Mechanism of How miR-145-5p Affects Asthma

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miR-145-5p promotes asthma by inhibiting KIF3A expression in airway epithelial cells

Background: MiR-145-5p was found to be remarkably increased in the asthma patients’ plasma. Ozone is an air pollutant that is associated with numerous adverse health effects that increased miR-145-5p expression in human bronchial airways in vivo at a concentration of 0.4 ppm for 2h. House dust mite (HDM) increases the expression of miR-145-5p, and inhibition of miR-145-5p reduced mucus hypersecretion, eosinophilic inflammation, TH2 cytokine production, and airway hyper-responsiveness. However, the underlying mechanism are still unclear.

Objective: To elucidate the underlying mechanism of how miR-145-5p affects asthma.

Methods: We established the asthma model according to the published method. MiR-145-5p was given via nasal infection. KIF3A and miR-145-5p expression levels were measured by immunohistochemistry, PCR and western blot. Tissue sections were stained with H&E or PAS. Eosinophils in lavage fluid was counted, and the levels of IL-4, 5, and 13 were determined. The airway hyper-responsiveness (AHR) was measured.  The expression of KIF3A was tested upon miR-145-5p overexpression or interference in airway epithelial cell line 16HBE 14o-. Finally, miR-145-5p and KIF3A co-transfection in 16HBE 14o- cells to study their effects on cytokine release, epithelial barrier dysfunction, and epithelial repair in HDM-exposed epithelial cells.

Results: KIF3A downregulation and miR-145-5p upregulation were detected in airway epithelial cells in HDM-exposed mice with asthma, while miR-145-5p antagonism improved asthma symptoms significantly. MiR-145-5p promotes HDM-induced release of chemokines and inflammatory factors, and regulates HDM-induced epithelial barrier dysfunction and epithelial repair by directly targeting KIF3A leading to the suppression of KIF3A expression.

Conclusion: MiR-145-5p influenced HDM-induced epithelial cytokine release and epithelial barrier dysfunction by regulating KIF3 expression and affect epithelial repair, which may lead to and exacerbate the HDM-induced Th2-type immune response in mice.

Keywords: miR-145-5p, KIF3A, asthma

Introduction

Asthma is a chronic inflammatory airway disease induced by inflammation, mucus overproduction, bronchial hyper-reactivity and airway obstruction. Allergic asthma is characterized by allergen-induced airway inflammation, hyper-responsiveness (AHR) and remodeling [1]. The airway epithelium is the first barrier against deposited aeroallergens and is a central player in the initiation of allergic responses [2-5]. It is demonstrated to be a critical controller of inflammatory and regenerative responses to allergens and environmental pollutants that contribute to asthma pathogenesis [6]. Many allergens, such as house dust mite (HDM) cause epithelial damage and induce asthma [7, 8]. The air pollutant such as ozone also alters respiratory immune response and causes adverse health effects [9]. The way to alleviate inflammatory response in airway epithelium is one of the effective ways to treat asthma. MicroRNAs (miRNAs) are short noncoding RNAs which are important regulators for posttranscriptional modulation of gene expression [10, 11].  MiRNAs regulate gene expression by inhibition of translation or destabilization of mRNA transcript through base pairing to the 3’-UTR regions of mRNAs [12]. MiRNAs have been reported in regulating a range of cellular activities and involved in various biological processes [13]. Importantly, miRNAs were reported to be a key role in the regulation of hematopoiesis and controlling inflammation [13-15]. Since airway inflammation is a main character of asthma, miRNAs may be potential important therapeutic targets to treat asthma. It has been reported that miR-145-5p is significantly increased in the plasma of chronic obstructive pulmonary disease (COPD) and asthma patients, indicating plasma miR-145-5p is a potential specific biomarker for related disease [16]. Ozone is an air pollutant that is associated with numerous adverse health effects that increased a serious of miRNAs including miR-145-5p expression in human bronchial airways in vivo at a concentration of 0.4 ppm for 2h [17]. Moreover, the miRNAs and their predicted targets were associated with inflammation and immune-related disease [17]. In the HDM-induced asthma mice models, results showed that HDM increases the expression of miR-145-5p, and inhibition of miR-145-5p reduced eosinophilic inflammation, mucus hypersecretion, TH2 cytokine production, and airway hyper-responsiveness [18]. However, the underlying mechanism are still unclear. We want to address how miR-145-5p affect immune response and find its direct target involved in immune regulation in a HDM-induced asthma model.

In this study, we identified and characterized the function and target of miR-145-5p during HDM-induced asthma development, while miR-145-5p antagonism remarkably improved asthma symptoms. We found miR-145-5p expression level was correlated with allergic airways inflammation and inhibited its direct target KIF3A expression. KIF3A overexpression significantly inhibit HDM-induced immune response by reducing inflammatory cells infiltration and chemokine release. Our study revealed that miR-145-5p influenced HDM-induced epithelial cytokine release and epithelial barrier dysfunction by regulating KIF3 expression and affect epithelium barrier and epithelial repair, which may lead to and exacerbate the HDM-induced Th2-type immune response in mice.

Materials and methods

Mice

BALB/c mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). The mice were maintained in specific pathogen-free conditions. All animal experiments were performed according to the Animal Care and Use Committee guidelines.

Induction of allergic airways disease

House dust mite (HDM, Greer Laboratories, Lenoir, NC, USA) was resuspended in sterile saline (SAL) and mice were treated through the nose with 50 g protein/50 L (Der p 1 constituted ~10% of protein weight) or vehicle sterile endotoxin-free saline on days 1, 2, and 3 for sensitization, which was followed by daily HDM exposure (5 g/50 L) on days 14, 15, 16, and 17 to induce allergic airways disease. Nonsensitized mice (vehicle-treated) were treated with sterile endotoxin-free saline only. Experiments were conducted on day 18, 24 hours after the last HDM exposure.

AHR measurement

Mice were anesthetized and mechanically ventilated, and AHR to nebulized acetylcholine (increased lung resistance) was measured as previously described. Responses to acetylcholine were expressed as a percentage change over control (baseline).

Antagomirs

Target miRNA sequences were downloaded from miRBase and specific antisense antagomirs designed and synthesized. 50 g antagomirs or ant-scrambled/50 l sterile saline intranasally on day 13 (24 hours before the first HDM re-exposure) and then every second day until mice were killed on day 8.

Cell culture and treatment

The human bronchial epithelial cell line 16HBE14o- (16HBE) was cultured in Eagle’s minimum essential media (EMEM, Life Technologies Europe BC, Bleiswijk, the Netherlands) supplemented with 10% FCS in collage-coated flasks. Cells were grown to 95-98% confluence and serum or hormone/growth factor deprived overnight before stimulation. Cells were stimulated with 50 g/ml HDM (Greer Laboratories, Lenoir, NC, USA) for 24 hours.

RNA extraction and q-PCR

Total RNA was extracted using Trizol reagent (Invitrogen). 1g RNA was reversely transcribed into cDNA with M-MLV Reverse Transcriptase (Promega). q-PCR was performed with SYBR Premix Ex Taq (Takara) on ABI 7500 fast real-time PCR system (Applied Biosystems). GAPDH mRNA was used as an endogenous control for mRNA.

Western blot analysis

The cells were suspended and lysed in RIPA buffer (Beyotime, Beijing, China) supplemented with protease inhibitor cocktail (Sigma). Protein extractions were separated by SDS-PAGE and transfected to a PVDF membrane (Millipore). The membrane was blocked with 5% (w/v) reagent-grade nonfat milk (Cell Signaling Technology) and incubated with primary antibodies at 4℃ overnight followed by secondary antibody incubation. The protein bands were visualized using ClarityTM Western ECL substrate (Bio-Rad). The protein level was quantified using Image J software normalized with GAPDH.

Histology and immunohistochemistry

Tissues were fixed overnight with 4% neutral formalin. Tissue sections were stained with haematoxylin and eosin for pathological evaluation. Periodic acid-Schiff (PAS) staining was performed using standardized protocols. For KIF3A immunehistological staining, sections were stained with anti-KIF3A antibody, then incubated with HRP-linked-secondary antibody using standardized IHC protocols.

Luciferase reporter assay

The luciferase assays were carried out using the Dual-luciferase Reporter Assay System (Promega, Madison, WI, USA). Briefly, cells were co-transfected with miR-145-5p mimics or miR-control and pMIR-reporter luciferase vector containing a specific sequence of wild-type or mutant KIF3A fragment, using Lipofectamine 2000 (Invitrogen). Cells were collected and lysed for luciferase detection 48 h after transfection. The relative luciferase activity was normalized against to the Renilla luciferase activity.

Differential cell counts in the BALF

Bronchoalveolar lavage was performed and total cell numbers in BALF were determined using a Coulter Counter (IG Instrumenten-Gesellschaft AG, Basel, Switzerland). Differential cell counts were performed on cytospins stained with Diff-Quick solution (Dade Behring, Siemens Healthcare Diagnostics, Deerfield, IL). Percentages of eosinophils and neutrophils were determined within a total count of 200 cells per sample.

Cytokine analysis

The levels of chemokines and inflammatory factors in cell culture supernatants were measured by ELISA. Baseline levels were set to 1, and mean levels (±SD) are shown.

Electric Cell-Substrate Impedance Sensing (ECIS)

Electrical properties of confluent or wounded epithelium were measured using electric ECIS as described previously[19]. Cell adhesion measurements were based on changes in resistance/capacitance to current flow applied at different frequencies (Applied Biophysics, Troy, NY, USA). Cells were inoculated at 75X103 cells/well in 400 l in duplicates and resistance/capacitance was measured at 400 and 40,000 Hz. Wounding was performed by electroporation using voltage pulses of 5 V and 40 kHz for 30s.

Epithelial cell migration assay

Cells were seeded and transfected with miR-145-5p mimics or with KIF3A. The cells were stained at 72 hours after transduction with di-8-ANEPPS (Biotium) diluted 1:500 in culture media. A scratch was made using a p200 pipette tip. The cells were washed with PBS and fed with culture media containing di-8-ANEPPS. A Nikon A1Rsi inverted laser scanning confocal microscope, equipped with a motorized x-y stage, Tokai Hit microplate incubator and Perfect Focus System, was used to document cell migration. Cells were imaged at preselected x-y coordinate points in each well, once every 10 min during a period of 16-22 hours. Cell migration was analyzed using a spot-tracking algorithm on Imaris software (Bitplane).

Statistical analysis

All data are presented as the mean ±SD and derived from at least three independent experiments. Statistical analysis was performed by SPSS 18.0 software (SPSS, Chicago, IL) and GraphPad Prism Software (GraphPad Software, InC., San Diego, CA). For all comparisons, differences were considered significant when P<0.05.

Results

KIF3A downregulation and miR-145-5p upregulation were detected in airway epithelial cells in animals with asthma, while miR-145-5p antagonism improved asthma symptoms significantly.

Mice were treated with HDM (50 g/50 l for the first 3 days, 5 g/50 l for day 14-17, i.n.) to induce asthma. miR-145-5p antagomir was given on day 13, 15, and 17. Mice were sacrificed and studied on day 18. The schematic working flow chart was shown in Figure 1A. HDM induced a strong upregulation of miR-145-5p expression compared with control measured by qRT-PCR (Figure 1B). While miR-145-5p antagomir significantly reduced HDM-induced miR-145-5p upregulation (Figure 1B). In HDM-induced asthma mice model, Kif3a expression was remarkably reduced both at mRNA and protein level (Figure 1C).  Interestingly miR-145-5p antagomir prominently reversed HDM-induced Kif3a downregulation (Figure 1C), suggested HDM-induced Kif3a downregulation was possibly mediated by miR-145-5p. We further studied the effect of miR-145-5p antagomir on Kif3a expression and asthma regulation by histological analysis. Immunochemistry staining revealed that Kif3a protein level was strongly reduced upon HDM induction, while reversed to the normal level when treated with miR-145-5p antagomir (Figure 1D). HDM exposure promoted inflammatory cell infiltration and higher mucus production in lung tissue indicated by H&E and PAS staining respectively (Figure 1D). However, the asthma characters were largely alleviated upon miR-145-5p antagomir treatment (Figure 1D). We then examined the inflammatory response induced by HDM. We found that HDM exposure significantly increased total inflammatory cell, eosinophils, and neutrophils cell numbers (Figure 1E).  Moreover, HDM also markedly elevated cytokine release, including IL-4, Il-5, and IL-13 (Figure 1F). Inhibition of miR-145-5p significantly reduced HDM-induced inflammatory response (Figure 1E and 1F). MiR-145-5p antagomir also remarkably reduced HDM-induced AHR response (Figure 1G). Taken together, miR-145-5p was upregulated and decreased Kif3A expression in mice with asthma, while inhibition of miR-145-5p strongly improved asthma symptoms.

MiR-145-5p regulates KIF3A expression in epithelial cells by direct targeting

We further studied how miR-145-5p regulates KIF3A expression in epithelial cells. MiR-145-5p mimics and inhibitor were transfected to the epithelial cells 16HBE14o- and qRT-PCR results showed they could efficiently increase or inhibit miR-145-5p expression (Figure 2A). Interestingly, miR-145-5p mimics significantly reduced Kif3a mRNA expression while miR-145-5p inhibitor strongly increased Kif3a expression (Figure 2B). The KIF3A protein expression also was decreased by miR-145-5p mimics, while increased upon miR-145-5p inhibitor treatment (Figure 2C). The putative miR-145-5p target binding sequence in Kif3a and its mutant of the binding sites was cloned into luciferase reporter construct (Figure 2D). miR-145-5p significantly reduced the luciferase activity of the reporter with the specific sequence of Kif3a but not that harboring the mutant sequence (Figure 2E), which means miR-145-5p could directly bind to Kif3a and suppressed its expression.

MiR-145-5p promotes HDM-induced release of chemokines and inflammatory factors by regulating KIF3A

Next, we elucidated how miR-145-5p and KIF3A affect HDM-induced immune response. We examined KIF3A overexpression efficiency and the effect of miR-145-5p on KIF3A expression. We found KIF3A overexpression vector greatly increased its protein level and reversed miR-145-5p-mediated KIF3A reduction (Figure 3A). MiR-145-5p mimics significantly increased HDM-induced immune response indicating by the release of chemokines and inflammatory factors, which were remarkably reduced by KIF3A overexpression (Figure 3B), indicating miR-145-5p promoted HDM-induced release of chemokines and inflammatory factors by suppressing KIF3A.

MiR-145-5p regulates HDM-induced epithelium barrier dysfunction and epithelial repair via KIF3A

We further studied the role of miR-145-5p and KIF3A in HDM-induced epithelial cell function. MiR-145-5p mimics significantly reduced cell connection related proteins E-cadherin, -caterin, and Claudin 1 protein levels upon HDM exposure, which were reversed by KIF3A overexpression (Figure 4A and 4B). We then tested whether this protein changes affected epithelial barrier. Epithelial resistance was measured at 400 Hz (by ECIS). We observed that the addition of HDM or miR-145-5p induced a rapid fall in low-frequency resistance, but not high-frequency capacitance (Figure 4C), indicating selective disruption of cell-to-cell adhesion. This temporary effect returned to its original values within 1h, but miR-145-5p mimics significantly postponed the recovery (Figure 4C). KIF3A overexpression alleviated the effect of miR-145-5p on HDM-induced epithelium barrier dysfunction (Figure 4C). To assess the role of miR-145-5p and KIF3A in cell proliferation and migration, we found miR-145-5p mimics suppressed the formation of primary cilia and markedly inhibited cell movement and migration in ‘scratch’ assay (Figure 4D). However, epithelial cells recovered their movement and migration when KIF3A was overexpressed (Figure 4D), indicating miR-145-5p inhibits epithelial repair via regulating KIF3A.

Discussion

Asthma is a common chronic respiratory disease. MiRNAs are emerging as important biomarkers in diseases and also as crucial regulators that are possibly essential in the pathogenesis of many illnesses. In our study, we demonstrated that miR-145-5p was up-regulated in the epithelium of HDM-induced asthma. It was previously shown that epithelial miR-145-5p was upregulated in patients with asthma and chronic obstructive pulmonary disease, and could be a biomarker for early asthma detection [16]. But the mechanism is still unclear. Our study firstly provided that miR-145-5p promoted HDM-induced release of chemokines and inflammatory factors, and regulated epithelium barrier dysfunction and epithelial repair, leading to asthma. The administration of miR-145-5p antagonism remarkably improved asthma symptoms by reducing inflammatory cell infiltration and chemokines release, indicating a therapeutic target for treating asthma. Besides our results, several other studies demonstrated that miRNAs are dysregulated in asthma. It was shown that decreased expression of miR-181b-5p in epithelial and plasma in asthma associated with airway eosinophilic inflammation [20]. In a miRNAs profiling study, miR-1248, miR-26a, Let-7a, and Let-7d were differently expressed in asthmatic patients [21]. Among them, miR-1248 regulates IL-5 expression, while Let-7 microRNA mediates the IL-13 regulation and allergic airway inflammation [5, 21]. In addition, aberrant microRNA-miR-143-3p expression in smooth muscle cells regulates the extracellular matrix protein production and contributes to asthma [22]. These findings collectively provide a key regulatory role for microRNA in asthma.

How miR-145-5p regulates immune response with the pathogenesis of asthma is still unclear. In our study, we further found that miR-145-5p directly targets KIF3A and inhibits expression at both mRNA and protein level. MiR-145-5p binds to 3’-UTR regions of KIF3A. KIF3A, the gene encoding kinesin family member 3A, is a human susceptibility gene associated with atopic dermatitis, rhinitis, and asthma [23, 24]. KIF3A is a component of a trimeric motor complex regulating microtubular function and transport and is required for formation and function of both motile cilia and nonmotile primary and sensory cilia [25, 26]. Genomic deletion of Kif3a in the mouse is embryonically lethal [23, 27, 28]. It was reported that KIF3A and its polymorphisms are associated with aspirin hypersensitivity in asthma [29]. Our data revealed that KIF3A was downregulated in the epithelial cells after HDM induction. KIF3A overexpression reduced HDM-induced immune response indicating by the release of chemokines and inflammatory factors upon miR-145-5p treatment. KIF3A overexpression alleviated the effect of miR-145-5p on HDM-induced epithelium barrier dysfunction and epithelium repair. Previous studies showed that KIF3A deletion resulted in enhanced AHR, goblet cell-associated gene expression, and Th2-mediated eosinophilic inflammation [29, 30]. Loss of Kif3a in airway epithelial cells impairs mucociliary clearance, epithelial repair following injury, and enhances Th2 inflammation that together may influence responses to aeroallergens [31]. Hence, our study present key functions of KIF3A in airway epithelial cells, including epithelial repair and innate immune responses, that may influence airway reactivity and Th2 inflammation.

For the first time, we found why KIF3A was downregulated in the epithelial cells suffering asthma. The main reason is the KIF3A is directly targeted by miR-145-5p, which was found to be up-regulated. Both of which play critical roles in immune response. Our study revealed that miR-145-5p influenced HDM-induced epithelial cytokine release and epithelial barrier dysfunction by regulating KIF3 expression and affect epithelium barrier and epithelial repair, which may lead to and exacerbate the HDM-induced Th2-type immune response in mice. Targeting miR-145-5p or KIF3A would be potential therapeutics for treating asthma.

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Figure legends

Figure 1. KIF3A downregulation and miR-145-5p upregulation were detected in airway epithelial cells in HDM-exposed mice with asthma, while miR-145-5p antagonism improved asthma symptoms significantly. (A) The schematic working flow chart for the experimental design. (B) miR-145-5p expression was measured by qRT-PCR. (C) KIF3a mRNA and protein expression determined by qRT-PCR and western blot respectively. (D) Lung tissue sections were stained with KIF3A IHC staining, H&E, and PAS staining. (E) Total cell numbers of eosinophils and neutrophils were quantified. (F) Levels of cytokines IL-4, Il-5, and IL-3 were measured. (G) Lung resistance is presented as a percentage change over baseline measured in response to inhaled acetylcholine. **P < 0.01, compared with control; ##P < 0.01, #P < 0.05, compared with Ant-scrambled.

Figure 2. MiR-145-5p regulates KIF3A expression in epithelial cells by direct targeting. (A, B) miR-145-5p and KIF3A mRNA expression was measured by qRT-PCR. (C) KIF3A protein expression was studied by western blot and quantified. (D) Sequence alignment of miR-145-5p and KIF3A was shown. (E) miR-145-5p reduced the activity of the luciferase reporter with KIF3A wild-type 3ʻ-UTR but not with the mutant 3ʻ-UTR. **P < 0.01, *P < 0.05, compared with control.

Figure 3. MiR-145-5p promotes HDM-induced release of chemokines and inflammatory factors by regulating KIF3A. (A) KIF3A protein expression was measured by western blot and quantified. *P < 0.05, compared with pCMV vector; ##P < 0.01, compared with mimics control; &&P<0.01, compared with mimics+pCMV vector. (B) The release of CCL20, CCL2, CCL17, CCL11, GM-CSF, IL-33, IL-25, and TSLP were measured by ELISA. **P < 0.01, *P < 0.05, compared with mimics control; ##P < 0.01, #P < 0.05 compared with miR-145-5p mimics.

Figure 4. MiR-145-5p regulates HDM-induced epithelial barrier dysfunction and epithelial repair via KIF3A. (A, B) Epithelial cell connection relative proteins were measured by western blot and quantified. (C) Absolute resistance values prior to (t=0) and 30, 60, 90, 120 min after HDM exposure are shown. (D) Cell migration was observed by videography for 16 hours and quantified. **P < 0.01, *P < 0.05, compared with mimics control; ##P < 0.01, #P < 0.05 compared with miR-145-5p mimics.

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