MicroRNA-21 Promotes Allergic Airway Inammation and AHR and Inhibits Mesenchymal Stem Cell Migration in Cockroach Allergen Induced Asthma Model

Background Mesenchymal stem cells (MSCs) have been used to treat asthma in a mouse model. However, the ecacy and mechanism of MSCs are not elucidated. MicroRNAs (miRNAs) play a key rolein asthma and related to the aim of this study was to illustrate the role of miR21 and its inuence on MSC migration in asthma model. Methods A mouse model of asthma was established using cockroach extract (CRE), and miR-21 expression was examined. A miR-21 lentivirus construct was used to investigate the role of miR-21 in vivo and in vitro in mouse bone marrow-derived (BM-) MSCs. A TOPFlash reporter gene assay was used to study the signaling downstream of miR-21. IL-4, IL-5, IL-13, IgE, and IgG1 levels in bronchoalveolar lavage uids were determined by enzyme-linked immunosorbent assays. Results MiR-21 was upregulated in CRE-induced asthmatic mice. MiR-21 promoted allergic airway inammation and airway hyperreactivity by inhibiting BM-MSC migration. β-Catenin was found to act downstream of miR-21 as a negative regulator of miR-21. Rescue experiments veried that miR-21 inhibited BM-MSC migration by suppressing Wnt/β-catenin signaling. MiR-21 promotes allergic airway inammation and AHR and inhibits BM-MSC migration through Wnt/β-catenin signaling, which may serve as an effective therapeutic target for asthma. mean ± three independent experiments. compared using a two-tailed paired Student’s t-test using the Chi-square test for categorical Taken together, our data indicate that miR-21 inhibits MSC migration by suppressing Wnt/β-catenin signaling. Our study demonstrated that miR-21 signicantly stimulates CRE-induced asthma by inhibiting MSC chemotactic migration both in vivo and in vitro. More importantly, we provided evidence that Wnt/β-catenin signaling is involved in the inhibitory effect of miR-21 on the migration of BM-MSCs. MiR-21 facilitates the migration of BM-MSCs, which is a potential target for MSC-based treatment of asthma. Other possible mechanisms involved in MSC migration with regard to the treatment of asthma remain to be explored.

Due to diverse pathogens and evolutionary antigens, the body produces stronger resistance, and some steroids are being developed for the treatment of asthma [11]. Although current drugs and allergen immunotherapies are effective to a certain extent, they have numerous side effects, including decreased bone mineral density, skin thinning and bruising, immunologic tolerance, rejection, and anaphylaxis [12,13]. And the morbidity of asthma is still increasing [14,15]. Therefore, novel therapeutic strategies targeting pathophysiological events need to be further explored.
Mesenchymal stem cells (MSCs) have attracted considerable attention for the treatment of various diseases, both in basic medicine and pre-clinical research, owing to their self-renewing and differentiating properties [16][17][18][19][20][21][22][23]. Murine bone marrow-derived (BM-) MSCs inhibited allergic responses in a mouse model of asthma [24], and human BM-MSCs suppressed chronic airway in ammation in a murine asthma model [25]. Increasing studies have shown that MSCs ameliorate asthma in both mouse models, and the mechanisms underlying the therapeutic effects of MSCs have been explored [26][27][28][29][30]. However, little is known about the mechanism underlying MSC migration to target sites to exert curative effects. Migration is one of the vital characteristics of MSCs and is crucial for MSC therapy targeting in ammatory sites. It is fundamental to the application of MSC-based asthma treatments in future clinical research.
MicroRNAs (miRs) are small, non-coding RNAs that play roles in transcriptional and post-transcriptional gene expression regulation by targeting the 3′untranslated regions of speci c mRNAs [31][32][33]. Recent evidence suggests that miRs play a critical role in the diagnosis and treatment of asthma [34][35][36].
Accumulating evidence indicates that MSCs ameliorate asthma via miRs that mediate relevant signaling pathways [37][38][39][40]. Additionally, some studies have shown interactions between MSCs and miRs in asthma [37,41,42]. MiR-21, which is related to cell apoptosis, invasion, and migration, is one of the most widely researched miRs [43,44]. It has been reported to suppress tumor cell migration into injured areas in several cancers [44][45][46]. Based on a literature review, we found that miR-21 is mostly overexpressed in asthma [1][2][3], which we validated in preliminary experiments. Therefore, we aimed to investigate the role and mechanism of miR-21 in asthma treatment with MSCs.
In this study, the miR-21 expression and its in uence on allergic airway in ammation and AHR and MSC migration were assessed in cockroach extract (CRE)induced mouse model of asthma. MiR-21 overexpression and silencing were used to evaluate the signaling pathway mediating MSC migration in vivo and in vitro.

Asthma mouse model establishment
Six-to eight-week-old C57BL/6 mice were purchased from SLAC Laboratories (Shanghai, China). The mice were housed under standard conditions with a 12h/12-h light/dark cycle at the Animal Center of Hunan Cancer Hospital (Changsha, China) and had free access to food and water. All animal experiments and procedures were performed according to the guidelines of the Center for Medical Ethics, Central South University. amount of CRE on D10-D13. Control mice received the same volume of PBS during the sensitization and challenge phases. Mice were randomly assigned to the two treatment groups (n = 6 each) and were analyzed in a double-blind manner. On D14, the mice were sacri ced. The lung tissues were dissected for histological analyses, and bronchoalveolar lavage uids (BALFs) were harvested to count total cells and in ammatory cells. A timeline of the mouse experiment is shown in Figure 1A.

Analysis of lung in ammation
Lung in ammation was assessed. Brie y, BALFs were centrifuged at 300 . g at 4°C for 10 min and washed with ice-cold PBS. Total cells and in ammatory cells including eosinophils (Eos), lymphocytes (Lym), macrophages (Mac), and neutrophils (Neu), were counted to evaluate the percentages of in ammatory cells in BALF by ow cytometry using a FACS Calibur cytometer (BD Biosystems).
IL-4, IL-5, and IL-13 in mouse BALFs were examined by ELISAs, using commercial kits (eBioscience, USA) according to the manufacturer's recommendations.
Serum CRE-speci c IgE and IgG1 were examined by ELISAs.
Small interfering (si) RNA targeting the β-catenin gene (Ctnnb1) was synthesized at GenePharma (Shanghai, China). An unrelated sequence was used as a negative control, according to our previous report [48]. Brie y, siRNAs were transfected into BM-MSCs during the logarithmic growth phase using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. During the challenge period (D10-13), mice were intranasally administrated 2 × 10 6 infectious units (IFUs) of miR-21 lentivirus, and an equal amount of empty lentiviral vector was used as a control.

BM-MSC migration assays in vivo and in vitro
To verify whether exogenous BM-MSCs can migrate to the lungs in asthmatic mice, 5 × 10 6 GFP + BM-MSCs in 0.2 mL of PBS were injected via the tail vein, and the same amount of PBS was injected as a control. To assay MSC migration in vitro, BM-MSCs (1 × 10 5 ) were transfected with a plasmid expressing miR-21, anti-miR-21, and β-catenin using Lipofectamine 2000 (Invitrogen) in serum-free minimum Eagle's medium at 37°C in a 5% CO 2 atmosphere for 48 h. Then, the cells were added to the upper chambers of Transwell inserts with 8.0-µm pores in serum-free medium, while normal growth medium was placed in the lower chambers. After incubation for 24 h, the MSCs were xed and stained with 20% methanol violet (Beyotime, China) and 0.1% crystal violet (Beyotime, China), and counted under a microscope.

Quantitative reverse-transcription (q-RT) PCR
To evaluate the expression of miR-21 and Ctnnb1 in BM-MSCs, total RNA was isolated from BM-MSCs using TRIzol reagent (Invitrogen, USA). The RNA was treated with RNase-free DNase I (Roche, Basel, Switzerland). cDNA was synthesized using the All-in-One TM First Strand cDNA Synthesis Kit (AORT-0050, Genecopoeia, USA) and miRNA First Strand cDNA Synthesis Kit (AMRT-0050, Genecopoeia). RT-qPCRs were run using the All-in-One TM qPCR mix (Genecopoeia) and All-in-One TM miRNA qRT-PCR Detection Kit (Genecopoeia) on an ABI 7300HT real-time PCR system (Applied Biosystems, USA). Primers for miR-21, Ctnnb1, Gapdh, and U6 were synthesized at Genecopoeia. Relative expression levels of miR-21 and Ctnnb1 were evaluated using the 2 -∆∆CT method [49,50]. U6 was used as an endogenous control for miR-21, and Gapdh was used as an internal control for Ctnnb1.

Western blotting
Lung tissues and BM-MSCs from the different treatment groups were collected and homogenized in RIPA lysis buffer (Beyotime, China) supplemented with 1 mM phenylmethylsulfonyl uoride (Beyotime). Protein concentrations were determined using a BCA Protein Assay Kit (Beyotime), and 25 μg protein was loaded per lane. Western blotting was conducted as described previously [48]. The primary antibodies used were anti-β-catenin (D10A8, CST, 1: 1000) and anti-GAPDH (14C10, CST, 1: 1000), and goat anti-rabbit IgG (Invitrogen) was used as the secondary antibody. Immunoreactive bands were visualized with enhanced chemiluminescence reagent (Bio-Rad, USA), using a Tanon-4500 digital image system (Tanon Science & Technology, China).

Immunostaining and immuno uorescence
For the histological assessment of lung in ammation, mouse lungs were collected and soaked in 10 mL of ice-cold PBS. The samples were xed in 4% formaldehyde (Sangon Biotech, Shanghai, China), and 5-μm sections were cut and stained with hematoxylin and eosin (HE) or with periodic acid Schiff reagent (Sigma). The sections were examined and photographed under a microscope (Nikon, Japan). Detailed immunostaining procedures for lung histology were described in our previous report [48].
For immuno uorescence, lung sections were blocked with protein-blocking serum-free solution (Dako, Denmark), permeabilized with 0.1% Triton X-100 for 10 min, and blocked in 1% BSA at room temperature for 30 min. The sections were incubated with primary antibody against β-catenin (1:100 dilution) at 4 °C overnight. The sections were washed and incubated with speci c secondary antibody in PBS (1:100 dilution) for 60 min. Nuclei were stained with DAPI (Beyotime) at room temperature for 10 min. Immuno uorescence was evaluated under a confocal microscope (Leica Microsystems, Germany). The intensity of co-staining was determined using image acquisition and analysis software (Image J), and the values are presented as mean uorescence intensity per square micro.

TOPFlash reporter gene assay
To evaluate the signaling downstream of miR-21, we used a pair of TOPFlash/FOPFlash luciferase reporter constructs (Upstate Biotechnology, USA). BM-MSCs were seeded in a 24-well plate (Corning) at 1 × 10 5 cells/well and allowed to settle for 24 h. TOPFlash or FOPFlash plasmid (500 ng), pRL-TK Renilla luciferase plasmid (150 ng; Promega, USA), and miR-21 mimic (50 nM) or miR-NC (50 nM) were cotransfected into the BM-MSCs. After 24 h of incubation, the cells were stimulated with 50 ng/mL recombinant murine Wnt3a (PeproTech, USA) for another 24 h. Then, the cells were harvested and luciferase reporter activity was measured in the reporter lysis buffer of the Luciferase Assay System (Promega). TOPFlash and FOPFlash signals were normalized to Renilla luciferase activity, and data are represented as normalized TOPFlash/FOPFlash activity values.

Statistical analysis
Data are reported as the mean ± SD derived from at least three independent experiments. Means were compared using a two-tailed paired Student's t-test or using the Chi-square test for categorical variables. P < 0.05 was considered statistically signi cant.

MiR-21 expression is increased in CRE-induced asthma model mice
MiR-21 expression was signi cantly higher in CRE-challenged mice than in PBS-treated mice ( Figure 1B). Further, miR-21 was signi cantly upregulated in miR-21-transfected CRE model mice as compared to miR-NC-transfected mice ( Figure 1C).

MiR-21 promotes lung in ammation in asthma
To understand the role of miR-21 in asthma, we conducted histological analysis. HE staining demonstrated that miR-21 accelerated in ammatory cell in ltration, goblet cell hyperplasia, and mucus overproduction (Figure 2A). Although the numbers of Lym, Mac, and Neu were not signi cantly different, there were signi cant increases in total cells and Eos in the miR-21+CRE group when compared with the miR-NC+CRE group ( Figure 2B and 2C). The ability of AHR to resist methacholine at 10 mg/mL and 30 mg/mL was increased in CRE-challenged miR-21 mice when compared to CRE-challenged miR-NC mice ( Figure  2D). Additionally, allergen-speci c IgE ( Figure 2E) and IgG1 ( Figure 2F) levels were signi cantly increased in CRE-challenged miR-21 mice. Th2-secreted cytokines, including IL-4 ( Figure 2G), IL-5 ( Figure 2H), and IL-13 ( Figure 2I), in BALFs were increased in CRE-challenged miR-21 mice. These results indicated that the aggravated Th2-dependent in ammatory reaction in asthma is related to miR-21 expression.
3.3 BM-MSCs improve allergic airway in ammation and AHR, whereas miR-21 aggravates in ammatory reactions by suppressing BM-MSC migration A schematic diagram of MSC administration in the CRE-induced mouse model was shown in Figure 3A. In miR-21-transfected asthmatic mice, GFP + BM-MSCs migrated into the lungs, but the number of BM-MSCs in the lungs was signi cantly lower than that in miR-NC-transfected asthmatic mice, indicating that miR-21 inhibits BM-MSC migration into the lungs ( Figure 3B). Mean uorescence intensities are shown in Figure S1. Next, we aimed to con rm that miR-21 promotes allergic airway in ammation and AHR by inhibiting BM-MSC migration. MiR-21-transfected asthmatic mice engrafted with GFP + MSCs exhibited decreased airway in ammation cell in ltration, and goblet cell secretion when compared with miR-21-transfected asthmatic mice administered PBS, suggesting that BM-MSCs inhibit airway in ammation and in ltration ( Figure 3C). These results were consistent with those of previous studies showing that MSCs suppress chronic/allergic airway in ammation in a murine asthma model [25,27]. Interestingly, in ammatory cell in ltration in the lungs was signi cantly decreased in the miR-21 + CRE + BM-MSCs group when compared with the miR-21 + CRE + PBS group ( Figure 3C). Although the numbers of Lym, Mac, and Neu showed no obvious differences, there were signi cant decreases in total cells and Eos cells after BM-MSC transplantation in asthmatic mice, whereas the numbers of total cells and Eos in the BALF were signi cantly increased in the miR-21-transfected group post BM-MSC injection ( Figure 3D and 3E). Furthermore, IgE ( Figure 3F) and IgG1 ( Figure 3G) levels were signi cantly decreased after BM-MSC transplantation in asthmatic mice, whereas these levels were enhanced in miR-21-transfected mice. IL-4 ( Figure 3H), IL-5 ( Figure 3I), and IL-13 ( Figure 3J) levels in the BALF were signi cantly decreased after BM-MSC transplantation in asthmatic mice, but were signi cantly increased in mice transfected with miR-21. These results were consistent with the histological examination ndings. Thus, these results corroborated that BM-MSC transplantation signi cantly improved the in ammatory reaction, whereas miR-21 aggravated the in ammatory reaction by inhibiting BM-MSC migration in asthma.

MiR-21 suppresses BM-MSC migration in vitro
MiR-21 expression in the different treatment groups was con rmed by RT-qPCR ( Figure 4A). First, we conducted wound-healing assays to evaluate the effect of miR-21 on the migration ability of BM-MSCs in vitro. Compared with the miR-NC group, BM-MSC migration was suppressed in the miR-21 group at 24 h and 48 h (Figure 4B), and when anti-miR-21 was added, BM-MSC migration was obviously enhanced. These results indicated that miR-21 inhibits BM-MSC migration in vitro. To assess the role of miR-21 in directed migration of BM-MSCs, we used a Boyden chamber to evaluate BM-MSC migration. As shown in Figure 4C, miR-21 suppressed BM-MSC migration, and the migration ability of BM-MSCs was signi cantly enhanced when anti-miR-21 was added. Detailed MSC numbers are shown in Figure 4D. Together, these data indicated that miR-21 suppresses BM-MSC migration in vitro.

MiR-21 inhibits BM-MSCs migration by suppressing Wnt/β-catenin signaling in vivo
After demonstrating the role of the Wnt/β-catenin pathway in the effect of miR-21 in suppressing BM-MSC migration in vitro, we further assessed the effects of miR-21 on BM-MSC migration in vivo. We found that Ctnnb1 mRNA expression was enhanced after BM-MSC transplantation in asthmatic mice when compared with the PBS control group, and it was obviously decreased in the miR-21 + CRE + BM-MSCs group as compared to the miR-NC + CRE + BM-MSCs group ( Figure 3A and 7A), suggesting that miR-21 negatively regulates BM-MSCs in asthma. Similar ndings were obtained at the protein level ( Figure 7B, 7C, and 7D). Collectively, these results indicated that miR-21 inhibits BM-MSC migration by suppressing Wnt/β-catenin signaling in CRE-induced asthma in vivo.

Discussion
Accumulating evidence demonstrates that transplanted MSCs can repair lung injury and improve in ammatory airway disorders [26,27,51]. Recently, researchers have found that miRNAs are involved in airway allergic in ammation in a mouse model of asthma after BM-MSC transplantation [37]. These ndings suggested that miRNAs may participate in MSC migration by targeting β-catenin signaling. In support of this hypothesis, we found that: (1) Figure 8. We found that miR-21 is involved in the regulation of β-catenin expression; however, the exact mechanism requires further study. The conventional mechanism of miRNA regulation of gene expression is the inhibition of translation. Sometimes, miRNAs regulate gene expression at both transcriptional and translational levels; in this way, both mRNA and protein expression will be inhibited. In addition, one miRNA can regulate multiple target genes, and target genes of other miRNAs form a complex regulatory network that is extensively involved in biological abnormalities [52]. Collectively, our ndings provide a novel insight into the molecular mechanisms underlying the regulation of MSC migration and provide a new therapeutic target for treating asthma.
Several miRNAs are implicated in asthma pathogenesis [34,53], and miRNA-21 is a key factor in allergic airway diseases [44,45,54]. Eosinophilic in ammation and IL-4 levels reportedly are reduced and accompanied by an increase in IFN-γ levels in ovalbumin-induced airway in ammation in miR-21knockout mice [55]. Further, miR-21 downregulates the expression of phosphatase and tensin homolog, which antagonizes phosphoinositol 3-kinase (PI3K) activity in a severe steroid-insensitive asthma mouse model [43]. Kim et al. showed that treatment with a miR-21-speci c antagomir or the PI3K inhibitor LY294002 reduced PI3K activity and restored HDAC2 levels, which led to inhibition of the AHR and restoration of steroid sensitivity in allergic airway disease, indicating that miR-21 is a novel therapeutic target for asthma [44]. More recently, Lee et al. found that inhibition of miR-21 ameliorated allergic in ammation in a mouse model of asthma [45]. In line herewith, we found that suppression of miR-21 can ameliorate allergic in ammation in asthma by reducing the in ltration of in ammatory cells, especially eosinophils, Th2 cytokines IL-4, IL-5, and IL-13 in the BALF, AHR, and speci c IgE production in the BALF.
MSCs have shown a therapeutic effect in models of acute lung in ammation and brosis by targeting several in ammatory cells, including mast cells, natural killer cells, B cells, T cells, T regulatory cells, and dendritic cells [56,57]. MiRNAs can serve as downstream adapters to regulate MSC migration and mediate different diseases [26,37]. Yue et al. found that miR-124 expression is markedly decreased in MSCs in response to hepatocyte growth factor stimulation via suppression of β-catenin signaling [58]. Lee et al. reported that miR-543 and miR-590-3p decrease AIMP3/P18 expression levels by modulating cellular aging in MSCs in vitro [42]. More recently, Qiu et al. demonstrated that miR-155 regulates oxidative stress and cyclooxygenase-2 in CRE-induced murine asthma [38]. Thus, several miRNAs can mediate MSCs to in uence asthma development and may serve as therapeutic targets.
It is well known that MSC migration into the injured site is crucial for MSC therapy in various diseases. How exactly miR-21 regulates the in ammatory response to exacerbate asthma is still unknown. In the present study, we found that miR-21 expression was markedly increased in the asthma model, and increased miR-21 expression was associated with suppressed MSC migration. MiR-21 could suppress MSC immigration both in vivo and in vitro. Our results strongly suggest that miR-21 promotes CRE-induced asthma by suppressing MSC migration. Therefore, miR-21 negatively regulates the chemotactic migration of BM-MSCs, which can serve as a potential target for treating asthma in future.
Wnt/β-catenin plays a key role in the development of airway in ammation. Activation of β-catenin signaling by both Wnt-dependent and Wnt-independent pathways has been demonstrated to contribute to airway remodeling [59]. Wnt/β-catenin signaling activation by Wnt3a or LiCl improves the migratory capacity of MSCs [60], and the Wnt inhibitor Dkk-1 promotes pathological Th2-mediated in ammation [61]. Activation of Wnt/β-catenin signaling in alveolar epithelial cells attenuates intercellular adhesion molecule 1/vascular cell adhesion molecule 1-mediated adhesion and inhibits lung in ammation [62]. The Wnt/β-catenin pathway is one of the major pathways associated with MSC migration [60]. The Wnt pathway is activated in in ammatory bowel disease and can be suppressed by the transplantation of MSCs, which differentiate into intestinal epithelium [63]. MiR-124 expression is markedly decreased in MSCs in response to hepatocyte growth factor stimulation [58]. It seems that Wnt/β-catenin signaling plays different roles in in ammatory diseases by targeting in ammatory cells and the immune microenvironment. In the present study, miR-21 was characterized as a novel regulator of the canonical Wnt/β-catenin signaling pathway, which is involved in the migration of MSCs. Based on our ndings, we conclude that miR-21 inhibits MSC migration by targeting Wnt/βcatenin signaling. This conclusion was supported by the following evidence. First, miR-21-treated CRE-induced asthma model mice showed decreased βcatenin expression compared to miR-NC-treated mice. Second, miR-21-treated asthmatic mice showed less MSC migration than miR-NC-treated mice. Finally, miR-21 was found to be associated with β-catenin expression in in uencing MSC migration both in vivo and in vitro. Taken together, our data indicate that miR-21 inhibits MSC migration by suppressing Wnt/β-catenin signaling.

Conclusions
Our study demonstrated that miR-21 signi cantly stimulates CRE-induced asthma by inhibiting MSC chemotactic migration both in vivo and in vitro. More importantly, we provided evidence that Wnt/β-catenin signaling is involved in the inhibitory effect of miR-21 on the migration of BM-MSCs. MiR-21 facilitates the migration of BM-MSCs, which is a potential target for MSC-based treatment of asthma. Other possible mechanisms involved in MSC migration with regard to the treatment of asthma remain to be explored.

Consent for publication
We con rm that the gures in the manuscript are original, and all authors agreed with publication.

Competing interests
The author declares no competing nancial interests.