The role of miR-497-5p in myofibroblast differentiation of LR-MSCs and pulmonary fibrogenesis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal fibrotic lung disease characterized by profound changes in stem cell differentiation, epithelial cell phenotypes and fibroblast proliferation. In our study, we found that miR-497-5p was significantly upregulated both during myofibroblast differentiation of lung resident mesenchymal stem cells (LR-MSCs) and in the lung tissues of a pulmonary fibrosis model. In addition, as determined by luciferase assays and Western blot analysis, reversion-inducing cysteine-rich protein with kazal motifs (Reck) was identified to be one of the target genes of miR-497-5p, and Reck could suppress the expression of matrix metalloproteinase-2 (Mmp2) and Mmp9, which could activate latent transforming growth factor-β1 (TGF-β1). To test the potential therapeutic significance of this miRNA, we modulated the expression of miR-497-5p in LR-MSCs and relevant animal models. The results demonstrated that upregulation of miR-497-5p could induce LR-MSCs to differentiate into myofibroblasts and promote pulmonary fibrogenesis, while inhibition of its expression could effectively retard these processes. In conclusion, our work supports that controlling pulmonary fibrogenesis via inhibition of miR-497-5p expression may provide a potential therapeutic strategy for IPF.

Scientific RepoRts | 7:40958 | DOI: 10.1038/srep40958 microRNAs (miRNAs) are small noncoding RNAs that are directly associated with the developmental process of cancer, diabetes, cardiovascular disease, and lung disease through regulating gene expression [15][16][17][18][19] . To date, a large number of miRNAs have been reported to play key roles in the development of IPF. Pandit et al. first reported that let-7d is mainly localized to the alveolar epithelium in normal lungs, but is significantly decreased in IPF lungs 15 . miR-21, which was first identified as an oncogenic miRNA targeting many tumor suppressor genes 20 , has been found to be highly increased in myofibroblasts, epithelial cells, as well as the cells surrounding the fibrotic foci of human IPF lungs 21,22 .
In our study, we observed that miR-497-5p was significantly higher during the myofibroblast differentiation of lung resident mesenchymal stem cells (LR-MSCs). And we also verified that miR-497-5p could target the 3′-UTR of reversion-inducing cysteine-rich protein with kazal motifs (Reck) which could suppress Mmp2 and Mmp9 synthesis and secretion, and impact on ECM integrity 23,24 . We further investigated whether this regulation was conserved in mice. In a bleomycin (BLM)-induced pulmonary fibrosis model, miR-497-5p was upregulated, and suppressing miR-497-5p expression using a lentiviral agent in vivo reduced the expression of fibrotic markers Mmp2, Mmp9 and Tgfb1 via enhancing the expression of Reck, suggesting augmented in vivo lung repair. These data reveal a potential new therapeutic approach for IPF and an intimate interplay among miR-497-5p, Reck, MMPs, TGF-β1 and pulmonary fibrosis.

Reck is suppressed in TGF-β1-treated LR-MSCs and the lung tissues of a pulmonary fibrosis model.
Reck is predicted to have a potential conserved binding site with miR-497-5p. We investigated the expression of Reck in TGF-β1-treated LR-MSCs and lung tissues administered with BLM. The results showed that Reck was dramatically suppressed ( Fig. 2A and B), indicating a potential relationship between Reck and miR-497-5p. miR-497-5p regulates the differentiation of LR-MSCs by targeting Reck. The seed sequence of miR-497-5p within the 3′-UTR sequence of mouse Reck was predicted to be a potential conserved binding site (Fig. 2C). To confirm whether miR-497-5p could regulate Reck expression, Reck 3′-UTR was cloned into a luciferase reporter system (the GV306 vector). A Reck 3′-UTR mutant with mutations in the predicted miR-497-5p site was also cloned into a GV306 vector. The constructs were subsequently transfected into 293 T cells with either LV-miR-497-5p or LV-NC. Co-transfection with LV-miR-497-5p, which contained WT-Reck but not MUT-Reck, evidently diminished the normalized luciferase activity indicating that miR-497-5p could bind with the 3′-UTR of Reck and suppress the transcription of luciferase (Fig. 2D).
To further explore if miR-497-5p upregulation was required for the myofibroblast differentiation of LR-MSCs, these cells were transfected with either LV-miR-497-5p or LV-NC followed by culture for 7 days. As shown in Fig. 3A and B, upregulating miR-497-5p could downregulate the expression of Reck, resulting in increased protein levels of Mmp2, Mmp9 and Tgfb1. In addition, overexpression of miR-497-5p could also induce αsmooth muscle actin (Acta2) and collagen I (Col1a1) expression at both mRNA and protein levels compared with LV-NC, along with increased protein levels of Vim ( Fig. 3A and B). When LR-MSCs were transfected with either LV-miR-497-5p-inhibitor or LV-NC-inhibitor and cultured for 72 h followed by treatment with TGF-β1 for another 7 days, downregulation of miR-497-5p in LR-MSCs could decrease TGF-β1-induced Acta2, Vim and Col1a1 expression by upregulating the expression of Reck, resulting in decreased levels of Mmp2, Mmp9 and Tgfb1 ( Fig. 4A and B). We also confirmed these findings using immunofluorescence staining (Fig. 4C). These data suggested that miR-497-5p was sufficient to modulate the differentiation of LR-MSCs and these cells play an important role in pulmonary fibrogenesis 26 . miR-497-5p induces myofibroblast differentiation of NIH/3T3 cells. As miR-497-5p was also increased in the lung tissues of the pulmonary fibrosis model, we also sought to illustrate whether miR-497-5p could induce myofibroblast differentiation of fibroblasts as this process is believed to play a key role in the development of IPF. In NIH/3T3 cells, miR-497-5p could also impair Reck expression and augment the levels of Acta2 and Vim, suggesting the myofibroblast differentiation of NIH/3T3 cells (Fig. 5A). The migration assay showed that miR-497-5p could promote the migration of NIH/3T3 cells (Fig. 5B). In order to further confirm the regulation of Reck by miR-497-5p, we transfected NIH/3T3 cells with miR-497-5p and a mutated form of Reck that had a disrupted 3′-UTR sequence followed by functional analysis. Our results found that such co-transfection suppressed miR-497-5p-induced downregulation of Reck and upregulation of Mmp2, Mmp9 and Tgfb1, counteracting miR-497-5p-induced myofibroblast differentiation of NIH/3T3 cells evidenced by reduced levels of Acta2 and Col1a1 (Fig. 5C). We also reproduced this finding in LR-MSCs (Fig. 5D). Taken together, these data strongly suggest that the profibrotic effect of miR-497-5p was meditated by binding with its target gene Reck.

Discussion
Crucial new insights into the diagnosis, treatment, prognosis and pathogenesis of various human diseases, including tissue fibrosis, have been provided by genome-wide approaches to miRNA expression profiling 17,27,28 . The application of these approaches in IPF has demonstrated that let-7d, miR-21 and miR-199a-5p make critical contributions to pulmonary fibrosis 15,21,29 . In our study, we explored the expression, regulation, and potential role of miRNAs in the myofibroblast differentiation of LR-MSCs. Among these significantly upregulated miRNAs, we focused on miR-497-5p and observed its high expression in the lung tissues of a pulmonary fibrosis model. miR-497-5p is one of the members of the miR-15/107 group with the seed sequence AGCAGC, which is an important determinant of target recognition 30 . It has previously been reported that miR-497-5p is closely related with several cancers 31,32 . However, the relationship between miR-497-5p and fibrosis remains unknown. In our investigation, we demonstrated that miR-497-5p could most likely bind with the 3′-UTR of Reck by the luciferase assay, strongly suggesting that Reck was a target gene of miR-497-5p. Additionally, we have demonstrated that upregulation of miR-497-5p induced myofibroblast differentiation of LR-MSCs and NIH/3T3 cells in vitro. We also found that the profibrotic function of miR-497-5p was mainly mediated through the activation of latent TGF-β1 anchored in ECM by targeting the 3′-UTR of Reck, which negatively regulates, at least three different Mmps, namely, membrane-type 1 matrix metalloproteinase (MT1-Mmp), Mmp2 and Mmp9 (Fig. 8). Secreted Mmps (e.g. MT1-Mmp, Mmp2 and Mmp9) have been implicated in the release and activation of TGF-β 13,33,34 , and may also be involved in the deregulation of the synthesis and degradation of ECM protein, a process that leads to enlarged ECM deposition in fibrosis 11 .   (Figs 3A and 4A), the expression of Acta2 and Col1a1 was measured by immunofluorescence. Acta2 and Col1a1were revealed with secondary 594-labeled antibodies; nuclei were revealed by DAPI staining. Pulmonary fibrosis is characterized by increased deposition of ECM and aberrant fibroblast proliferation 35 . LR-MSCs undergo injury-induced phenotypic modulation to become α-SMA positive myofibroblasts, which is a crucial step in the repair process that facilitates collagen secretion following lung injury. The collagen deposition and the proliferation of myofibroblasts at the site of injury result in scar formation, which helps to maintain alveolar structural integrity and function 36 . Activation of the TGF-β1 pathway is a significant event in the fibrogenic response, as it contributes to myofibroblast differentiation of LR-MSCs and pulmonary fibroblasts, and triggers the synthesis of ECM proteins 37,38 . In vivo, intratracheal inhalation of a miR-497-5p hairpin inhibitor was sufficient to induce the expression of Reck and decrease the release of activated TGF-β1 by suppressing Mmp2 and Mmp9 expression, which attenuated the fibrotic process. In contrast, upregulating the expression of miR-497-5p could induce the expression of Acta2 and Col1a1 and contribute to the phenotype of pulmonary fibrosis by activating the Mmps/TGF-β pathway.
Moreover, as miRNAs could regulate more than one mRNA, some other miR-497-5p-predicted target genes, such as Smad7, a negative regulator of TGF-β signaling 39 , were also implicated in the phenotype of pulmonary fibrosis 40 . Considerable data have suggested that miRNAs play key roles in the development of pulmonary fibrosis and may be explored as promising therapeutic targets for pulmonary fibrosis. For example, Liu et al. demonstrated that miR-21 is overexpressed in the lungs of both mice with BLM-induced fibrosis and patients with IPF 21 . Pandit et al. found that let-7d is downregulated in the lungs of IPF patients and the number of epithelial cells that express let-7d is correlated with pulmonary function 15 . In addition, miR-29, miR-31 and miR-200 each plays an anti-fibrotic role in the lungs [41][42][43] . However, prior to our study, no experimental evidence had shown that miR-497-5p could contribute to lung fibrosis. To our knowledge, our current work represents the first report arguing that administration of a miR-497-5p inhibitor may attenuate the fibrotic processes both in vitro and in vivo. Our data supported that miR-497-5p modifying drugs may have a therapeutic benefit for IPF.  miRNA microarray. Total RNA was isolated from LR-MSCs incubated with TGF-β1 (PeproTech, Rocky Hill, NJ) for 7 days using Trizol Reagent (Ambion, Foster, CA). miRNA profiling was performed using a low-density miRNA Taqman array service (Invitrogen, Shanghai, China). The miRNAs exhibiting an expression fold change (log 2 ) greater than 1 or less than -1 were deemed to be differentially expressed. We validated a series of abundantly and differentially expressed miRNAs via quantitative reverse transcription polymerase chain reaction (Q-RTPCR).  Antibodies. Mouse monoclonal antibody against mouse β-actin (ab8277), mouse monoclonal antibody against mouse Mmp2 (ab86607), rabbit polyclonal antibody against mouse Mmp9 (ab38898), rabbit polyclonal antibody against mouse Acta2 (ab5694), rabbit monoclonal antibody against mouse Vim (ab92547), rabbit polyclonal antibody against mouse Col1a1 (ab34710), mouse monoclonal antibody against mouse Tgfb1 (ab27969), and rabbit polyclonal antibody against mouse Fn1 (ab2413) were purchased from Abcam (Cambridge, MA). Rabbit monoclonal antibody against mouse Reck (D8C7) was purchase from Cell Signaling Technology (Beverly, MA).

Materials and Methods
Cell culture. Mouse fibroblast cells (NIH/3T3) was obtained from the American Type Culture Collection (Manassas, VA). The cells that were frozen down at an early passage were cultured for a maximum of eight passages. The cells were maintained at 37 °C with 5% v/v CO 2 in Dulbecco's modified Eagle's medium (DMEM, Life Technologies/Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco).
Luciferase assays. For luciferase assays, the sequence of the Reck 3′-UTR and the Reck 3′-UTRs in which the putative binding sites had been mutated were amplified with specific primers, and verified by DNA sequencing. These gene fragments were then subcloned into the GV306 vector (GENECHEM) to generate the wild-type Reck plasmid (WT-Reck) and the mutant Reck plasmid (MUT-Reck). 293 T cells were transiently co-transfected with 125 ng GV306 vector and 5 × 10 7 TU/ml LV-miR-497-5p or LV-NC lentivival vector. Luciferase assays were performed 48 h later using the Dual-Luciferase Reporter System (Promega, Madison, WI). The Renilla and firefly luciferase signals were detected using a GloMax ® -Multi + Detection System (Promega). The activity of an internal firefly luciferase was normalized by the Renilla luciferase activity.
Induction and treatment of pulmonary fibrosis. All animal procedures were conducted in accordance with humane animal care standards approved by the Nanjing University Ethics Committee (Nanjing, China) and maintained under specific pathogen-free conditions. The animals were acclimated to the environment for 1 week prior to treatment.
The mice were administered with BLM (Nippon Kayaku, Tokyo, Japan) intratracheally at a dose of 5 mg/kg dissolved in a total of 50 μl sterile saline. The control group was similarly treated with 50 μl of sterile saline. The lentiviral vector of LV-miR-497-5p and LV-NC (LV-miR-497-5p-inhibitor and LV-NC-inhibior, respectively) were purchased from GENECHEM. Mice were administered with the lentiviral vector intratracheally at a dose targets Reck, which leads to myofibroblast differentiation of lung resident mesenchymal stem cells and pulmonary fibrosis through activating latent TGF-β1. of 2 × 10 8 TU/ml diluted by sterile saline. Seven days later, the LV-miR-497-5p-inhibitor and LV-NC-inhibitor groups were administered with BLM intratracheally at a dose of 5 mg/kg dissolved in a total of 50 μl sterile saline. The LV-miR-497-5p and LV-NC groups were similarly treated with 50 μl sterile saline. The mice were sacrificed for lung collection at day 14 after BLM administration (n = 6 for each time point).

SDS-PAGE and immunoblotting.
Briefly, whole cell or tissue lysates were separated on 12% SDS-polyacrylamide gels and transferred to a polyvinylidene fluoride (PVDF) membrane (Roche, Germany) by standard procedures. Membranes were blocked by incubation for 1 h with 5% non-fat milk in PBS containing 0.5% Tween-20 (PBST) and blotted with specific antibodies at 4 °C for 12 h. After three washes in PBST, the membranes were incubated with the secondary antibody at 37 °C for 1 h. Immunoreactive protein bands were detected using an Odyssey Scanning System (LI-COR, Lincoln, NE).

Q-RTPCR.
For Q-RTPCR analysis of mRNAs and mature miRNAs, total RNA was extracted using Trizol Reagent (Ambion). An amount of 0.05 μg total RNA was reverse-transcribed using the Taqman MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster, CA). Comparative quantitative PCR (Q-PCR) was performed in triplicate using Taqman Universal PCR Master Mix (Applied Biosystems) on the 7300 Real-Time PCR System (Applied Biosystems). Mature miR-497-5p probes were alternatively obtained from Applied Biosystems. Normalization was performed by using RNU6B probes (Applied Biosystems). Relative expression was calculated by using the comparative Ct (ΔΔCt) method.
For analysis of mRNA, HiScript 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) was used for reverse transcription polymerase chain reaction (RT-PCR). Q-PCR was performed using the SYBR Green Q-PCR Kit (Roche, Germany). Specific primers for mRNAs are listed in Table 1. The Ct values were analyzed using the ΔΔCt method and relative changes of mRNA levels were obtained by normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) relative to the control.
Histopathology. The mouse lungs were inflated with a neutral buffered formalin solution overnight and embedded in paraffin before sectioning into 5 μm-thick slices. The sections were stained with hematoxylin-eosin (H&E) and Masson's trichrome to assess the degree of fibrosis.
Immunohistochemistry. Five μm-thick paraffin-embedded sections were deparaffinized with xylene (twice for 5 minutes each) before being rehydrated in water using an ethanol gradient. After washing with water, antigen retrieval was performed in a steamer using citrate buffer (pH 6.0, DAKO) for 20 minutes, and the samples were then cooled to room temperature. The sections were then washed with PBST, incubated with 3% H 2 O 2 for 10 minutes and blocked with the avidin/biotin blocker and the serum-free blocking reagent. The sections were subsequently incubated with mouse anti-Mmp2, rabbit anti-Mmp9, rabbit anti-Abcg2 or rabbit anti-Acta2 antibodies overnight at 4 °C. The DAB Substrate System (DAKO) was used to reveal the immunohistochemical staining. Immunofluorescent staining. The immunofluorescence analysis was performed as described 44 . Rabbit anti-Col1a1 and rabbit anti-Acta2 were employed as the primary antibody. Alexa Fluor 594-conjugated goat anti-rabbit IgG (Invitrogen) was used as the secondary antibody. Nuclei were stained with 1 μg/ml DAPI (Sigma). The images were captured using a confocal fluorescence microscope (Olympus, Tokyo, Japan). Statistical Analysis. The data are presented as mean values ± SD. Differences were analyzed for significance (P < 0.05) by one-way ANOVA using SPASS for windows version 11.0 (SPASS, Chicago, IL). Supplement files for low-density miRNA Taqman array. The raw data of profiled miRNA in lung resident mesenchymal stem cells following TGF-β1-induced myofibroblast differentiation was uploaded in the supplement file.