Introduction

Recently, childhood asthma has become one of the prevalent chronic illnesses in pediatric patients.¹ Airway irritation tends to induce mucus accumulation, airway inflammatory response, and remodeling, leading to the onset of asthma.² Asthma etiology is linked to the increased number of airway smooth muscle cells (ASMCs) along with their migration, and secreted adhesion molecules, cytokines, matrix components and metalloproteinases secreted by ASMCs; these secretions promoted the proliferation of ASMCs and aggravated airway remodeling.^3,4 Therefore, the inhibition of pathobiological processes in ASMCs provides a therapeutic approach for childhood asthma.

Highly conserved microRNAs (miRNAs) are small non-coding RNAs that are responsible for regulating the post-transcriptional expression of genes. Research studies have shown the vital role of miRNAs in regulating ASMCs malignant phenotypes during asthma.⁵ For instance, miR-221 regulated the expression of p21^WAF1 and p27^kip1 to enhance the aberrant proliferation of ASMCs.⁶ In contrast, miR-217 interacted with zinc finger E-box binding homeobox 1 (ZEB1) to repress abnormal ASMCs survival and migration.⁷ The protective effects of miR-506 in progression of different cancers, especially in lung cancer, were reported in previous studies, which raised our concern.^8–10 A research uncovered that miR-506-3p inhibitor contributed to non-small cell lung cancer tumorigenesis by boosting the proliferative, migratory as well as invasive properties of cells.¹¹ In addition, overexpression of miR-506 was reported to downregulate Tubby-like protein 3, leading to an inhibitory pathological process in lung cancer.¹² Moreover, miR-506 was apparently downregulated and its overexpression inhibited pulmonary fibrosis by reducing apoptosis and improving inflammation in a lung fibrosis model.¹³ However, the link between miR-506 and asthma etiology still needs to be explained.

Polypyrimidine tract-binding protein 1 (PTBP1), a member of the heterogeneous ribonucleoprotein subfamily, participates in pre-mRNA processing, and mRNA metabolism and transport.^14–16 PTBP1 has been verified to play an oncogenic function in multiple cancers, including lung cancer.^17,18 Furthermore, abnormal expression of PTBP1 was involved in controlling integral metabolism and proliferation in pulmonary hypertension and fibrosis.^19,20 PTBP1 was markedly upregulated in a vascular smooth muscle cells (VSMCs) injury model, and PTBP1 knockdown resulted in a reduced proliferation of VSMCs.²¹

Here, we established a transforming growth factor--beta1 (TGF-β1)-stimulated airway smooth muscle cells (AMSCs) model of asthma in vitro. In this model, we investigated the protective properties of miR-506 and the modulatory regimes involved. Our study offers novel insights for improving pediatric asthma treatment.

Materials and Methods

Clinical tissues

In this study, 50 asthma patients with a balanced gender ratio of 1:1 were recruited; patients’ varied between 5 and 12 years, with an average age of 8.2 ± 3.5 years. The control group included children who were diagnosed as having no history of respiratory and allergy-related diseases within 1 month by the respiratory asthma department of Xi’an Children’s Hospital. A total of 12 boys and 13 girls were enrolled in the control group of this study. The age of these children ranged from 6 to 13 years, with a mean age of 8.2 ± 3.1 years. All tissue samples were extracted by performing biopsy of the subjects according to a previous study,²² and cryopreserved for subsequent experiments.

The study was conducted in accordance with the Declaration of Helsinki, and was authorized by the Ethics Committee of Xi’an Children’s Hospital. Informed consent was taken for all subjects.

Cell culture and treatment

Human-derived ASMCs were obtained from Procell Life Science & Technology Co. Ltd. (Cat. No. CP-H014; Wuhan, China) and cultivated in Dulbecco’s modified Eagles Medium (DMEM; Cat. No. D5796; Sigma-Aldrich, St Louis, USA) enriched with 10% fetal bovine serum under the condition of 5% CO₂ and 37°C. We developed an airway remodeling cell model as described by Cao et al.⁷ In short, ASMCs were exposed to 10-ng/mL TGF-β1 (Cat. No. 75362; Cell Signaling Technology, Shanghai, China) for 24 h. Besides, ASMCs were treated with 2.5-μM wingless-related integration (Wnt)/ β-catenin inhibitor (IWR-1; Cat. No. I0161; Sigma-Aldrich) for 24 h.

Cell transfection

In order to enhance expression of PTBP1, the amplified DNA fragment of PTBP1 was cloned into plasmid cloning (pc) DNA3.1 (Cat. No. V79020; Invitrogen, Carlsbad, CA, USA). MiR-506 mimic, miR-NC, anti-miR-506, and anti-miR-NC were obtained from GenePharma Co. (Shanghai, China). The specific sequences of mimics were as follow: miR-506 mimic (5'-UAAGGCACCCUUCUGAGUAGA-3'), miR-NC (5'-UUCUCCGAACGUGUCACGUTT-3'), anti-miR-506 (5'-UCUACUCAGAAGGGUGCCUUA-3'), and anti-miR-NC (5'-CAGUACUUUUGUGUAGUACAA-3'). The transfections of the above-mentioned plasmids and miRNAs in ASMCs were conducted with Lipofectamine 3000 (Cat. No. L3000015;, Invitrogen) in compliance with -manufacturer’s protocol.

Real-time quantitative polymerase chain reaction (RT-qPCR)

Using Trizol (Cat. No. 15596018; Invitrogen), total RNA was extracted from ASMCs. M-MLV (Cat. No. 28025021; Invitrogen) reverse transcriptase kit was used to obtain complementary DNA (cDNA). RT-qPCR was performed on a 7500 real-time PCR machine (Cat. No. 4351107; Applied Biosystems, Shanghai, China) with a TB Green Premix Ex Taq II (Cat. No. RR820Q; Takara, Dalian, China) and the relative mRNA level was quantified by the 2^−ΔΔCt method. Reference controls for miRNAs and mRNAs were U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), respectively. The sequences of primers are listed in Table 1.

Western blot analysis

Radioimmunoprecipitation assay (RIPA) buffer (Cat. No. 89900; Sigma-Aldrich) was applied to lyse ASMCs to obtain total proteins. Then, protein quantification was executed with a bicinchoninic acid (BCA) protein assay kit (Cat. No. P0012S; Beyotime, Shanghai, China). Next, the proteins were separated by electrophoreses and transferred onto polyvinylidene fluoride (PVDF) membranes (Cat. No. VVLP02500; Millipore, MA, USA). The membranes were treated with 5% skimmed milk for 1 h. The following antibodies were used as primary antibodies: anti-PTBP1 (1:1000; ab5642; Abcam, Shanghai, China), anti-collagen I (1:1000; ab138492; Abcam), anti--collagen III (1:1000; ab6310; Abcam), anti-β-catenin (1:1000; ab32572; Abcam), anti-c-MYC (1:1000; ab32072; Abcam), anti-cyclin D1 (1:1000; ab16663; Abcam), and anti-GAPDH (1:5000; sc-47724; Santa Cruz Biotechnology, TX, USA). Incubation of primary antibodies was carried out overnight at 4°C; then, the membranes were washed and treated with horseradish peroxidase-conjugated secondary antibody (1:5000; sc-2004; Santa Cruz) for 1 h at room temperature. Eventually, the results were visualized using enhanced -chemiluminescence (ECL) reagents (Cat. No. 32209; Millipore). Using Image J (NIH, MD, USA), GAPDH was -utilized to normalize protein bands.

Luciferase reporter assay

An online website TargetScan 7.2 (http://www.targetscan.org/ vert_72/) predicted possible correlation between PTBP1 and miR-506. The 3’ untranslated regions (3-UTR) of PTBP1 mutant (MUT) and wild-type (WT) bound to miR-506 were inserted into psiCHECK (Cat. No. C8021; Promega, Madison, WI, USA). Co-transfection of these recombinant plasmids into ASMCs with miR-506 mimics or miR-NC was accomplished by using Lipofectamine 3000 (Invitrogen). Following 48-h transfection, renilla luciferase activity was utilized to standardize luciferase reporter activity on a Dual Luciferase Reporter Assay System (Promega).

Cell counting kit (CCK)-8 assay

First, ASMCs were added into 96-well plates (2 × 10⁴ cells/well). Subsequently, the proliferative activity of ASMCs was determined with CCK-8 solution (Cat. No. CK04; Dojindo Molecular Technologies, Shanghai, China) as directed by the manufacturer. Finally, analysis of absorbance at 450 nm was recorded using a microplate reader (Cat. No. VL0000D2; Thermo Fisher Scientific, Waltham, MA, USA).

Enzyme-linked immunosorbent serological assay (ELISA)

The measurements of interleukin (IL)-1β and i nterferon gamma (IFN-γ) levels in cell supernatant were conducted using commercial ELISA kits (Cat. No. PI305 and PI511; Beyotime) according to instruction manual of the manufacturer. IL-1β and IFN-γ contents were also calculated based on the absorbance values at 450 nm.

Wound healing assay

ASMCs were planted into 24-well plates (5 × 10⁴ cells per well). After cell confluency reached 90%, a sterile disposable pipette was used to make a scratch in the center of cell monolayer. Images were captured under a microscope (×100 magnification; Cat. No. CX43/33; Olympus, Tokyo, Japan) at 0 h and 48 h after scratching to quantify and assess the ability of migration.

Transwell assay

In addition, an 8-μm pore size Transwell chamber (Cat. No. 430157; Costar, Suzhou, China) was used to evaluate cell migration. After serum starvation for 24 h, resuspended ASMCs in serum-free medium was put in the top of 24-well Transwell chamber. The lower part of Transwell chamber was loaded with 10% DMEM. After being cultured for 48 h, the unmigrated cells were removed gently, and the migrated cells in the bottom of the chamber were fixed with formaldehyde and stained with crystal violet. Cell migration rate was calculated in five randomly selected fields of view with a light microscope (×100 magnification; Cat. No. CX43/33; Olympus).

Statistical analysis

All experiments were carried out thrice independently. SPSS 22.0 was used for all statistical analyses. The results were presented as mean ± standard error of mean (SEM). In order to identify the significance of variations between groups, Student’s t-test and ANOVA were applied. Spearman’s Rank test was applied to ascertain correlations between miR-506 and PTBP1 in asthmatic tissues. P < 0.05 was considered as statistically significant difference.

Results

miR-506 was poorly expressed in asthmatic tissues and TGF-β1-induced ASMCs

We first identified miR-506 expression in asthmatic tissues and a TGF-β1-stimulated in vitro cell model of asthma. We observed that miR-506 expression was markedly attenuated in asthmatic tissues, compared to normal tissues (Figure 1A). Likewise, a remarkable decline in miR-506 expression was discovered in TGF-β1-stimulated ASMCs, compared to untreated ASMCs. Meanwhile, the results showed that miR-506 mimic dramatically upregulated TGF-β1-induced miR-506 expression whereas anti-miR-506 effectively attenuated the role of TGF-β1 in miR-506 expression (Figure 1B).

miR-506 inhibited TGF-β1-stimulated aberrant cellular proliferation and inflammatory responses of ASMCs

Subsequently, ASMCs were exposed to TGF-β1 to imitate asthma environment in vivo. CCK-8 assay showed that exposure to TGF-β1 elevated cell viability considerably. Interestingly, overexpression of miR-506 remarkably suppressed the abnormal proliferation of ASMCs evoked by TGF-β1 whereas anti-miR-506 exacerbated the malignant growth of ASMCs (Figure 1C). In addition, we found that TGF-β1 treatment elevated IL-1β levels and inhibited IFN-γ production in the supernatant of ASMCs culture system, while the upregulation of miR-506 reversed this effect, and anti-miR-506 aggravated TGF-β1-stimulated abnormal production of pro-inflammatory factors (Figures 1D and 1E). The findings revealed that miR-506 had an inhibitory effect on TGF-β1-stimulated aberrant cellular proliferation and inflammation of ASMCs.

miR-506 repressed TGF-β1-stimulated aberrant migration and collagen deposition of ASMCs

Next, we addressed the involvement of miR-506 in TGF-β1-stimulated aberrant migration of ASMCs. Administration of TGF-β1 dramatically increased the migratory capacity of ASMCs, while miR-506 mimic markedly depressed this effect (Figures 2A and 2B). Meanwhile, the downregulation of miR-506 induced elevation in the migratory capacity of ASMCs evoked by TGF-β1. Besides, excessive collagen deposition is also an essential factor leading to airway remodeling.²³ Further validation experiments revealed that expression of collagen I and collagen III was remarkably enhanced via TGF-β1 treatment. In addition, anti-miR-506 markedly promoted this effect whereas miR-506 mimic mitigated this effect (Figure 2C). Altogether, these findings supported that miR-506 attenuated TGF-β1-stimulated aberrant ASMCs migration and collagen deposition.

miR-506 directly targeted to PTBP1

A recent study has demonstrated that PTBP1 was involved in the aberrant inflammation and migration of ASMCs evoked by TGF-β1.²⁴ Interestingly, we used TargetScan 7.2 to identify PTBP1 as a potential target for miR-506 and predicted the matched regions between miR-506 and PTBP1 (Figure 3A). Further investigation revealed that miR-506 mimic markedly diminished the Luciferase activity of PTBP1-WT (Figure 3B). Moreover, PTBP1 expression was enhanced in TGF-β1-induced ASMCs, which was dramatically diminished by miR-506 mimic (Figures 3C and 3D). In addition, PTBP1 expression was dramatically elevated in asthma tissues, compared to healthy tissues and had a negative regulatory relationship with miR-506 expression (Figures 3E and 3F). The above data indicated that miR-506 directly targeted to PTBP1 and served as a negative regulator of PTBP1.

miR-506/PTBP1 axis repressed TGF-β1-stimulated aberrant cellular proliferation and inflammatory responses of ASMCs by regulating Wnt/β-catenin signaling pathway

Wnt/β-catenin signaling pathway exerts a profound effect on pathogenesis of asthma.²⁵ Therefore, we tried to unveil the correlation between miR-506 and Wnt/β-catenin signaling pathway. The result pointed out that TGF-β1 treatment significantly enhanced expression of key target proteins (β-catenin, c-MYC, and cyclin D1) in Wnt signaling pathway. miR-506 overexpression significantly suppressed the TGF-β1-triggered activation of Wnt signaling pathway, while PTBP1 overexpression reversed these effects (Figure 4A).

Rescue assays were performed in TGF-β1-induced ASMCs using IWR-1 inhibitor. As depicted in Figures 4B–D, miR-506 mimic markedly repressed TGF-β1-stimulated aberrant proliferative and migratory activity of ASMCs whereas the upregulation of PTBP1 dramatically abated these effects. Additionally, miR-506 overexpression suppressed the generation of IL-1β and enhanced the secretion of IFN-γ in asthmatic cell model whereas opposite effects were observed in the PTBP1 overexpression groups. Moreover, the repressive role of miR-506 mimics in proliferation, migration as well as inflammatory responses of TGF-β1-stimulated ASMCs was greatly counteracted by PTPB1 overexpression, which was markedly reversed by exogenous IWR-1 (Figures 4B–F). Overall, miR-506/PTBP1 axis inhibited proliferation, migration, and cellular inflammatory responses of TGF-β1-stimulated ASMCs through inactivated Wnt/β-catenin pathway.

Discussion

The present treatment for severe or gentle asthma is primarily focused on inhalation of corticosteroids, long-acting β2-agonists, and leukotriene antagonists. Although traditional therapy has made great progress, treatment of persistent and uncontrolled asthma in children is still a problem.²⁶ Molecular targeted therapy has become a novel alternative for asthma patients, especially in controling airway inflammatory responses and remodeling. Our data indicated that miR-506 modulated the alterations of proliferation, migration, inflammation, and collagen deposition in a TGF-β1-induced asthmatic cell model by targeting PTBP1 to inactivate the Wnt/β-catenin pathway.

Growing research suggests that miRNAs is instrumental in controlling the progression of asthma.^27–29 For example, miR-142-5p and miR-130a-3p have been shown to be engaged in pulmonary inflammation and airway phenotypic alterations.³⁰ MiR-30a inhibited autophagy, and hence reduced airway fibrosis in a mouse model.³¹ MiR-506 is a newly identified miRNA involved in multiple biological processes, mostly reported in cancer. Notably, the sequences that contained single nucleotide polymorphism rs3809865, significantly associated with asthma, was predicted to bind to the seed regions of miR-506,³² which raised our speculation that miR-506 may be associated with asthma. At the same time, Wang et al. observed that miR-506-3p expression was declined in ovalbumin (OVA)-based asthma model, and its overexpression reduced growth and induced apoptosis of ASMCs.³³ Consistently, our data suggested that upregulated miR-506 suppressed ASMCs proliferation and migration during asthma progression. Simultaneously, upregulated miR-506 reduced inflammation and collagen deposition in an asthmatic in vitro cell model evoked by TGF-β1. Additionally, a previous research confirmed a positive role of miR-506 in the fibrosis and inflammatory response of nonalcoholic fatty liver disease.³⁴ Therefore, it implied that miR-506 presented a protective role in the development of asthma.

miRNA-mediated gene regulation has a variety of regulatory modes by targeting different regions, such as coding regions,³⁵ 3'-UTR,³⁶ or 5'-UTR.³⁷ Among them, the binding of miRNAs to the 3'-UTR of target genes is deemed as a classic way to participate in the regulatory mechanism of airway remodeling.³⁸ For example, Sun et al. documented that miR-874 inhibited TNF-α-stimulated human fetal ASMCs remodeling by targeting Signal transducer and activator of transcription 3 (STAT3).³⁹ In a study conducted by Chen et al., miR-23b directly bound to Smad3 to suppress ASMCs proliferation.⁴⁰. Interestingly, PTBP1 was considered as a crucial RNA-binding protein in asthmatic tissues and a common miRNA-target.^41,42 However, there were only two studies regarding the effects of PTBP1 in asthma etiology. In our work, the direct target of miR-506 was identified as PTBP1. Consistent with our findings, another research demonstrated that miR-506-3p mediated the direct regulation of PTBP1 in gastric cancer.⁴³ Besides, our research indicated that PTBP1 was greatly upregulated in asthmatic tissues and promoted abnormal biological behavior in TGF-β1-stimulated ASMCs. Similarly, PTBP1 silencing was reported to inhibit the malignant phenotype of VSMCs and lung cancer cells.^18,21 Additionally, we observed a promotive impact of PTBP1 on TGF-β1-stimulated inflammatory responses in ASMCs. Consistently, a recent study has shown that the overexpression of PTBP1 enhanced ASMCs proliferation, migration, and inflammation by upregulating neuro-oncological ventral antigen 1.²⁴

As a well-known pathway, Wnt/β-catenin pathway is extremely relevant for cell growth and development, and has been connected to recent findings of asthma. For instance, in TGF-β1-induced bronchial smooth muscle cell, miR-188 mediated the blockage of Wnt/β-catenin signaling pathway to dampen cell proliferation.⁴⁴ Induction of Wnt/β-catenin signaling pathway mediated by miR-21 exacerbated airway inflammation and oxidative damage in asthma.⁴⁵ Therefore, this further aroused us to speculate whether Wnt/β-catenin signaling took part in miR-506 modulation of asthma pathogenesis. Previous study has indicated that a positive regulatory relationship existed between PTPB1 and Wnt/β-catenin signaling in glioma.⁴⁶ Furthermore, several evidences indicated that miR-506 regulated the tumorigenesis of osteosarcoma via Wnt/β-catenin signaling pathway.⁴⁷ Our data pointed out that overexpression of miR-506 inactivated Wnt/β-catenin pathway to mitigate TGF-β1-mediated adverse progression of asthma, while PTBP1 exacerbated TGF-β1-stimulated airway remodeling and inflammation in ASMCs by triggering the Wnt/β-catenin signaling pathway.

The limitation of this study was its sample size, which was limited; moreover, the animal model of asthma was not created. Further investigation of mechanism is required to verify the findings of this study, which would help to provide a theoretical basis to develop new agents for asthma therapy.

Conclusion

To conclude, this research implied that miR-506 targeted PTPB1 to alleviate TGF-β1-induced airway inflammatory response and remodeling in ASMCs by suppression of Wnt/β-catenin pathway. This would figure out the possibility of miR-506 in treating asthma.