Long non‐coding RNA MFAT1 promotes skeletal muscle fibrosis by modulating the miR‐135a‐5p‐Tgfbr2/Smad4 axis as a ceRNA

Abstract Fibrosis after skeletal muscle injury is common in sports and can cause irreversible damage to the biomechanical properties of skeletal muscle. Long non‐coding RNAs (lncRNAs) have been validated to act as important modulators in the fibrosis of various organs. Here, we reported a novel lncRNA (the skeletal muscle fibrosis‐associated transcript 1, lnc‐MFAT1), which was highly expressed in skeletal muscle fibrosis. We demonstrate that lnc‐MFAT1 knockdown can reduce TGFβ‐induced fibrosis in vitro and attenuate skeletal muscle fibrosis after acute contusion in mice. Further study showed that lnc‐MFAT1 acted as a competitive endogenous RNA of miR‐135a‐5p. Besides, the miR‐135a‐5p inhibition obviously promoted TGFβ‐induced fibrosis in vitro via enhancing its target genes Tgfbr2/Smad4. Moreover, we discovered that lnc‐MFAT1 regulates Tgfbr2/Smad4 expression by sponging miR‐135a‐5p to exert competing endogenous RNA function, resulting in TGFβ pathway activation. In conclusion, our study identified a crucial role of lnc‐MFAT1‐miR‐135a‐Tgfbr2/Smad4 axis in skeletal muscle fibrosis, providing a promising treatment option against skeletal muscle fibrosis.

of fibrosis in various organs, including liver, lung, heart, kidney and skeletal muscle. 10,11 As a competing endogenous RNA (ceRNA), lncRNAs typically participated in post-transcriptional regulation by interacting with miRNAs or mRNAs in the cytoplasm. 12,13 However, the function and involvement of lncRNA-miRNA-TGFβ/Smad pathway crosstalk remains unclear and might be a key point in finding the potential therapeutic targets against skeletal muscle fibrosis.
In current study, we first reported a novel lncRNA (the skeletal muscle fibrosis-associated transcript 1, lnc-MFAT1), which was markedly up-regulated in a skeletal muscle fibrosis mouse model and correlated with poor recovery. Through a series of in vitro and in vivo experiments, we demonstrated that silencing lnc-MFAT1 can alleviated skeletal muscle fibrosis after acute contusion. lnc-MFAT1 functioned as a ceRNA of miR-135a-5p (miR-135a) and subsequently regulated Tgfbr2/Smad4 expression. The present study identified a crucial role of lnc-MFAT1-miR-135a-Tgfbr2/Smad4 axis in skeletal muscle fibrosis, providing a promising treatment option against skeletal muscle fibrosis.

| Animal experiments
C57BL/6J mice aged 10 weeks were purchased from Vital Co. and were maintained in a 12-h light/dark cycle with free access to food and water. All animal experiments procedures were carried out following the technical guidelines and approved by the Animal Care and Use Committee of Fudan University. The acute contusion-induced mouse skeletal muscle fibrosis model of the right tibialis anterior (TA) was established following the previous method. 7 Mice were anaesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.5 mL/100 g) and immobilized with its right hindlimb taped to a plate. After that the TA was exposed and hit by a stainless-steel ball (2 cm in diameter, 15 g in weigh) dropping from a height of 1 m. The left TA served as control.

| In vivo adeno-associated virus (AAV) particle administration
Thirty-six skeletal muscle fibrosis mice models were randomly assigned into three groups: acute contusion without injection
Donkey anti-rabbit IgG and donkey anti-mouse IgG served as secondary antibodies for immunohistochemical analysis, whereas goat anti-rabbit IgG for Western blot (Table S2).

| Cell transfection
The transfection was carried out using the Lipofectamine 3000 Reagent (Invitrogen) following the manufacturer's protocol. The small interfering RNAs against lnc-MFAT1 (si-MFAT1), negative control (si-NC) and the pcDNA3.1 vector targeting lnc-MFAT1 were all acquired from Sangon Biotech. To knockdown or overexpress miR-135a, miR-135a mimics, miR-135a inhibitors and its negative controls were purchased from Sangon Biotech and were transfected into C2C12 cells. The sequences are listed in Table S2.

| Western blot
Total proteins were prepared in RIPA buffer (Beyotime) supplemented with protease and phosphatase inhibitor cocktail (Pierce, Thermo Fisher Scientific). Total protein quantity was determined using BCA protein assay (BCA Protein Assay Kit; Pierce Thermo Fisher Scientific). Then, samples were normalized and separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore).
After that, the membrane was blocked with 5% skim milk in 1×TBST for 1 hour at room temperature and then immunoblotted in primary antibodies solution at 4°C overnight. After that, the membranes were washed in 1×TBST and incubated with goat anti-rabbit IgG for 1 hour at room temperature. The proteins were visualized using ECL reagent (Beyotime) and imaged using Tanon

| Quantitative real-time PCR (qRT-PCR) analysis
Total RNA from cells was isolated using TRIzol reagent (Invitrogen). Afterwards, cDNA was synthesized using Advantage RT-for-PCR Kit (TaKaRa). qRT-PCR was implemented with SYBR Green I Master (Roche) on a 7500 Real-Time PCR System (Applied Biosystems).
GAPDH and U6 were used as endogenous control for lncRNA/ mRNA and miRNA, respectively.
Expression fold change was calculated using the 2−ΔΔCt method.
All primer sequences are listed in supplemental Table S1.

| Cytoplasmic and nuclear protein fractionation
Cytoplasmic and nuclear RNA isolation were carried out with PARIS™ Kit (Invitrogen) according to the manufacturer's instruction.
Then, qRT-PCR was used for measurement of nuclear and cytoplasmic RNA. β-Actin and U6 were used as cytoplasmic and nuclear controls, respectively.

| Dual-Luciferase reporter assay
The putative miR-135a binding sites in lnc-MFAT1, Tgfbr2-3ʹ-UTR or Smad4-3ʹ-UTR and the mutant binding sites were constructed and cloned to the pSI-Check2 luciferase vector (Hanbio), immediately downstream of the luciferase gene. HEK293T cells were cotransfected with the pSI-Check2 reporter plasmids and the miR-135a-5p mimics or miR-135a-5p inhibitor, respectively. Twenty-four hours after transfection, the fluorescence intensity was detected with the Dual-Luciferase Reporter Assay System (Promega). And the Renilla luciferase activity normalized to firefly luciferase activity. The experiments repeated independently three times, and the data are represented as mean ± SD.

| Immunohistochemical staining
After fixation for 24 hours in 4% paraformaldehyde, the tissues were dehydrated, embedded in paraffin and sectioned perpendicularly to the direction of the muscle fibre into 5μm-thick slices. Then, slices were incubated for 12 hours in a-SMA antibody followed by secondary antibody conjugated with HRP. Subsequently, immunohistochemical staining was conducted by 3,3ʹ-diaminobenzidine and haematoxylin detection. All histological slices were visualized with inverted light microscopy (Olympus) and digitalized with DP72 Manager (Olympus). Digital quantification of α-SMA positive areas was conducted with software Image-Pro (Meida Cybernetics).

| Immunofluorescence (IF)
C2C12 cells were seeded on glass slides in six-well plates and incubated in DMEM until 80%-90% confluent. After washed with PBS, cells were fixed with 4% paraformaldehyde for 10 minutes followed by permeabilization in PBS with 0.5% Trion-X-100 for 15 minutes and 3% BSA blockage for 30 minutes at room temperature.
Immunofluorescence was then carried out with overnight Col 1 and α-SMA antibodies incubation at 4°C and then with the corresponding secondary antibodies labelled with FITC for 45 minutes at 37°C.
DAPI was added to stain the nuclei. Finally, images were taken with immunofluorescence microscope (Olympus).
The subcellular localization of lnc-MFAT1 was evaluated using a FISH kit (RiboBio). Confocal section images were captured using a confocal laser scanning microscope (FV1000, Olympus). The specific target probe is listed in Table S1.

| Masson staining
After 24 hours fixation in 4% paraformaldehyde, the tissues were routinely dehydrated, embedded in paraffin and sectioned at 5μm thickness. Sections were incubated at 37°C overnight, dewaxed and stained with Masson trichrome according to standard procedures. 14 Under Masson trichrome staining, the collagen fibres were stained to blue and skeletal muscle fibres to red. Digital pictures were taken using identical exposure settings for all sections. The blue area was measured as fibrotic areas with Image-Pro (Meida Cybernetics).

| Microarray and data analysis
In brief, 4 paired skeletal muscle fibrosis samples and normal control samples were collected. lncRNAs were chosen for further data analysis after quantile normalization of the raw data. Differentially expressed lncRNAs between the two groups (fold change ≥ 2, P < .05) were identified from microarray data. Finally, Hierarchical Clustering and combined analysis were performed to show the differentially expressed lncRNAs between the two groups using in-house scripts.

| Functional group analysis of mRNAs
The differentially expressed mRNAs were mapped with the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to investigate the biological functions using online bioinformatics tool DAVID6.8 (https://david.ncifc rf.gov/). A significant P value (hypergeometric P value) indicates a correlation between a pathway and the conditions.
The recommend P value cut-off is .05.

| Statistical analysis
Data are presented as the mean ± SD. For the relative lncRNA, miRNA, mRNA, protein and luciferase activity quantification, the means of the control groups are taken as 1. Statistical analysis was performed by GraphPad Prism software (v6). To determine statistical significance of differences between groups, the Student t test was performed. A two-side P values < .05 were considered statistically significant.

| lnc-MFAT1 was up-regulated in the expression profile of lncRNAs induced by skeletal muscle fibrosis
The acute contusion-induced mice skeletal muscle fibrosis model was established according to previous study. 7 As shown by Masson staining, acute contusion led to a significantly larger collagen deposition area ( Figure 1A). In line with it, immunohistochemical staining revealed significantly increased α-SMA positive areas in the acute contusion group ( Figure 1A). Col 1, VIM, and α-SMA, served as fibrosis indicators, were all remarkably elevated at both mRNA and protein levels in acute contusion group ( Figure 1B,C).
The lncRNA microarray was acquired from four pairs of samples between the acute contusion group and normal control group. After screening (fold change ≥ 2, P < .05), the levels of differentially expressed lncRNAs in the samples were displayed using volcano plot ( Figure 1D) and scatter plot ( Figure 1E). The expression profiling data suggested 513 differentially expressed lncRNAs in total, with 455 up-regulated and 58 down-regulated ( Figure 1F). The top 20 mostly changed lncRNAs (fold change ≥ 2, P < .05) were shown in Figure 1G and Table 1 Figure 1G, Table 1) and qRT-PCR results ( Figure 1H). We next examined the expression profile of lnc-MFAT1 and found that lnc-MFAT1 expression was detected in skeletal muscle and heart, but not in any other tissues ( Figure S1). In addition, the coding ability of lnc-MFAT1 was predicted using Ensembl Genome Browser (http://asia.ensem bl.org/), prediction software and the NCBI ORF finder (https://www.ncbi.nlm.nih.gov/orffi nder/); the results showed no protein-coding capability for lnc-MFAT1, indicating it a non-coding RNA ( Figure S2). Furthermore, KEGG pathway enrichment analysis was applied to identify the differentially expressed pathways after skeletal muscle fibrosis ( Figure 1I). Among the upregulated signalling pathways, the TGFβ signalling pathway (Rich factor = 0.074, P = .0069) and the ECM receptor interaction (Rich factor = 0.115, P = .0004), both of which play critical roles in the fibrosis process ( Figure 1I), appeared to be most enriched pathways.

| lnc-MFAT1 was up-regulated in C2C12 cells stimulated with TGF-β1
TGF-β1 stimulation induces the activation of C2C12 myoblasts into fibroblasts in vitro, which has been well-established and widely validated in previous studies. 7,15,16 Consistent with previous studies, TGF-β1 stimulation remarkably up-regulated lnc-MFAT1 expression level (Figure 2A,B), correlating with an increase of Col 1, VIM and α-SMA at both mRNA and protein levels (TGF-β1, 10 ng/mL, 12 hours) ( Figure 2C,D). Additionally, an increased expression of α-SMA (4.3fold) was detected after incubated with TGFβ, suggesting that fibroblasts differentiated into myofibroblasts ( Figure 2E). Consistent with this, an increased expression of Col 1 (6.0-fold) was confirmed in the TGF-β1 stimulation group, representing more extracellular matrix formation ( Figure 2E).

| Knockdown of lnc-MFAT1 attenuated the fibrosis of C2C12 cells
To
The binding sites between lnc-MFAT1 3ʹ-UTR and miR-135a were predicted using bioinformatics analysis ( Figure 4F). After that, lnc-MFAT1 3ʹ-UTR wild-type or mutated binding sites were cloned into pSI-Check2 vector and luciferase activity was detected after cotransfection of miR-135a and luciferase plasmids. As predicted, cotransfection of lnc-MFAT1 3ʹ-UTR wild-type luciferase plasmids and miR-135a mimics significantly attenuated the luciferase activity, whereas miR-135a inhibitor increased the luciferase activity. Yet, cotransfection of the lnc-MFAT1 3ʹ-UTR mutant luciferase plasmids and miR-135a mimics or inhibitor had no effect on the luciferase activity ( Figure 4G).

| miR-135a inhibition increased the expression of fibrosis-related proteins through Tgfbr2/Smad4 signalling
Combined with previous studies 6,7 and bioinformatics analysis by TargetScan (http://www.targe tscan.org/vert_72/), Tgfbr2 and Smad4 were selected for miR-135a candidate prediction. The predicted binding sites between Tgfbr2 3ʹ-UTR, Smad4 3ʹ-UTR and miR-135a were shown in Figure 5A. The results showed that the luciferase activities remarkably attenuated by cotransfection of Tgfbr2 3ʹ-UTR, Smad4 3ʹ-UTR wild-type luciferase plasmids and miR-135a mimics and increased by miR-135a inhibitor. By contrast, cotransfection of the mutant luciferase plasmids and miR-135a mimics or inhibitor had no effect on the luciferase activity ( Figure 5B).
A decrease in Col1, VIM and α-SMA protein expression was noticeable after transfecting of with miR-135a mimics ( Figure S3).
Meanwhile, the opposite effects of miR-135a inhibitor were also displayed ( Figure S3). qRT-PCR and Western blot analysis demonstrated that miR-135a mimics transfection attenuated Tgfbr2 and Smad4 expression ( Figure 5C,D), whereas miR-135a inhibitor  F I G U R E 5 miR-135a inhibition increased the expression of fibrosis-related proteins through Tgfbr2/Smad4 signalling. A, Schematic diagram of the predicted miR-135a binding sites in Tgfbr2 3ʹ-UTR or Smad4 3ʹ-UTR, respectively. B, Dual-luciferase reported assay was conducted by cotransfection with miR-135a mimics/inhibitors and luciferase plasmids containing WT/MUT Tgfbr2 3ʹ-UTR or WT/MUT Smad4 3ʹ-UTR in HEK293T cells. C,D, qRT-PCR and Western blot analysis showing the mRNA and protein levels of Tgfbr2 and Smad4 in C2C12 cells transfected with miR-135a mimics/inhibitors/negative control, respectively. E-H, qRT-PCR and Western blot analysis showing the mRNA and protein levels of Col 1, VIM and α-SMA. C2C12 cells transfected with miR-135a inhibitor, si-Tgfbr2, si-Smad4 or their negative control. All experiments were repeated three times independently, and data are expressed as the mean ± SD. *P < .05, **P < .01, ***P < .001

| D ISCUSS I ON
In this study, lnc-MFAT1 was confirmed to be substantially upregulated in skeletal muscle fibrosis in vivo and in vitro, and its si-  20 Moreover, lncRNA ITPF was up-regulated in idiopathic pulmonary fibrosis, which could promote pulmonary fibrosis by targeting hnRNP-L depending on its host gene ITGBL1. 19 Consistent with previously reported lncRNAs, lnc-MFAT1 was shown to be up-regulated in skeletal muscle fibrosis in our study (Figures 1 and 2). Additionally, lnc-MFAT1 silencing attenuated the expression of the fibrotic indicators and immunohistochemically positive area both in vitro and in vivo (Figures 3 and 7).
In addition, the current study showed that lnc-MFAT1 could regulate skeletal muscle fibrosis by acting as a miR-135a ceRNA.
It have been proposed that the biological function of lncRNA was closely related to its subcellular location. 21 Typically, lncRNAs localized in the cytoplasm regulate signalling pathways and mRNA stability or translation, whereas lncRNAs localized in the nucleus participate in RNA processing, transcriptional regulation and chromatin interactions. The lncRNAs localized in the cytoplasm were suggested to regulate the target mRNA by sponging to miRNAs.
For example, lnc Kcnq1ot1 alleviates fibrosis through sponging miR-214-3p to regulate TGF-β1/Smads pathway in high glucosetreated cardiac fibroblasts. 8 Additionally, lncRNA PFAR promotes lung fibroblast activation and fibrosis by targeting miR-138 to regulate the YAP1-Twist axis. 22 In the current study, we demonstrated that lnc-MFAT1 predominately localized in the cytoplasm ( Figure 4). Subsequent bioinformatics analysis and dual-luciferase reporter assays showed that both lnc-MFAT1 and Tgfbr2/Smad4 contain binding sites of miR-135a (Figure 4), whose anti-fibrotic effects were reported in pulmonary 23 and cardiac 24 fibrosis. In current study, miR-135a up-regulation and down-regulation could, respectively, attenuate and promote the level of fibrotic indicators, indicating the inhibitory effect of miR-135a on skeletal muscle fibrosis ( Figure 6).
Further, Tgfbr2/Smad4 was proved to be target genes of miR-135a and played an important role in mediating the fibrosis function of lnc-MFAT1/miR-135a. TGFβ exerts its effects by binding to Tgfbr1/2, leading to phosphorylation of Smad2/3. Subsequently, activated Smad2/3 binds to Smad4 and translocates into the nucleus, which initiates TGFβ signalling pathway. 25 Previous studies discovered that several lncRNAs interacted with TGFβ/Smads in the promotion of liver 9,26 and atrial fibrosis. 27 In this study, the profibrotic effect of miR-135a inhibitor transfection was attenuated by Tgfbr2 or Smad4 silencing, suggesting that the anti-fibrotic role of miR-135a was mediated by Tgfbr2/Smad4 ( Figure 5). Additionally, lnc-MFAT1 silencing suppressed the expression of Tgfbr2/Smad4, and miR-135a inhibition could restore the expression ( Figure 6).
These results suggest that Tgfbr2/Smad4 is crucial in mediating the fibrosis function of lnc-MFAT1/miR-135a.
In summary, the newly identified lnc-MFAT1 was found to be upregulated in skeletal muscle fibrosis tissues. Through integrating in vivo and in vitro experiments, we illustrated that lnc-MFAT1 acts as an miR-135a sponge to regulate Tgfbr2/Smad4, resulting in skeletal muscle fibrosis (Figure 8). This study provides novel insights into the regulation of skeletal muscle fibrosis by lncRNAs and suggests lnc-MFAT1 as a promising therapeutic target.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation of China (No. 81772419 to JC).

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests.