MicroRNA-15b-5p inhibits tumor necrosis factor alpha-induced proliferation, migration, and extracellular matrix production of airway smooth muscle cells via targeting yes-associated protein 1

ABSTRACT The excessive proliferation and the deposition of extracellular matrix (ECM) of airway smooth muscle (ASM) cells facilitates airway remodeling in asthma. This study explores how microRNA-15b-5p (miR-15b-5p) functions in modulating the proliferation, migration, inflammatory response, and ECM deposition of ASM cells. MiR-15b-5p and yes-associated protein 1 (YAP1) mRNA expression levels in tumor necrosis factor alpha (TNF-α)-induced ASM cells were, respectively, examined by real-time quantitative polymerase-chain reaction. Besides, the proliferative ability and migrative potential of ASM cells were examined by cell counting kit-8 assay, 5-bromo-2 ‘-deoxyuridine assay, and transwell assays, respectively. Interleukin-6 and interleukin-8 levels in ASM cells were detected by enzyme-linked immunosorbent assay. YAP1, collagen I, and collagen III expressions in ASM cells were detected by Western blot. With dual-luciferase reporter gene assay, the relations between miR-15b-5p and YAP1 3ʹUTR in ASM cells was examined. MiR-15b-5p expression level was reduced in ASM cells treated with TNF-α. MiR-15b-5p repressed TNF-α-initiated growth and migration of ASM cells and also suppressed IL-6 and IL-8 secretion, and inhibited collagen I and collagen III expressions in ASM cells. Furthermore, it was validated that YAP1 was a downstream target of miR-15b-5p in ASM cells. Notably, YAP1 overexpression attenuated the inhibitory effects of miR-15b-5p up-regulation on the proliferation, migration, and inflammatory response, as well as ECM deposition of TNF-α-induced ASM cells. In conclusion, miR-15b-5p/YAP1 axis modulates the growth, migration, inflammatory response, and ECM deposition of ASM cells, thus participating in the pathogenesis of asthma.


Introduction
Asthma is a common respiratory disease characterized by chronic airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling, affecting 400 million people worldwide [1,2]. Multiple inflammatory cytokines are pertinent to airway inflammation and AHR in asthma [3]. Among them, tumor necrosis factor α (TNF-α) significantly highly expressed in the lung tissue of asthmatic mice and bronchoalveolar lavage fluid of asthmatic children, has been reported to induce the proliferation, migration, and extracellular matrix (ECM) deposition of airway smooth muscle (ASM) cells [4,5]. ASM cells are the main structural components of the airways, and their excessive proliferation causes airway remodeling and can exacerbate the airway inflammatory response by secreting pro-inflammatory cytokines/chemokines, such as interleukin-6 (IL-6) and interleukin-8 (IL-8) [6]. However, the molecular mechanisms of ASM cell hyperproliferation are not fully clarified.
Yes-associated protein 1 (YAP1) is a pivotal protein in Hippo pathway and is involved in intracellular signal transduction and transcriptional coactivation of downstream genes [16]. YAP1 is vital in regulating biological processes, including cell proliferation, apoptosis, and differentiation [17]. Importantly, YAP1 is also an important modulator of asthma pathogenesis [11,18]. Fibulin-5 can promote ASM cell growth and migration via modulating Hippo-YAP/TAZ signaling pathway [18]. Additionally, YAP1 enhances the proangiogenesis of ASM cells by activating STAT3 and VEGF signaling pathways [11]. However, the interaction of YAP1 with miR-15b-5p in ASM cells has not been elucidated.
In this work, we hypothesized that miR-15b-5p played an important role in TNF-α-induced dysfunction of ASM cells. The main objective of this study was to investigate how miR-15b-5p regulates the proliferation, migration, inflammatory response, and ECM deposition of ASM cells. In our study, we found that miR-15b-5p was inhibited in ASM cells treated by TNF-α, and miR-15b-5p could repress the dysfunction of ASM cells by down-modulating YAP1 expression.

RNA extraction and real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted using the RNAiso kit (Takara, Dalian, China). RNA was treated with DNase (Solarbio, Beijing, China) before cDNA synthesis. RNA was reversely transcribed into cDNA by the miScript reverse transcription kit (Qiagen, Hilden, Germany) and M-MLV reverse transcriptase kit (Promega, Madison, WI, USA). PCR amplification was accomplished on the ABI7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc., Foster City, CA, USA) with the SYBR Green PCR kit (TaKaRa, Dalian, China), with U6 and β-actin as internal controls. The relative expressions of miR-15b-5p and YAP1 mRNA were calculated with 2 −ΔΔCt method [20]. The primer sequences are displayed in Table 1.

Cell proliferation assay
Cell counting kit 8 (CCK-8, Dojindo, Kumamoto, Japan) was adopted to assess the proliferation of ASM cells. ASM cells were seeded into 96-well plates at 2 × 10 4 cells/well and cultured at 37°C overnight. 24 h later, the cells were followingly treated with 10 ng/ml of TNF-α for 24 h. Next, 10 μL of CCK-8 reagent was loaded into each well at different time (24,48,72, and 96 h) and then incubated for 2 h at 37°C. Subsequently, the values of optical density (OD) 450 nm were assessed by a Multiscan MS spectrophotometer (Labsystems, Stockholm, Sweden).

5-bromo-2 ′ -deoxyuridine (BrdU) assay
The viability of ASM cells was also detected with a BrdU kit (Invitrogen, Carlsbad, CA, USA). Briefly, the transfected ASM cells were inoculated into 96-well plates at 2 × 10 3 cells/well and treated with 10 ng/ml of TNF-α for 24 h. Next, the cells were then rinsed with phosphate buffered solution (PBS; Beyotime, Shanghai, China) and 20 μL of BrdU solution was dripped into each well. The cells were followingly incubated for 6 h at 37°C and fixed with 4% paraformaldehyde for 10 min at ambient temperature. After rinsing with PBS, the above cells were incubated with anti-BrdU antibody (Abcam, Cambridge, Massachusetts, USA) for 8 h at 4°C. After that, the nuclei were then stained with 10 μmol/L Hoechst 33342 (Beyotime, Shanghai, China) for 2 h in darkness. Then, cells were then observed under a fluorescence microscope. Cell proliferation rate = numbers of BrdUstained positive cells/total number of cells × 100%.

Transwell assay
Transwell migration assays were accomplished to test the migration ability of ASM cells with Transwell inserts (8 µm pore size, Corning Incorporated, NY, USA). Briefly, 24 h after cell transfection, the cells were treated with 10 ng/ml of TNF-α for 24 h. Then, the cells were immersed twice in PBS and subsequently resuspended in serum-free medium (1 × 10 5 cells/mL). Moreover, 200 μl of cell suspension was loaded into the upper compartment of each Transwell insert, and 600 μL of medium with 20% FBS was dripped into the lower compartment, and the cells were cultured at 37°C for 24 h. After that, the cells retained in the upper were removed with a cotton swab and the migrated cells on the lower surface of the filter were fixed with 4% formaldehyde (Sigma-Aldrich, Shanghai, China) for 30 min and subsequently stained with 0.5% crystal violet (Sigma-Aldrich, Shanghai, China) for 20 min. Ultimately, the migrated cells were counted in five randomly selected fields (magnification ×  250) under a light microscope (Olympus, Tokyo, Japan).

Enzyme -linked immunosorbent assay (ELISA)
Transfected ASM cells (4 × 10 5 /per well) were inoculated in 12-well plates and treated with 10 ng/ml of TNF-α for 24 h. These cells, together with the medium, were harvested and followingly centrifuged at 1000 × g for 10 min at 4°C. The supernatant was then obtained. Subsequently, IL-6 and IL-8 contents in the samples were measured with the corresponding ELISA kits (TaKaRa, Dalian, China).

Dual-luciferase reporter gene assay
The luciferase reporter vectors carrying the fragments of YAP1 3'-UTR containing wild type (WT) or mutated (MUT) binding sites of miR-15b-5p were available from RiboBio (Guangzhou, China). ASM cells (5 × 10 5 cells/well) were subsequently inoculated in 24-well plates and cultured for 24 h. Next, the reporter vectors were co-transfected into ASM cells with miR-15b-5p mimics (or mimics-NC) by Lipofectamine TM 3000 (Invitrogen, Carlsbad, CA, USA). 48 h later, the luciferase activity was assessed by a dual-luciferase reporter kit (Promega, Madison, WI, USA), with the renilla luciferase reporter vector as an internal control.

Statistical analysis
All data analysis was performed by SPSS 18.0 software. All experiments were independently repeated 3 times. Experimental data were displayed in the form of mean ± standard deviation. The differences between two groups were analyzed by student's t-test; one-way ANOVA with post-hoc Tukey test, was used to compare the differences among multiple groups. Statistically, P < 0.05 is meaningful.

Results
This work focused on exploring the expression characteristics of miR-15b-5p and investigating its biological function in regulating ASM cell proliferation, migration, inflammatory response, and ECM deposition, with TNF-α-stimulated ASM cells as a model. In addition, the interaction of miR-15b-5p with YAP1 in ASM cells was also investigated. This study showed that miR-15b-5p could inhibit the proliferation, migration, inflammation, and ECM deposition of TNF-α-induced ASM cells by targeting YAP1.

TNF-α suppresses miR-15b-5p expression in ASM cells
We first used TNF-α to treat human ASM cells to establish an in-vitro asthma model and then detected miR-15b-5p expression in ASM cells by RT-qPCR, revealing that miR-15b-5p expression was inhibited in TNF-α-stimulated ASM cells compared with the blank group (Figure 1(a)). Thus, it might be involved in the TNF-α-induced dysfunction of ASM cells. To delve into the biological role of miR-15b-5p in ASM cells, we transfected ASM cells with miR-15b-5p mimics, along with mimics-NC as a negative control (Figure 1(b)).

Upregulating miR-15b-5p restrains TNF-αmediated ASM cell growth and migration
Next, the impact of miR-15b-5p on TNF-α-induced proliferation and migration of ASM cells was investigated. CCK-8, BrdU and Transwell assays suggested that TNF-α stimulation significantly accelerated the proliferation and migration of ASM cell, while miR-15b-5p up-regulation weakened these effects (Figure 2(a-c)). Collectively, miR-15b-5p could probably suppress the dysfunction of ASM cells in asthma.

miR-15b-5p up-modulation inhibits TNF-αinduced inflammatory response and ECM deposition
To figure out the impact of miR-15b-5p on the inflammatory response induced by TNF-α, we performed ELISA and observed that IL-6 and IL-8 levels were markedly elevated in ASM cells treated with TNF-α, while those were declined followed by transfecting with miR-15b-5p mimics (Figure 3 (a)). Additionally, Western blot indicated that the expression levels of collagen I and collagen III proteins were markedly up-regulated in ASM cells in the TNF-α treatment group as against the blank group, while transfecting with miR-15b-5p mimics significantly decreased those levels as against the TNF-α + mimics-NC group (Figure 3 (b)). Collectively, up-regulation of miR-15b-5p impeded TNF-α-initiated inflammatory response and ECM deposition in ASM cells.

YAP1 is a direct target of miR-15b-5p in ASM cells
As to the hidden mechanism of miR-15b-5p in regulating the phenotypes of ASM cells, we conducted bioinformatics analysis and found that the 3'-UTR of YAP1 harbors two binding sites for miR-15b-5p (Figure 4(a)). To clarify whether miR-15b-5p targets YAP1, dual-luciferase reporter gene assay was performed, and it highlighted that miR-15b-5p upregulation remarkably repressed the luciferase activity of YAP1 3ʹUTR WT1 and WT2, but the luciferase activity of YAP1 3ʹUTR MUT1 and MUT2 were not significantly affected (Figure 4(b and c)). These data suggested that both of the two potential-binding sites were functional. Furthermore, we demonstrated that up-regulation of miR-15b-5p markedly suppressed YAP1 mRNA and protein expression levels ( Figure 4(d and e)). In addition, YAP1 mRNA and protein expression levels were dramatically increased in TNF-α-activated ASM cells compared with those of the blank group ( figure 4(f and g)). Collectively, miR-15b-5p could target the YAP1 3 '-UTR and inhibit YAP1 expression.

YAP1 restoration abrogates the effect of miR-15b-5p on ASM cells
To investigate whether YAP1 is implicated in miR-15b-5ʹs function in regulating ASM cell proliferation, migration, inflammatory response, and ECM deposition, we co-transfected miR-15b-5p mimics (or mimics-NC) and YAP1 overexpression plasmids (or empty plasmids) into ASM cells. Western blot indicated that the impact of miR-15b-5p overexpression on YAP1, collagen I and collagen III protein expression levels in ASM cells was counteracted by YAP1 overexpression (Figure 5(a)). Additionally, YAP1 overexpression mitigated the impact of miR-15b-5p overexpression on TNF-α-mediated ASM cell viability and migration ( Figure 5(b-d)). ELISA indicated that the effect of transfection of miR-15b-5p mimics on TNF-α-activated IL-6 and IL-8 production in ASM cells were dramatically counteracted by YAP1 overexpression (Figure 5(e)). In short, miR-15b-5p impeded TNF-α-induced ASM cell growth, migration, inflammatory response, and ECM deposition by downregulating YAP1 expression.

Discussion
Airway remodeling in asthma is closely associated with submucosal fibrosis, abnormal proliferation, and migration of ASM cells, and excessive deposition of ECM [22,23]. ASM cell is one of the important sources of inflammatory factors, chemokines, and growth factors [24]. Various cytokines, including TNF-α, transforming growth factor-β (TGF-β), IL-1β, and platelet-derived growth factor (PDGF-BB), have been found to be important mediators of airway remodeling and fibrotic responses, and they can also initiate and drive the inflammatory response in asthma [5,[24][25][26]. As reported, the combined effect of interferon-gamma (IFN-γ), IL-1β, and TNF-α accelerates the release of chemokine CXCL10 in asthmatic ASM cells [27]. Notably, TNF-α stimulation alone can also induce the dysfunction of ASM cells [5,19,28,29]. Here, we found that TNF-α strengthened the proliferative and migrative (a) The binding sites between miR-15b-5p and YAP1 mRNA 3 ′ -UTR were predicted by StarBase database. (b and c) Dual-luciferase reporter gene assay was adopted to validate the targeting relationship between miR-15b-5p and the YAP1 mRNA 3' -UTR. (d-g) The expression levels of YAP1 mRNA and protein in ASM cells transfected with miR-15b-5p mimics or stimulated with TNF-α were detected by RT-qPCR and Western blot. Data are displayed as mean ± standard deviation (n = 3 independent experiments). **P < 0.01 versus mimics-NC group, ***P < 0.001 versus mimics-NC group or blank group. capabilities of ASM cells; TNF-α could raise the secretion IL-6 and IL-8 of ASM cells, and it increased the expression of ECM-associated proteins (collagen I and collagen III proteins). In other words, TNF-α induces the inflammatory response and ECM deposition in ASM cells, which is consistent with the previous reports [5,19,28,29].
In-vitro experiments showed that miR-15b-5p upregulation could reverse the impact of TNF-α stimulation on the growth and migration of ASM cells, and restrain TNF-α-induced inflammatory response and ECM deposition. Collectively, miR-15b-5p may be a protective modulator in asthma pathogenesis.
As a vital signal transducer of Hippo pathway, YAP1 is extensively expressed in diverse tissues and organs of mammals, and in normal tissues. YAP1 is regulated by other upstream proteins of Hippo pathway such as Lats1/2, Mst1/2, Sav1, and MOB1/2, leading to the cascade amplification [35]. After entering the nucleus, YAP1 can bind to transcriptional factors such as transcriptional enhancerrelated domain (TEAD) to function as a transcriptional enhancer, and thus the transcription of downstream genes is promoted [36]. YAP1, as reported, is highly expressed in tumors such as pancreatic ductal adenocarcinoma, colorectal, and bladder cancer, and it expedites the proliferation, migration, and invasion of cancer cells [37][38][39]. YAP1 is also widely expressed in respiratory epithelial cells, and the Hippo signal pathway is vital in regulating the growth and differentiation of the epithelial cells [40]. As reported, the dysregulation of YAP1 is pertinent to the pathogenesis of asthma [11,18,41]. YAP1 is observed to be up-regulated in bronchial airway tissue of a mouse model with chronic asthma [41] and it can enhance the viability and migrative potential of ASM cells [11]. In addition, YAP1, reportedly, is modulated by multiple miRNAs, such as miR-375, miR-126-5p, miR-582-3p and miR-27b-3p [11,38,42,43]. Here we found that YAP1 was a downstream target of miR-15b-5p in ASM cells; YAP1 was highly expressed in ASM cells stimulated by TNF-α. Here we proved that the impacts of miR-15b-5p up-regulation on ASM cell growth, migration, inflammatory response and ECM deposition could be rescued by YAP1 overexpression. We therefore summarized that miR-15b-5p/ YAP1 axis mediated the TNF-α-initiated ASM cell growth, migration, inflammatory response, and ECM deposition.
Notably, there are some limitations of this study: first, only TNF-α-induced ASM cells were used as a model, and whether miR-15b-5p/YAP1 axis is involved in other pro-inflammatory factorsinduced dysfunction of ASM cells remains to be explored in the following work; secondly, in-vivo studies are lacking in the present work; and finally, other downstream targets of miR-15b-5p need to be identified in the future.

Conclusion
On all accounts, miR-15b-5p expression is declined in the TNF-α-initiated ASM cells and miR-15b-5p is involved in repressing the proliferation, migration, inflammatory response as well as ECM deposition of ASM cells by targeting YAP1.

Data Availability Statement
The data used to support the findings of this study are available from the corresponding author upon request.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Ethics statement
Our study was approved by the Ethics Review Board of Yantai Yuhuangding Hospital.

Funding
The present study was supported by Natural Science Foundation of China (No. 81700030).