA muscles from mdx mice were injected with NC or miR-29 mimics oligos. Quantification of the MyoD+/SMA+ cells on the above muscles were performed on a minimal of 15 sections for each group. N = 5 mice per group. Data are plotted as mean ± SD.*P < α-SMA, α-smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole; mRNA, messenger RNA; NC, negative control; TA, tibialis anterior; UTR, untranslated region; WT, wild-type.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Elevated TGF-β signaling downregulates miR-29 in dystrophic muscles and mdx myoblasts. (a) Expressions of TGF-β mRNAs in TA muscles of WT or mdx mice at indicated ages. (b) Expressions of miR-29 in TA muscles of mdx mice injected with decorin (+) or PBS control (‐). (c) Expressions of collagens and Mfap5 in TA muscles of mdx mice injected with decorin (+) or PBS control (‐). (d,e) Primary myoblasts from mdx muscles were treated with or without TGF-β and examined for expressions of miR-29 or collagens and Mfap5. mRNA, messenger RNA; PBS, phosphate-buffered saline; TA, tibialis anterior; TGF-β, transforming growth factor-β; WT, wild-type.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Systemic delivery of miR-29 oligos reduces fibrosis in mdx diaphragm. (a) Administration scheme for miR-29 injection. NC or miR-29 oligos formulated with liposome were injected into mdx mice through tail vein at day (d) 0, 4, and 7. Mice were killed and diaphragm muscles were harvested at day 21 for histology and immunostaining. n = 5 mice for each treatment group. (b) Expressions of miR-29 in various tissues collected at day 3 after the injection. (c) Trichrome staining of the fibrotic areas in NC and miR-29 injected diaphragm muscles. The positively stained areas were quantified with Image-Pro Plus from a minimum of 15 sections. (d) IHC staining of the above muscles with collagen 1. The positively stained areas were quantified as the above. (e) IF staining of the above muscle with collagen 1. (f) Expressions of Pax7, MyoD, myogenin, and YY1 mRNAs in the above treated diaphragm muscles. (g) H&E staining of the above NC or miR-29 injected mdx muscles. (h) Damaged areas were identified as fibrotic and fatty areas and quantified as the above. **P < 0.01, ***P < H&E, hematoxylin and eosin; IF, immunofluorescence; IHC, immunohistochemistry; mRNA, messenger RNA; NC, negative control; SI, small intestine; TA, tibialis anterior; YY1, Yin Yang 1.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

9. Figure 8
A model of the role of miR-29 in dystrophic muscles. The model depicts the critical roles of miR-29 in muscle regeneration and muscle fibrogenesis. The model depicts the roles of the TGF-β–miR-29 regulatory axis in transdifferentiation of myoblasts into myofibroblasts. In the normal myogenesis, NF-κB-YY1 regulated miR-29 promotes the differentiation through downregulating YY1, which leads to successful myogenic differentiation and muscle regeneration. In dystrophic muscles, miR-29 is downregulated by increased TGF-β signaling, leading to overexpression of ECM genes including collagens, which drives the conversion of myoblasts into myofibroblasts thus contributing to muscle fibrogenesis and defective regeneration. Arrows: activation; blunted arrows: repression. ECM, extracellular matrix; MFAP, microfibrillar-associated protein; NF-κB, nuclear factor-κB; TGF-β, transforming growth factor-β; YY1, Yin Yang 1.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions