Studies performed previously have indicated that long non-coding RNAs (lncRNAs) may be involved in skeletal muscle regeneration; however, the roles of lncRNAs during the repair of skeletal muscle contusion remain unclear. The present study established a mouse skeletal muscle contusion injury model to identify the roles of lncRNAs that are specifically enriched in the skeletal muscle, namely metastasis-associated lung adenocarcinoma transcript 1 (Malat1), H19, myogenesis-associated lnc (lnc-mg), long intergenic non-protein coding RNAs (linc)-muscle differentiation 1 (linc-MD1), linc-yin yang 1 (linc-YY1) and sirtuin 1-antisense (Sirt1-AS). Morphological analyses revealed that fibrotic scars and regenerating myofibers were formed in the muscle following contusion injury. Gene expression was analyzed by reverse transcription-quantitative polymerase chain reaction. The data revealed that the expression of inflammatory cytokines, myogenic regulatory factors and angiogenic factors increased significantly following skeletal muscle contusion. Additionally, various lncRNAs, including Malat1, H19, lnc-mg, linc-MD1, linc-YY1 and Sirt1-AS were also upregulated. Correlation was also observed between lncRNAs and regulatory factors for skeletal muscle regeneration including transforming growth factor-β1, myogenic differentiation, myogenin, myogenic factor 5 (myf5), myf6, hypoxia-inducible factor-1α and angiopoietin 1. In conclusion, lncRNAs may serve important roles in the regeneration of skeletal muscle following contusion injury, which provides a promising therapy avenue for muscle injury.
Skeletal muscle injury is a common injury in daily life and/or during physical exercise. Skeletal muscle has the remarkable ability to self-regenerate following injury. The mechanism of skeletal muscle repair is one of the major issues surrounding the field of sports medicine. In particular, skeletal muscle contusion is a common form of injury. It is a contact injury caused mainly by an acute, relatively large blunt trauma that is characterized by intact skin and no external damage. The repair of damaged skeletal muscle is a complex process which mainly consists of the inflammatory response, myofiber regeneration, angiogenesis and fibrosis (
Effective repair of damaged skeletal muscle requires the coordinated action of several cell types and a variety of factors. For example, macrophages serve complex roles in damaged skeletal muscle, and may be involved in all phases of skeletal muscle regeneration mentioned above (
In previous years, the roles of long non-coding RNAs (lncRNAs) have become the focus of research. lncRNAs, which can, are non-coding RNAs with a transcript length of >200 nucleotides, which have emerged as an important class of regulators of gene expression, and localize to the nucleus and the cytosol (
A total of 40, 8 week old C57BL/6 male mice weighing 18.2–22.9 g, purchased from JiesiJie-Lab Animal Research Center (Shanghai JiesiJie Experimental Animal Co., Ltd.), were housed at 21±2°C and 50±5% humidity on a 12 h light/dark cycle, and received water and food ad libitum. Following acclimatization to the local environment for 7 days, the mice were randomly divided into two groups: The uninjured control group (group C) and the muscle contusion group (group M). Mice from group M were used for the induction of contusion injury. All experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of Shanghai University of Sports (approval no. 2016006).
A simple and reproducible muscle contusion model in mice was applied as previously described with little modification (
At days 3, 6, 12 and 24 following muscle contusion, the right GM was harvested, fixed in 4% paraformaldehyde at 4°C for 24 h and then embedded in paraffin (n=6 mice/group). Cross sections cut at 4 µm were produced from the GM, which were subsequently stained with H&E to evaluate the general morphology using a method described previously (
To visualize fibrosis in the muscle injury sites, Total collagen staining was performed to detect fibrosis in injured muscle via Masson's trichrome staining (total collagen staining; Servicebio, Inc.). The procedure was as follows: GM tissue samples were cut into 4-µm-thick sections and stained with hematoxylin for 5 min, 1% hydrochloric acid alcohol for 5 sec, Biebrich scarlet-acid fuchsin for 8 min, Phosphomolybdic acid aqueous solution for 4 min, Aniline blue solution for 5 min, and 1% glacial acetic acid for 1 min. All staining was performed at room temperature. Following Masson's trichrome staining, images were captured for each muscle section viewed under a bright-field microscope (magnification, ×400; Labophot-2; Nikon Corporation). The ratio of the fibrotic area to the total cross-sectional area of the muscle was calculated to estimate the extent of fibrosis formation using Image Pro 6.0 (Media Cybernetics, Inc.). A total of six different fields of view (magnification, ×400) were randomly selected from each section.
Total RNA from the GM was extracted using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.), and the concentration and purity were determined by measuring the absorbance at 260 and 280 nm with a microplate reader (Model 550 Microplate Reader; Bio-Rad Laboratories, Inc.). Total RNA (2 µg) was subsequently reverse transcribed into complementary cDNA (cDNA) using the Revertaid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). The temperature protocol for RT was as follows: 25°C for 5 min followed by 42°C for 60 min, termination at 70°C for 5 min and cooling at 4°C. The qPCR reaction system included SYBR Green (Fermentas; Thermo Fisher Scientific, Inc.), nuclease-free water, upstream and downstream primers (designed and synthesized by Shanghai Shenggong Biology Engineering Technology Service, Ltd.; primer sequences presented in
All data were analyzed using the SPSS 22.0 software (IBM Corp.) and are presented as the mean ± standard deviation of at least three experiments. Statistical analysis was carried out using one-way analysis of variance, and post-hoc multiple comparisons were performed using the Bonferroni test. Image Pro 6.0 software was used to assess fibrosis, which was compared using an independent samples t-test. Correlations were calculated according to Pearson's correlation coefficient. P<0.05 was considered to indicate a statistically significant difference.
Following H&E staining, the histological appearance of the skeletal muscle was compared between the uninjured control group and the muscle contusion group. Skeletal muscles that were not injured exhibited cells that were arranged regularly with the nuclei, stained blue-black, located primarily in the cell periphery (
Following Masson's trichrome staining, the tissue in the injured area of the GM was assessed. Fibrotic scar tissues, in the form of collagen, were stained in blue, whereas skeletal muscle cells were stained in red (
The mRNA levels of specific markers of macrophages in muscle were evaluated. Compared with the uninjured control group, the mRNA levels of CD68, which is a specific marker of M1 macrophages (
The present study evaluated the expression of inflammatory cytokines (IL-1β, IL-6, TNF-α, INF-γ, IL-10 and TGF-β1) in isolated GM samples. The mRNA levels of proinflammatory cytokines IL-1β, TNF-α, and IFN-γ increased significantly in skeletal muscle samples on days 3 and 6 following contusion compared with control (all P<0.01;
The expression of myogenic regulatory factors including MyoD, myogenin, myf5 and myf6 was investigated in GM samples following contusion injury. The data revealed that MyoD, myogenin, myf5 and myf6 displayed similar gene expression patterns. Their mRNA levels were elevated significantly at 3 and 6 days after injury compared with uninjured control, which returned to normal 24 days after injury (
Next, the expression of angiogenic factors was evaluated in the skeletal muscle tissues isolated following muscle contusion injury. Vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1α (HIF-1α) and angiopoietin-1 (Angpt-1) exhibited differential expression patterns. The mRNA levels of VEGF did not appear to be significantly altered during the healing process following muscle injury (
The expression levels of lncRNAs (Malat1, H19, lnc-mg, Sirt1 AS, linc-MD1 and linc-YY1) during GM regeneration were subsequently determined using RT-qPCR. The expression levels of linc-MD1 and Sirt1 AS were significantly increased compared with the uninjured control group at 3, 6 and 12 days following injury (all P<0.01), and returned to normal levels 24 days after injury (
To assess the association between lncRNAs and specific markers of macrophages, inflammatory cytokines, myogenic regulatory factors and angiogenic factors, Pearson's correlations analysis was performed. The results of this analysis are summarized in
Skeletal muscle retains the ability to regenerate following damage. The present study employed a mouse skeletal muscle contusion injury model which can induce inflammatory responses with macrophage infiltration as one of the signatures, followed by regeneration. Histologically, a large number of inflammatory cells and factors infiltrated the injured area in the early stages of skeletal muscle injury. The infiltration patterns of inflammatory cells were consistent with a previous study (
Fibrosis may occur when skeletal muscles experience severe injury, which is characterized by the accumulation of fibroblasts and myofibroblasts, and high levels of extracellular matrix deposition (
Despite the rapidly increasing number of studies investigating the functions of lncRNAs, their specific roles in myogenesis remain poorly defined. The findings presented in this study provided a comprehensive analysis of lncRNA (Malat1, H19, lnc-mg, Sirt1 AS, linc-MD1 and linc-YY1) expression during skeletal muscle regeneration following contusion injury. In addition, their association with the expression levels of lincRNAs and macrophage markers, inflammatory cytokines, myogenic factors and angiogenic factors was elucidated. Malat1 and Sirt1 AS are lincRNAs that are expressed in high abundance in proliferating and differentiating myoblasts (
Additionally, lnc-mg has also been suggested to be a skeletal muscle-enriched lncRNA (
Although several lncRNAs have been demonstrated to serve a number of roles in skeletal muscle cell differentiation and myogenesis
Myogenic factors Myf5 and Myf6 are essential for muscle regeneration and can promote myoblast differentiation (
During the skeletal muscle repair process, lncRNAs have also been reported to be involved in the regulation of the skeletal muscle inflammatory response, angiogenesis and fibrosis (
Vascular regeneration is part of the complete regeneration of damaged skeletal muscle. In the present study, the expression of HIF-1α and Angpt1 was markedly increased following muscle contusion, which correlated positively with Malat1. Michalik
Recently, a growing body of evidence suggested that lncRNAs are also involved in tissue fibrosis in several organs, including the lungs, liver and heart (
lncRNAs such as Malat1 serve important roles in the inflammatory response and angiogenesis of injured skeletal muscle. To the best of our knowledge, only a small number of studies have evaluated the role of lncRNAs in the inflammatory response and angiogenesis following skeletal muscle injury (
This present investigation was the first to demonstrate that lncRNAs are associated with the regeneration of contused skeletal muscle. The changes in the expression of a number of candidate lncRNAs at multiple timepoints following skeletal muscle contusion, as well as their association with other physiological factors, were assessed. Results illustrated in the present study support the hypothesis that lncRNAs may play important roles in the regeneration of contused skeletal muscle, but further research is needed to elucidate the underlying mechanism. However, there are several limitations to the study; for example, knockdown or overexpression experiments on the lncRNAs were not performed. Although Pearson's correlation analysis indicated correlations between lncRNAs and macrophage infiltration, inflammation and angiogenesis, this did not reveal the mechanism underlying the role of lncRNAs in contused muscle regeneration. Nevertheless, this present investigation do lay the foundation for further research into the functional role of lncRNAs in skeletal muscle regeneration.
In conclusion, the expression of inflammatory cytokines, myogenic regulatory factors and angiogenic factors were demonstrated to be significantly increased following the induction of skeletal muscle contusion, along with lncRNAs including Malat1, H19, lnc-mg, linc-MD1, linc-YY1 and Sirt1 AS. There was a correlation between lncRNAs and a variety of established regulatory factors (TGF-β1, MyoD, myogenin, myf5, myf6, HIF-1α and Angpt1) during the skeletal muscle regeneration process. These results suggest that lncRNAs may serve important roles in the regeneration of damaged skeletal muscle. Effective muscle regeneration is essential for the treatment of muscle diseases including muscle atrophy, muscular dystrophy and sporting injuries. Therefore, these findings serve as a basis for the effective treatment of muscle atrophy and muscular dystrophy.
The present study was previously presented at a conference (
The present study was supported by the National Natural Science Foundation of China (grant nos. 31271273 and 31300975), Shanghai Natural Science Fund Project (grant no. 18ZR1437100) and Shanghai Key Laboratory of Human Movement Development and Protection (Shanghai University of Sport; grant no. 11DZ2261100).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
LZ analyzed the results and drafted the manuscript. LZ performed histological staining and PCR. XL assisted with PCR. PC and WX designed the current study and provided funds. All authors reviewed and critiqued the manuscript and agreed to the final submission of the manuscript. All authors read and approved the final manuscript.
The present study was approved by the Ethics Review Committee for Animal Experimentation of Shanghai University of Sports (approval no. 2016006).
Not applicable.
The authors declare that they have no competing interests.
Representative images from the hematoxylin and eosin staining of gastrocnemius muscle tissues from the (A) uninjured control group, and (B) 3, (C) 6, (D) 12 and (E) 24 days after injury induction. Thin arrows indicate inflammatory cells, bold arrows indicate central nucleation. Scale bars, 50 µm.
Histological evaluation of scar tissue formation in the injured and uninjured GM by Masson's trichrome staining. Scar tissues are indicated in blue and muscle tissue in red. Representative images of the (A) uninjured control group, (B) muscle contusion group (12 days post-injury) and (C) muscle contusion group (24 days post-injury). (D) Quantification of the scar tissue area following GM injury. Data are presented as the mean ± standard deviation. Scale bars, 100 µm. d, days; GM, gastrocnemius muscle.
Expression of macrophage-specific markers in gastrocnemius muscle samples after muscle contusion. (A) mRNA expression of CD68, (B) CD163 (marker of M2 macrophages) and (C) CD206. Data are presented as the mean ± standard deviation (n=8). *P<0.05, **P<0.01 vs. Con. Con, control; d, days; CD, cluster of differentiation.
Expression of inflammatory factors in gastrocnemius muscle samples following muscle contusion. mRNA expression levels of (A) IL-1β, (B) IL-6, (C) TNF-α, (D) IFN-γ, (E) IL-10 and (F) TGF-β1. Data are presented as the mean ± standard deviation (n=8). *P<0.05, **P<0.01 vs. con. Con, control; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; IL-10, interleukin-10; TGF-β1, transforming growth factor-β1.
Expression of myogenic regulatory factors in gastrocnemius muscle samples following muscle contusion. mRNA expression levels of (A) MyoD, (B) myogenin, (C) myf5 and (D) myf6. Data are presented as the mean ± standard deviation (n=8). *P<0.05, **P<0.01 vs. Con. Con, control; MyoD, myogenic differentiation 1; myf5, myogenic factor 5; myf6, myogenic factor 6.
Expression of angiogenic factors in gastrocnemius muscle samples following muscle contusion. mRNA expression levels of (A) VEGF, (B) HIF-1α and (C) Angpt1. Data are presented as the mean ± standard deviation (n=8). **P<0.01 vs. Con. Con, control; HIF-1α, hypoxia-inducible factor-1α; VEGF, vascular endothelial growth factor; Angpt1, angiopoietin 1.
Expression of long non-coding RNAs in gastrocnemius muscle samples following muscle contusion. mRNA expression levels of (A) Malat1, (B) H19, (C) lnc-mg, (D) Sirt1 AS, (E) linc-MD1 and (F) linc-YY1. Data are presented as the mean ± standard deviation (n=8). **P<0.01 vs. Con. Con, control; Malat1, metastasis associated lung adenocarcinoma transcript 1; lncRNA, long non-coding RNA; lnc-mg, myogenesis-associated long non-coding RNA; Sirt1 AS, sirtuin 1-antisense; linc-MD1, long intergenic non-protein coding RNAs-muscle differentiation 1; linc-YY1, long intergenic non-protein coding RNA-yin yang 1.
Primers used for reverse transcription-quantitative PCR.
Target gene | Forward primer sequence | Reverse primer sequence |
---|---|---|
CD68 | 5′-CAAAGCTTCTGCTGTGGAAAT-3′ | 5′-GACTGGTCACGGTTGCAAG-3′ |
CD163 | 5′-GCAAAAACTGGCAGTGGG-3′ | 5′-GTCAAAATCACAGACGGAGC-3′ |
CD206 | 5′-GGATTGTGGAGCAGATGGAAG-3′ | 5′-CTTGAATGGAAATGCACAGAC-3′ |
IFN-γ | 5′-GCTTTGCAGCTCTTCCTCAT-3′ | 5′-GTCACCATCCTTTTGCCAGT-3′ |
TNF-α | 5′-CTTCTGTCTACTGAACTTCGGG-3′ | 5′-CACTTGGTGGTTTGCTACGAC-3′ |
IL-1β | 5′-TGACGTTCCCATTAGACAACTG-3′ | 5′-CCGTCTTTCATTACACAGGACA-3′ |
IL-6 | 5′-GAACAACGATGATGCACTTGC-3′ | 5′CTTCATGTACTCCAGGTAGCTATGGT-3′ |
TGF-β1 | 5′-TGCGCTTGCAGAGATTAAAA-3′ | 5′-CGTCAAAAGACAGCCACTCA-3′ |
IL-10 | 5′-CAAGGAGCATTTGAATTCCC-3′ | 5′-GGCCTTGTAGACACCTTGGTC-3′ |
Myo D | 5′-GAGCGCATCTCCACAGACAG-3′ | 5′-AAATCGCATTGGGGTTTGAG-3′ |
Myogenin | 5′-CCAGTACATTGAGCGCCTAC-3′ | 5′-ACCGAACTCCAGTGCATTGC-3′ |
Myf5 | 5′-GGAATGCCATCCGCTACATT-3′ | 5′-CGTCAGAGCAGTTGGAGGTG-3′ |
Myf6 | 5′-CCTCAGCCTCCAGCAGTCTT-3′ | 5′-TTCTCCACCACCTCCTCCAC-3′ |
VEGF | 5′-TAACAGTGAAGCGGAGTG-3′ | 5′-TTTGACCCTTTCCCTTTCCTCG-3′ |
HIF-1α | 5′-GGCGAGAACGAGAAGAAAAAGATGA-3′ | 5′-GCTCACATTGTGGGGAAGTGG-3′ |
Angpt1 | 5′-AACCGGATTCAACATGGGCA-3′ | 5′-GAGCGTTGGTGTTGTACTGC-3′ |
Malat1 | 5′-CACTTGTGGGGAGACCTTGT-3′ | 5′-TGTGGCAAGAATCAAGCAAG-3′ |
H19 | 5′-TGACTTCATCATCTCCCTCCTGTC-3′ | 5′-GGGTAAATGGGGAAACAGAGTCAC-3′ |
lnc-mg | 5′-CTGCATCACGGAAGGAGATA-3′ | 5′-AACAATCCATCCTCATTGGC-3′ |
Sirt1 AS | 5′-AATCCAGTCATTAAACGGTCTACAA-3′ | 5′-TAGGACCATTACTGCCAGAGG-3′ |
linc-MD1 | 5′-GCAAGAAAACCACAGAGGAGG-3′ | 5′-GTGAAGTCCTTGGAGTTTGAGCA-3′ |
Linc-YY1 | 5′-AGTTACAGGGAAGTTTGGGCTAC-3′ | 5′-AGGCAAAGGACGGCTGTGAG-3′ |
GAPDH | 5′-ACTCCACTCACGGCAAATTC-3′ | 5′-TCTCCATGGTGGTGAAGACA-3′ |
IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; IL-10, interleukin-10; TGF-β1, transforming growth factor-β1; MyoD, myogenic differentiation 1; myf5, myogenic factor 5; myf6, myogenic factor 6; HIF-1α, hypoxia-inducible factor-1α; VEGF, vascular endothelial growth factor; Angpt1, angiopoietin 1; Malat1, metastasis associated lung adenocarcinoma transcript 1; lnc-mg, myogenesis-associated long non-coding RNA; Sirt1 AS, sirtuin 1-antisense; linc-MD1, long intergenic non-protein coding RNAs-muscle differentiation 1; linc-YY1, long intergenic non-protein coding RNA-yin yang 1.
Correlation between the lncRNAs and the specific markers of macrophages, inflammatory cytokines, myogenic regulatory factors and angiogenic factors.
lncRNA | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Malat1 | H19 | lnc-mg | Sirt1 AS | linc-MD1 | linc-YY1 | |||||||
Gene | r | P-value | r | P-value | r | P-value | r | P-value | r | P-value | r | P-value |
CD68 | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. |
CD163 | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | 0.793 | 0.015 | N.S. | N.S. |
CD206 | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | 0.862 | 0.002 | N.S. | N.S. |
TGF-β1 | 0.916 | 0.029 | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | 0.912 | <0.001 | N.S. | N.S. |
IL-10 | 0.986 | <0.001 | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | 0.896 | <0.001 | N.S. | N.S. |
IL-6 | 0.598 | 0.005 | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | 0.850 | 0.024 | N.S. | N.S. |
IL-1β | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. |
TNF-α | 0.886 | 0.046 | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | 0.906 | 0.034 | N.S. | N.S. |
IFN-γ | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | 0.884 | 0.037 | N.S. | N.S. |
MyoD | 0.558 | 0.003 | N.S. | N.S. | N.S. | N.S. | 0.563 | <0.001 | 0.825 | <0.001 | 0.474 | 0.003 |
myogenin | 0.600 | 0.012 | 0.470 | 0.003 | N.S. | N.S. | 0.535 | <0.001 | 0.773 | <0.001 | 0.423 | 0.007 |
Myf5 | N.S. | N.S. | 0.797 | 0.001 | N.S. | N.S. | 0.703 | <0.001 | 0.782 | <0.001 | N.S. | N.S. |
Myf6 | N.S. | N.S. | 0.674 | 0.007 | N.S. | N.S. | 0.620 | 0.001 | 0.897 | 0.039 | N.S. | N.S. |
VEGF | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. |
HIF-1α | 0.785 | 0.016 | 0.504 | 0.001 | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. |
Angpt1 | 0.653 | 0.040 | 0.593 | <0.001 | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. | N.S. |
N.S., not significant; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; IL-10, interleukin-10; TGF-β1, transforming growth factor-β1; MyoD, myogenic differentiation 1; myf5, myogenic factor 5; myf6, myogenic factor 6; HIF-1α, hypoxia-inducible factor-1α; VEGF, vascular endothelial growth factor; Angpt1, angiopoietin 1; Malat1, metastasis associated lung adenocarcinoma transcript 1; lnc-mg, myogenesis-associated long non-coding RNA; Sirt1 AS, sirtuin 1-antisense; linc-MD1, long intergenic non-protein coding RNAs-muscle differentiation 1; linc-YY1, long intergenic non-protein coding RNA-yin yang 1.