miR-483-5p plays a protective role in chronic obstructive pulmonary disease
- Authors:
- Published online on: May 18, 2017 https://doi.org/10.3892/ijmm.2017.2996
- Pages: 193-200
Abstract
Introduction
microRNAs (miRNAs or miRs) are a family of small non-coding RNAs which modulate gene expression by binding to complementary sequences of target mRNAs in the coding or non-coding region such as the 3′ untranslated region (3′UTR) and 5′UTR (1). The mature miRNAs cause post transcriptional gene repression by increasing mRNA degradation or by inhibiting translation (2). In the human body, miRNAs play important roles in the responses to injury or adaptation to chronic stress. More and more studies have revealed that specific miRNAs can serve as biomarkers during disease progression and development, such as disorders of the lung, by regulating cell proliferation and differentiation (3–6).
Chronic obstructive pulmonary disease (COPD) is considered as a type of airway disorder and respiratory disease, which is associated with persistent inflammation (7). It may become the third leading cause of death by 2020 globally (8). Usually this type of chronic condition is influenced by a combination of environmental, genetic and epigenetic components and physiological changes. Different signaling pathways and important molecular biomarkers involved in the progression of chronic inflammation in lung disorders have been presented in miRNA studies (9,10). miRNAs, such as miR-218 and miR-128b, are regulators of smoking-induced gene expression alterations in human airway epithelium (11). Scientists have also found that cigarette smoke condensate increases the expression of miR-31 in airway cells (12), and let-7d is involved in idiopathic pulmonary fibrosis (13). Recently the expression of let-7c and miR-125b in sputum samples from patients with COPD was demonstrated to be much lower than levels in healthy controls (14). In lung tissue samples from patients with COPD, high expression of miR-199a-5p and miR-34a is associated with downregulation of HIF-1α protein expression which is important during the progression of COPD (15). We believe that numerous functional miRNAs associated with COPD are still unknown.
Thus, a completed profile of alternative miRNAs in lung samples from COPD patients and healthy donors was detected by microarray analysis in this study. According to the results, 15 miRNAs with high expression and 15 with low expression were selected and validated in vitro using real-time PCR. Finally, miR-483-5p was selected as a study candidate and, importantly, miR-483-5p transfection significantly inhibited the transforming growth factor-β (TGF-β)-mediated decrease in cell proliferation, and α-smooth muscle actin (α-SMA) and fibronectin expression in BEAS-2B and HFL1 cells in vitro. Our results suggest that miR-483-5p plays an important and protective role in patients with COPD.
Materials and methods
Patient characteristics, clinical features and serum harvest
This study was approved by the Third Xiangya Hospital, Central South University (Hunan, China); and an informed consent form (ICF) was provided by each participant. All of the patients gave informed consent to have their tissues banked.
Lung tissue samples from 20 patients were included in this study and were divided into two groups according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification. Ten samples were collected from patients with normal lung function (no COPD, n=10); the rest of the samples were from patients with diagnosed COPD (n=10; stage I/II/III, GOLD classification). Table I summarizes the patient characteristics and clinical features. The diagnosis of emphysema was made by a pathologist based on histological examination, and all of the lung tissue samples collected from patients with COPD had centrilobular emphysema.
Table IDemographic, clinical and biological data of the COPD patients and healthy controls in the miRNA screen study. |
All lung tissue samples were maintained at −80°C until the processing of total RNA isolation.
Chemicals
TGF-β was purchased from Sigma (St. Louis, MO, USA). Other chemicals were commercially available and purchased as reagent grade from Sinopharm (Shanghai, China).
Cell culture and treatment
Human normal pulmonary epithelial BEAS-2B cells obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured in growth media containing Roswell Park Memorial Institute-1640 (RMPI-1640) medium, supplemented with no serum and 1% penicillin-streptomycin (Mediatech, Herndon, VA, USA) at 37°C in a humidified atmosphere of 5% CO2 in air. Within the same culture condition, human normal lung fibroblast HFL1 cells (ATCC) were cultured in growth media containing Ham's F12K medium (F12K).
Before being diluted into single-cell suspensions and seeded in 12-well plates (1×105 cells/ml), the cells were treated with or without TGF-β (1 mg/ml) for 24 h, and then transfected with or without different miRNA mimics. Finally, the cells were harvested for total protein isolation. The cells receiving no treatment served as the negative control group, and cells with TGF-β only treatment served as the positive control group.
Total RNA isolation and reverse transcription
Total RNA from the lung sample short RNAs (<200 bp) was harvested and extracted using an RNA Mini Elute kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions. RNA quality was ascertained using Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). One microgram of total RNA was reverse-transcribed and the product (11 μl) was pre-amplified using Megaplex PreAmp Primers and DBI Bestar® qPCR RT kit (Applied Biosystems, Foster City, CA, USA) in a 20-μl PCR reaction. The pre-amplification cycling conditions were 37°C for 60 min and 98°C for 10 min. The pre-amplified cDNA was diluted with 0.1X TE (pH 8.0) to 10 μl and then 1 μl diluted cDNA was used in each plate for real-time PCR reactions.
miRNA microarray labeling and hybridization
Extracted RNA was quantitated using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and monitored by agarose gel electrophoresis. Then, the samples were labeled and hybridized on Affymetrix GeneChip miRNA arrays 3.0 (Affymetrix, Santa Clara, CA, USA) according to the manufacturer's protocol. Samples were denatured at 99°C for 5 min followed by 45°C for another 5 min, injected into the array chips and hybridization was allowed for 17 h at 48°C in an Affymetrix Hybridization Oven 645 in constant movement at 60 rpm. The raw intensity of the image was read using GenePix Pro V6.0. The intensity of the green signal was calculated after background subtraction, and four replicated spots for each probe on the same slide were averaged. The median normalization method was used to obtain ʻnormalized data' [Normalized data = (foreground − background)/median]. The median was defined as the 50% quantile of miRNA intensity that was >50 in all samples after background correc tion. The statistical significance of the differentially expressed miRNAs was analyzed using the Student's t-test.
Quantitative RT-PCR of mature miRNAs
The solution contained 1 μl of RT product, 5 μl of 2X SYBR®-Green Mix, 0.5 μl of each primer and 3 μl nuclease-free water. The reactions were performed in a 96-well optical plate at 94°C for 2 min, followed by 40 cycles of 94°C for 20 sec, 58°C for 20 sec and 72°C for 20 sec, and the fluorescence signal was collected by ABI PRISM® 7900HT system (Applied Biosystems). All reactions were run in triplicate. All primers used are listed in Table II.
Cell proliferation detection
After TGF-β treatment for 24 h, miR-483-5p mimics and miRNA mimic negative control (NC) (1 μg; GenePharma, Shanghai, China) were transfected into BEAS-2B and HFL1 cells using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA). Then, after transfection for 24, 48 and 72 h, 100 μl Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) solution was added into each well and the plates were incubated in a incubator for 1 h. The absorbance was measured at a wavelength of 450 nm using a microplate reader.
Protein isolation and western blot analysis
To evaluate the target gene expression change in vitro affected by miR-483-5p, the protein extracted from the cells was lysed using RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM PMSF, 1X Roche complete mini protease inhibitor cocktail, Roche PhosSTOP phosphatase inhibitor cocktail) and then determined using BCA kit (Pierce, Rockford, IL, USA) and 20 μg protein lysates were separated on 10% SDS-PAGE gels followed by transfer to nitrocellulose membranes. Western blot analysis was performed as previously described (16), and the signal was visualized using the Odyssey Imaging system (LI-COR Bioscience, Lincoln, NE, USA). Antibodies used in this study included anti-human α-SMA (1:4,000), anti-human fibronectin (1:2,000) and anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:10,000) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).
Data analysis
The miRNA microarray data used the total gene signal, which was proportional to the total number of targets bound by the probes targeting each miRNA. Differentially expressed signals were determined by one-way ANOVA with P<0.01. To compare qPCR-array and microarray assays, the log2 of microarray signals was used.
Real-time PCR assay was used to determine the changes in the expression of the target miRNAs in cells or lung tissue samples. The change in amplification was normalized to the expression of U6 RNA. The fold-change in expression was calculated for each sample using 2−ΔΔCt. 2−ΔΔCt >1.5 or <0.67 was indicative of miRNAs that were differentially expressed.
Results
Differentially expressed miRNAs in COPD patients
A total of 3,000 miRNAs were identified. Among these, 138 miRNAs were differentially expressed with a >2-fold-change in the lung tissues between COPD patients and healthy donors, and 203 miRNAs had significantly downregulated expression (Fig. 1).
Validation of miRNA microarray results in clinical samples by real-time PCR
In order to confirm the results obtained from the miRNA microarray, 15 high-expression candidates and 15 low-expression candidates with at least a 2-fold difference and P<0.05 were randomly selected from the result identified by the microarray, and the expression of these miRNAs was analyzed by real-time PCR. As shown in Fig. 2A, hsa-miR-24-3p, hsa-miR-101-3p, hsa-miR-125a-5p, hsa-miR-30c-5p, hsa-let-7b-5p, hsa-miR-146-5p,hsa-miR-193a-3p, hsa-miR-200c-3p, hsa-miR-140-3p, hsa-miR-22-3p, hsa-miR-195-5p, hsa-miR-16-5p, hsa-miR-141-3p, hsa-miR-4328 and hsa-miR-191-5p were upregulated and hsa-miR-4451, hsa-miR-204-3p, hsa-miR-340-5p, hsa-miR-3611, hsa-miR-665, hsa-miR-483-5p, hsa-miR-4644, hsa-miR-485-3p, hsa-miR-4698, hsa-miR-185-5p, hsa-miR-659-3p, hsa-miR-378a-3p, hsa-miR-3653-3p, hsa-miR-4421, and hsa-miR-663a were downregulated in each lung sample (Fig. 2A), which was consistent with the results from the miRNA microarray (Fig. 2B and C). Among these candidates, we chose miR-483-5p as our target for the following detections.
Effect of miR-483-5p on cell proliferation in vitro
Furthermore, we examined the effects of miR-483-5p on TGF-β-treated BEAS-2B and HFL1 cell proliferation after transfection for 24, 48 and 72 h. As shown in Fig. 3A, miR-483-5p, which was significantly downregulated in COPD samples, exhibited an inhibitory effect against the TGF-β-induced decrease in cell proliferation compared to the negative control group in the BEAS-2B and HFL1 cells (Fig. 3B).
Effect of miR-483-5p on expression of COPD-related proteins
In order to explore the effect of miR-483-5p in COPD, we harvested BEAS-2B and HFL1 cells after TGF-β treatment for 24 h followed by miR-483-5p mimic transfection. As a result, we found that the protein levels of α-SMA and fibronectin were decreased 48 h post-transfection (Fig. 4).
Discussion
COPD is a debilitating lung disease that generally affects older individuals, owing to the duration of smoking (17). miRNAs may be quiescent while lung homeo stasis is maintained after development, but may become perturbed in early states of COPD involving cell differentiation and inflammation (18). In this study, we report that COPD, rather than smoking, has a significant impact on the miRNA expression profile based on miRNA microarray analysis using lung samples from patients with and without COPD. Consistent with the microarray results, expression levels of certain miRNAs were validated in vitro by real-time PCR.
Recently, miR-483-3p has been reported to be involved in the occurrence of many diseases, such as esophageal squamous cell carcinoma (19), cardiomyocyte apoptosis (20) and gastric cancer (21). It has also been shown to be dysregulated and associated with poorer disease-specific survival in various cancers (22–26). Although Song et al (24) reported that miR-483-5p can serve as a negative regulator of lung cancer metastasis suppressors RhoGDI1 and ALCAM, the role of miR-483 in lung disease particularly COPD and the molecular mechanisms by which miR-483 regulates such diseases are still not clear. Meanwhile, Soeda et al (27) also found that miR-483-5p was significantly downregulated in the plasma from COPD patients when compared with normal smokers by TaqMan low-density array screening. Therefore, in order to clarify the role of miR-483-5p in COPD, miR-483-5p was selected as the target in this study. Based on the results of microarray and RT-PCR, the miR-483-5p expression was significantly decreased (~2.5-fold reduction) in COPD compared to the healthy controls.
Cigarette smoke or other inhaled irritants activate epithelial cells to release growth factors, such as TGF-β and fibroblast growth factor (FGF) which induce fibroblast proliferation, resulting in small-airway inflammation and fibrosis (28,29). Studies have shown that myofibroblasts can be transdifferentiated from fibroblasts in vitro by their exposure to the fibrogenic cytokine TGF-β (30-32). Furthermore, others have shown that TGF-β is a potent stimulus for myofibroblast differentiation and induction of pulmonary fibrosis in vivo (33,34). In addition, Burgess et al (35) used TGF-β-treated human lung fibroblasts to study pulmonary myofibroblast differentiation. Therefore, in our study, in order to imitate COPD in vitro, TGF-β treatment was used to induce a similar condition of COPD in BEAS-2B and HFL1 cells. Our results showed that miR-483-5p transfection significantly abrogated the TGF-β-mediated decrease in cell proliferation, and α-SMA and fibronectin expression in BEAS-2B and HFL1 cells. This indicates that the abrogation of TGF-β-decreased cell proliferation by miR-483-5p may be associated with the expression of α-SMA and fibronectin.
α-SMA and fibronectin play important roles during COPD progression. A high molecular weight glycoprotein, fibronectin, is present in the human body as two major isoforms: an insoluble extracellular matrix isomer and a soluble form in the blood (36). The primary function of blood fibronectin is to heal wounds by inducing the reticulo-endothelial system and by mediating cellular adhesion, motility, differentiation, apoptosis and hemostasis (37). This raises the possibility that fibronectin may play an important role in predicting the clinical outcomes in a cohort of patients with mild-to-moderate COPD. Meanwhile, although smooth muscle and endothelial cells also express this marker, α-SMA is still the most commonly used but not a specific marker for myofibroblasts (38-41), whose expression is mainly intracellular also in spindle-shaped cells (42). α-SMA-positive cells are increased in the airways of COPD patients (43), which suggests that most α-SMA-positive cells reveal typical expression profile of myofibroblasts being positive for α-SMA, vimentin and negative for desmin (44). Based on our findings, we hypothesis that, during COPD progression, low expression of miR-483-5p may upregulate the expression of these two important proteins by decreasing the chance of binding them directly. In further research, the molecular mechanism through which miR-483-5p regulates α-SMA and fibronectin needs to be clarified before miR-483-5p can be developed as a potential molecular biomarker for COPD patients.
In conclusion, we found a new miRNA named miR-483-5p with relatively low expression in COPD patients, which may protect human lung cells by promoting cell growth and activating important proteins such as α-SMA and fibronectin. Our findings can provide clues for future functional studies aimed at determining the role of miR-483-5p suppression, as observed in COPD patients, in modulating the adaptive immune balance.
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