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Depletion of miR-380 mitigates human bronchial epithelial cells injury to improve chronic obstructive pulmonary disease through targeting CHRNA4
Journal Pre-proof Depletion of miR-380 mitigates human bronchial epithelial cells injury to improve chronic obstructive pulmonary disease through targeting CHRNA4 Xiujun Wu PII:
S0890-8508(19)30395-0
DOI:
https://doi.org/10.1016/j.mcp.2019.101492
Reference:
YMCPR 101492
To appear in:
Molecular and Cellular Probes
Received Date: 10 October 2019 Revised Date:
4 December 2019
Accepted Date: 4 December 2019
Please cite this article as: Wu X, Depletion of miR-380 mitigates human bronchial epithelial cells injury to improve chronic obstructive pulmonary disease through targeting CHRNA4, Molecular and Cellular Probes (2020), doi: https://doi.org/10.1016/j.mcp.2019.101492. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Depletion of miR-380 mitigates human bronchial epithelial cells injury to improve chronic obstructive pulmonary disease through targeting CHRNA4 The running title: miR-380/CHRNA4 works in COPD Xiujun Wu Department of Respiration, the People’s Hospital in Xintai, Shandong Province Correspondence to: Xiujun Wu, the People’s Hospital, No. 1329 Xinfu Road, in Xintai, Shandong Province, 271200, P. R. China Email: [email protected]
Abstract Background: Chronic obstructive pulmonary disease (COPD) not only causes respiratory damage, but also affects circulatory function, which can be life-threatening in severe cases. Therefore, it is very essential to reveal the molecular mechanism of its pathogenesis. Methods: Human Bronchial Epithelial cells were exposed to 20% cigarette smoke extract (CSE) condition to simulate the cells in COPD patients. GEO database was applied to analyze the expression of miR-380 in COPD patients. The expression level of miR-380 in CSE model was determined with qRT-PCR. Cells proliferation, apoptosis, and inflammation response-related factors were detected with cell counting kit 8, flow cytometry, and western blot, respectively. A correlation between miR-380 and Cholinergic Receptor Nicotinic Alpha 4 subunit (CHRNA4) was predicted with bioinformatics software, and confirmed by dual luciferase assay. Rescue assay was applied to explore further relationship between miR-380 and CHRNA4. Results: miR-380 showed a tendency of high expression in COPD patients and CSE models. Overexpression of miR-380 promoted the inhibitory effect of cells proliferation, and promotion effects of cells apoptosis and inflammation response, which were caused by CSE. CHRNA4, which was lower expressed in COPD patients, was affirmed as a target of miR-380 and negatively modulated by miR-380. Rescue assay indicated that exhausting of CHRNA4 attenuated the moderating effects of miR-380 inhibitor on cells damage induced by CSE. Conclusions: Depletion of miR380 alleviated cells damage caused by CSE through targeting CHRNA4, suggesting that miR-380/CHRNA4 may serve as novel therapeutic targets for COPD treatment. Key words: chronic obstructive pulmonary disease, CHRNA4, miR-380, CSE, apoptosis
1. Introduction Chronic obstructive pulmonary disease (COPD) is a chronic lung disease accompanied by incomplete reversible airflow limitation and inflammation response [1,2], which is usually caused by tobacco exposure [3]. Currently, COPD is the leading cause of mortality and morbidity worldwide due to poor treatment outcomes [4]. Despite years of research, the health care and social burden of COPD have increased and breakthroughs have not yet been made in the treatment of COPD [5]. This is mainly because the potential mechanisms that promote the progression of COPD are not fully understood. Therefore, it is indispensable to explore the underlying molecular mechanisms of COPD. MiRNA, as a 21-23 nucleotides, has been confirmed to be involved in the regulation of various physiological and pathological processes, including COPD. Research from Kara et al. illustrated that differentially expression patterns of miRNAs were exhibited in normal and COPD patients, including miR-29c and miR-126. A recent study stated that miR-29b was connected with the airway inflammation in COPD via targeting bromodomain protein 4 (BRD4) [6]. It has been previously observed that depletion of miR-126 promoted DNA damage response in cigarette smokers and patients with COPD [7]. In addition, data from Conickx et al. suggested that miR-218-5p played a vital role in the pathogenesis of COPD. And miR-145-5p has been reported to be connected with smoke-related COPD through targeting KLF5 [8]. Moreover, miR-212-5p executed a protective effect in COPD [9]. All these information emphasized the importance of miRNAs in COPD. As a novel miRNA, miRNA-380 has very limited reports on its function except for its high expression in acute myocardial infraction [10]. Furthermore, miR-380-5p has been reported to regulate cell viability via suppressing p53, as well as associated with poor prognosis
in mycn-amplified neuroblastoma [11]. However, research about the function of miR380 in COPD was limited. In our research, miR-380 has been confirmed to be highly expressed in COPD patients and cells. Moreover, miR-380 suppressed cells proliferation and promoted cells apoptosis and inflammation response. Cholinergic Receptor Nicotinic Alpha 4 subunit (CHRNA4), which was lower expressed in COPD patients, was affirmed as a target of miR-380 and negatively regulated by miR-380. Depletion of CHRNA4 blocked the protective effects of miR-380 inhibitor on cells damage caused by CSE, indicating that miR-380/CHRNA4 might be potential therapeutic targets for COPD treatment. 2. Materials and methods 2.1 Data acquisition Differentially expression levels of miR-380 and CHRNA4 were determined by analyzing GEO database with accession number GSE61741 (including 94 normal and 47 COPD) and GSE106986 (containing 5 normal and 14 COPD). 2.2 Preparation of cigarette smoke extract (CSE) One cigarette (Yuxi, China) was smoked by a syringe and 500 mL smoke was dissolved into 30 mL serum-free RPMI 1640 medium (Hyclone, Tauranga, New Zealand). The PH was adjusted to 7.4 and the obtained CSE was filtered by a 0.22mm filter as the initial solution for CSE. CSE was prepared freshly before each assay and diluted to 20% as working concentration. 2.3 Cell culture and transfection
Chinese Academy of Science Cell Bank (Shanghai, China) supplied Human Bronchial Epithelial (HBE) cells. RPMI-1640 medium (Hyclone, Tauranga, New Zealand) was applied to culture cells accompanied by 10% fetal bovine serum (Gibco, ThermoFisher Scientific, USA), 100 mg/mL streptomycin, and 100 U/mL penicillin (Sigma, USA) at 37 °C incubator with 5% CO2. After 24 h of culture, the cells were exposed to 20% cigarette smoke extract (CSE) condition for 24 h to simulate the cells in COPD. GenePharma Co. (Shanghai, China) provided all small fragments including miR380
mimic/inhibitor,
negative
GCTCATCGAGTCCATGCATAA-3’),
control and
(NC),
si-CHRNA4 si-con
(5’(5’-
CGAACUCACUGGUCUGACC-3’). All small fragments were transfected into cells by Lipofectamine 2000 reagent (Invitrogen, USA) to alter the expression of miR-380 and CHRNA4. 2.4 RNA extraction and Quantitative Real-time PCR (qRT-PCR) Entire RNA was extracted by TRIzol (Invitrogen, USA), and PrimeScript RT Reagent Kit (Takara, Japan) was applied for reverse transcription of mRNA into cDNA. Then, real-time PCR was carried out on the 7900HT real-time PCR system using SYBR Premix Ex Taq II (TaKaRa, Japan). GAPDH was regarded as an internal standard for mRNA detection. MiScript Reverse Transcription kit (Qiagen) was utilized to inverse transcribe miRNA into cDNA, and MiScript SYBR-Green PCR kit (Qiagen) was used for real-time PCR to detect miRNA expression. U6 was considered as an internal control for miRNA analysis. The relative expression of miR-380 and CHRNA4 were calculated by 2-∆∆Ct method. The sequences of primers synthesized by GenePharma were attached below:
miR-380: F: 5’-GGTTGACCATAGAACATG-3’, R: 5’-GAACATGTCTGCGTATCTC-3’; U6: F: 5’-CTCGCTTCGGCAGCA CATATACT-3’, R: 5’-ACGCTTCACGAATTTGCGTGTC-3’; CHRNA4: F: 5’-CTCCGAGCTCATCTGGCG-3’, R: 5’-GGAGCCGAATTTCATGGTGC-3’; GAPDH: F: 5’-TGAAGGTCGGAGTCAACGGATTTGGT-3’, R: 5’-CATGTGGGCCATGAGGTCCACCAC-3’. 2.5 Cell proliferation detection Cell proliferation was analyzed by cell counting kit 8 (CCK8, Dojindo, Japan). The treated cells were implanted into 96-well plates at the density of 1000 cells/well, and cultured in CO2 incubators. The cell activity was measured at 0 h, 24 h, 48 h, and 72 h. Before each detection, the cells were added into 10 µL of CCK-8 reagent and cultured for 1.5 h. Finally, the optical density (OD) values were measured by microplate reader at the 450 nm wavelength, and the proliferation curve was drawn with GraphPad Prism 5.0.
2.6 Cell apoptosis detection Cell apoptosis was measured by Annexin V/Propidium iodide (PI) double staining. Cells were collected into a centrifuge tube, which was centrifuged at 1000 rpm for 5 min. Pre-cooled PBS was added to suspend the cells, and the cells were centrifuged again. Suspended the cells with 1× binding buffer, and adjusted the cell density to 1-5 × 106 cells/mL. Put 100 µL of cell suspension and 5 µL of Annexin V/FITC (Invitrogen, USA) into 5 mL flow tube, and the mixture was cultured in the dark at room temperature for 5 min. Before detection, 10 µL of PI (Invitrogen, USA) and 400 µL of PBS were added. The results were analyzed by Flowjo software. 2.7 Luciferase reporter assay
The downstream target of miR-380 was predicted by bioinformatics software, and further confirmed by dual luciferase assay. Firstly, wild-type (WT) 3’ UTR of CHRNA4 or mutant (MUT) 3’ UTR of CHRNA4 was cloned into luciferase vector pGL3. Subsequently, miR-380 mimic or miR-380 mimic NC and WT-CHRNA4 or MUT-CHRNA4 were transfected into cells by Lipofectamine 2000 reagent (Invitrogen, USA). The transfected cells were implanted in 96-well plates at a density of 1 × 104 cells/well, and cultured for another 48 h. Finally, collected and lysed the cells, and the fluorescence intensity was measured through dual-luciferase report analysis (Promega, Madison, WI, USA). 2.8 Western blot RIPA lysate including protease inhibitor was used to lyse and extract protein. BCA method was applied to determine protein concentration. 20 µg protein was added into each well of the vertical electrophoresis tank, separated by SDS-polyacrylamide gel
electrophoresis, and transferred to PVDF membrane. Then, the membranes were blocked with 5% non-fat milk for 1 h, and incubated with primary antibodies (Abcam, UK) including CHRNA4 (ab41172, 1:800), IL-10 (ab133575, 1:1000), TNF-α (ab6671, 1:1000), IL-6 (ab178013, 1:1000) at 4 °C over 16 h. The membranes were washed and incubated with secondary antibody for 1 h at room temperature. Eventually, after washing, the membranes were used to develop the signals with the help of ECL (BioVision, USA). GAPDH was regarded as an internal control. QUANTITY ONE software was used to scan the protein bands. The relative expression of each protein was calculated by target protein/internal reference. 2.9 Statistical analysis The experimental data, which were obtained by repeating the experiment at least three times, was processed with SPSS 19.0 and GraphPad Prism 5.0. The difference between two groups was analyzed with student’s t test, and ANOVA was applied to analyze the comparison among multiple groups with post hoc test Dunnett and Bonferroni. P value < 0.05 was regarded as statistically significant. 3. Results 3.1 High expression of miR-380 was observed in COPD patients and CESinduced cells To gain insight into the function of miRNA in the development of COPD, microarray-based analysis was carried out according to the samples from GEO database including 47 COPD patients and 94 normal people. Among all detected miRNAs, miR-380 was selected due to its high expression and limited research in COPD. As shown in Figure 1A, miR-380 exhibited a high expression trend in COPD patients compared with the normal. Subsequently, HBE cells were applied to simulate
the cells in COPD by exposure to 20% CSE condition. The data from Figure 1B indicated that miR-380 was significantly up-regulated in CSE condition. High expression of miR-380 in COPD patients and CSE-induced cells suggested that miR380 has an important relationship with the development of COPD. 3.2 Depletion of miR-380 attenuated cells damage caused by CSE In our study, miR-380 inhibitor and mimic were used to regulate the expression of miR-380. The results from Figure 2A exhibited that the expression of miR-380 could be repressed or promoted by miR-380 inhibitor or mimic transfection. Subsequently, CCK-8, flow cytometry, and western blot were performed to explore functions of miR-380 on CSE-induced cells. After CSE treatment, cells OD values were reduced about 40%, cells apoptosis rate was elevated nearly tripled, as well as the inflammatory response was enhanced accompanied by the increase of TNF-α and IL-6 and decrease of IL-10. These phenomena indicated that CSE treatment can induce cells damage. And overexpression of miR-380 promoted cells damage caused by CSE, while inhibition of miR-380 alleviated cells damage induced by CSE (Figure 2B-F). All data suggested that inhibition of miR-380 attenuated cells damage caused by CSE, manifesting the importance of miR-380 in COPD. 3.3 CHRNA4 functioned as a direct target of miR-380 and was down-regulated in COPD patients Using prediction websites (miRanda, miRWalk, TargetScan, miRDB) to analyze the possible target genes of miR-380, a total of 2128 genes were obtained. According to GEO dataset, 400 differentially expressed genes in COPD patients were acquired, including 222 up-regulated genes and 178 down-regulated genes. The predicted target genes of miR-380 were intersected with down-regulated genes in COPD patients, and
8 genes were obtained. Based on literature review, CHRNA4 was selected as a target of miR-380 for further exploration. Subsequently, dual luciferase assay was performed to detect the relationship between miR-380 and CHRNA4. The binding sites between miR-380 and CHRNA4 were presented in Figure 3A. And the data in Figure 3B exhibited that overexpression of miR-380 reduced the luciferase activity in WT-CHRNA4 group, but has no impact on MUT-CHRNA4 group. Moreover, the data from GEO database indicated that CHRNA4 was significantly down-regulated in COPD patients (n=14) compared with the normal (n=5, Figure 3C). Subsequently, the expression of CHRNA4 at mRNA and protein levels were analyzed after different treatment. The interference efficiency of si-CHRNA4 was presented in Figure 3D. Compared with the control and si-con, the expression of CHRNA4 was profoundly decreased after si-CHRNA4 treatment. In CSE condition, the expression of CHRNA4 was significantly reduced compared with control. However, after CSE+miR-380 inhibitor treatment, the expression of CHRNA4 was remarkably increased compared with the cells in CSE. While, after CSE+miR-380 inhibitor+si-CHRNA4 treatment, the expression of CHRNA4 was declined sharply in contrast with the CSE+miR-380 inhibitor group (Figure 3E-G). All data indicated that CHRNA4, which was lower expressed in COPD patients, was a target of miR-380 and negatively modulated by miR-380, providing the basis for the following experiments. 3.4 Knockdown of CHRNA4 attenuated the remission effects of miR-380 inhibitor on cells damage caused by CSE To explore functions of miR-380/CHRNA4 on COPD, CCK-8 and flow cytometry assays were employed after co-transfected miR-380 inhibitor and siCHRNA4 into CSE-induced cells. The data from Figure 4A showed that the OD values were significantly elevated after knockdown of miR-380 in CSE condition,
however, depletion of miR-380 and CHRNA4 attenuated the cells OD values under same condition. Whilst, the data from flow cytometry indicated that the apoptosis rate of cells reduced from 21.45% to 10.79% after silencing of miR-380 in CSE condition, but the apoptosis rate of cells increased from 10.79 to 20.68 after knockdown of miR380 and CHRNA4 under same condition (Figure 4B-C). The results suggested that knockdown of CHRNA4 inhibited cells proliferation and promoted cells apoptosis, suggesting that silencing of CHRNA4 suppressed the alleviation effects of miR-380 inhibitor on cells damage caused by CSE. 4. Discussion COPD, as a multifactorial disease, is determined by both environment and genetic factors [12]. Currently, few research has successfully uncovered the pathogenetic mechanisms of COPD [13]. In the study, we discovered that miR-380 was significantly up-regulated in COPD patients. Subsequently, HBE cells were exposed to CSE condition to simulate the cells in COPD. Similarly, high expression of miR-380 was observed in CSE-induced cells. And the cells in CSE condition showed decreased proliferation and increased apoptosis. Moreover, overexpression of miR-380 promoted the influences on cells behaviors induced by CSE, and knockdown of miR-380 suppressed the impacts on cells behaviors caused by CSE. Through bioinformatics software, CHRNA4 was predicted as a target of miR-380. Then, the prediction was affirmed by dual luciferase assay. CHRNA4 showed a lower expression level in COPD patients and was negatively modulated by miR-380. Further experiments indicated that silencing of CHRNA4 suppressed the mitigation effects of miR-380 inhibitor on cells behaviors induced by CSE.
As a novel gene expression regulator, miRNA plays a central role in different pathophysiological processes, including COPD [14]. At present, several research indicated that multiple miRNAs were involved in COPD patients [15]. For example, miR-186 has been illustrated to conduce to the pathogenesis of COPD by regulating HIF-1α [16]. Moreover, research from Jiang et al. indicated that miR-190a-5p might regard as a biomarker of diagnosis and prognosis in COPD patients(2018). In our research, miR-380 was picked out for exploration in COPD according to bioinformatics analysis. Meanwhile, miR-380 showed a high expression level in both COPD patients and CSE-induced cells. In addition, overexpression of miR-380 suppressed cells proliferation, and promoted cells apoptosis and inflammation response. While, inhibition of miR-380 accelerated cells growth, and limited cells apoptosis and inflammation response. These findings underscored the importance of miR-380 in COPD. Differently, in neuroblastoma, miR-380 acted as an oncogene by promoting the growth of tumor [11]. Generally speaking, miRNAs regulated gene expression and protein coding through base pairing with the 3’UTR of target mRNAs [17]. On the basis of bioinformatics software prediction and literature analysis, CHRNA4 was selected as a target of miR-380. CHRNA4, which encodes a nicotinic acetylcholine receptor, has been reported to be associated with smoking [18], alcoholism [19], and family epilepsy [20]. In our study, CHRNA4 was affirmed as a target of miR-380, and significantly down-regulated in COPD patients and CES-induced cells. Furthermore, CHRNA4 was illustrated to be negatively regulated by miR-380. And depletion of CHRNA4 suppressed the protective effects of miR-380 inhibitor on cells damage caused by CSE. These data indicated that depletion of miR-380 could attenuate cells damage through targeting CHRNA4.
COPD, as an inflammatory disease, is commonly associated with an abnormal inflammatory response of lungs [21]. Recent studies have exhibited increase in different pro-inflammatory cytokines containing TNF-α and IL-6 [22-24], and decrease in anti-inflammatory IL10 in COPD patients [25]. Importantly, these makers of systemic inflammation were changed in our research. After being treated with CSE, the expression of TNF-α and IL-6 increased, while IL-10 decreased. Whilst, overexpression of miR-380 could strengthen the effects of TNF-α, IL-6, and IL-10 expression, which were caused by CSE. While, knockdown of miR-380 has an opposite result. All data indicated that depletion of miR-380 attenuated CSE-induced cells damage through limiting inflammation response. In summary, depletion of miR-380 alleviated the cells damage caused by CES through targeting CHRNA4, suggesting that miR-380/CHRNA4 might serve as potential molecular targets for the diagnosis and treatment of COPD. Acknowledgments None Declaration of interest statement Author states that there is no conflict of interest.
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Figure legends Figure 1. miR-380 was over expressed in COPD patients and CSE-induced cells. A. Data from GEO database showed that the expression of miR-380 was up-regulated in COPD patients compared with the normal, P < 0.0001. B. The expression of miR380 was increased in HBE cells after being treated with CSE, **P < 0.01. Figure 2. Cells damage induced by CSE was alleviated by miR-380 inhibitor. A. The expression of miR-380 was decreased or increased after transfected with miR380 inhibitor or mimic,
**
P < 0.01 vs. miR-380 mimic NC,
##
P < 0.01 vs. miR-380
inhibitor NC. B. Cells activity was reduced after exposed to CSE condition, and the descending trend of cells activity was promoted or inhibited by miR-380 mimic or inhibitor,
**
P < 0.01 vs. control,
##
P < 0.01 vs. CSE. C. The apoptosis of cells was
increased in CSE condition, and the rising trend of apoptosis was increased or decreased after transfected with miR-320 mimic or inhibitor. D. The apoptosis rate was calculated,
**
P < 0.01 vs. control,
##
P < 0.01 vs. CSE. E. The expression of
inflammatory factors were changed after being treated with miR-380 mimic or inhibitor. F. The statistics of gray value of protein bands, **P < 0.01 vs. control, ##P < 0.01 vs. CSE. Figure 3. CHRNA4 was as a target of miR-380 and down-regulated in COPD patients. A. The sequences of WT-CHRNA4 including miR-380 binding site, MUTCHRNA4 excluding miR-380 binding site and miR-380 were exhibited. B. The luciferase activity was detected after transfected with miR-380 mimic, **P < 0.01 vs. mimic NC. C. The expression of CHRNA4 in COPD patients was analyzed on the basis of GEO database, P = 0.0103. D. The expression of CHRNA4 was significantly decreased after si-CHRNA4 treatment,
**
P < 0.01 vs. control,
##
P < 0.01 vs. si-con.
The mRNA (E) and protein (F) expression of CHRNA4 were evaluated after different treatment, G was the statistic of F, **P < 0.01 vs. control, ##P < 0.01 vs. CSE,
&&
P<
0.01 vs. CSE+miR-380 inhibitor. Figure 4. Silencing of CHRNA4 reduced the protective effects of miR-380 inhibitor on cells damage caused by CSE. A. Cells proliferation was measured after transfected with miR-380 inhibitor and si-CHRNA4. B. Cells apoptosis was detected by flow cytometry after being treated with miR-380 inhibitor and si-CHRNA4. C. The apoptosis rate was calculated, **P < 0.01 vs. control, ##P < 0.01 vs. CSE, vs. CSE+miR-380 inhibitor..
&&
P < 0.01
1. miR-380 was overexpressed in COPD patients and CES-induced cells. 2. Depletion of miR-380 attenuated cell damage caused by CSE. 3. CHRNA4, as a direct target of miR-380, was down-regulated in COPD patients. 4. Knockdown of CHRNA4 promoted the protective effect of miR-380 inhibition on cell injury caused by CSE.
S0890-8508(19)30395-0
DOI:
https://doi.org/10.1016/j.mcp.2019.101492
Reference:
YMCPR 101492
To appear in:
Molecular and Cellular Probes
Received Date: 10 October 2019 Revised Date:
4 December 2019
Accepted Date: 4 December 2019
Please cite this article as: Wu X, Depletion of miR-380 mitigates human bronchial epithelial cells injury to improve chronic obstructive pulmonary disease through targeting CHRNA4, Molecular and Cellular Probes (2020), doi: https://doi.org/10.1016/j.mcp.2019.101492. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Depletion of miR-380 mitigates human bronchial epithelial cells injury to improve chronic obstructive pulmonary disease through targeting CHRNA4 The running title: miR-380/CHRNA4 works in COPD Xiujun Wu Department of Respiration, the People’s Hospital in Xintai, Shandong Province Correspondence to: Xiujun Wu, the People’s Hospital, No. 1329 Xinfu Road, in Xintai, Shandong Province, 271200, P. R. China Email: [email protected]
Abstract Background: Chronic obstructive pulmonary disease (COPD) not only causes respiratory damage, but also affects circulatory function, which can be life-threatening in severe cases. Therefore, it is very essential to reveal the molecular mechanism of its pathogenesis. Methods: Human Bronchial Epithelial cells were exposed to 20% cigarette smoke extract (CSE) condition to simulate the cells in COPD patients. GEO database was applied to analyze the expression of miR-380 in COPD patients. The expression level of miR-380 in CSE model was determined with qRT-PCR. Cells proliferation, apoptosis, and inflammation response-related factors were detected with cell counting kit 8, flow cytometry, and western blot, respectively. A correlation between miR-380 and Cholinergic Receptor Nicotinic Alpha 4 subunit (CHRNA4) was predicted with bioinformatics software, and confirmed by dual luciferase assay. Rescue assay was applied to explore further relationship between miR-380 and CHRNA4. Results: miR-380 showed a tendency of high expression in COPD patients and CSE models. Overexpression of miR-380 promoted the inhibitory effect of cells proliferation, and promotion effects of cells apoptosis and inflammation response, which were caused by CSE. CHRNA4, which was lower expressed in COPD patients, was affirmed as a target of miR-380 and negatively modulated by miR-380. Rescue assay indicated that exhausting of CHRNA4 attenuated the moderating effects of miR-380 inhibitor on cells damage induced by CSE. Conclusions: Depletion of miR380 alleviated cells damage caused by CSE through targeting CHRNA4, suggesting that miR-380/CHRNA4 may serve as novel therapeutic targets for COPD treatment. Key words: chronic obstructive pulmonary disease, CHRNA4, miR-380, CSE, apoptosis
1. Introduction Chronic obstructive pulmonary disease (COPD) is a chronic lung disease accompanied by incomplete reversible airflow limitation and inflammation response [1,2], which is usually caused by tobacco exposure [3]. Currently, COPD is the leading cause of mortality and morbidity worldwide due to poor treatment outcomes [4]. Despite years of research, the health care and social burden of COPD have increased and breakthroughs have not yet been made in the treatment of COPD [5]. This is mainly because the potential mechanisms that promote the progression of COPD are not fully understood. Therefore, it is indispensable to explore the underlying molecular mechanisms of COPD. MiRNA, as a 21-23 nucleotides, has been confirmed to be involved in the regulation of various physiological and pathological processes, including COPD. Research from Kara et al. illustrated that differentially expression patterns of miRNAs were exhibited in normal and COPD patients, including miR-29c and miR-126. A recent study stated that miR-29b was connected with the airway inflammation in COPD via targeting bromodomain protein 4 (BRD4) [6]. It has been previously observed that depletion of miR-126 promoted DNA damage response in cigarette smokers and patients with COPD [7]. In addition, data from Conickx et al. suggested that miR-218-5p played a vital role in the pathogenesis of COPD. And miR-145-5p has been reported to be connected with smoke-related COPD through targeting KLF5 [8]. Moreover, miR-212-5p executed a protective effect in COPD [9]. All these information emphasized the importance of miRNAs in COPD. As a novel miRNA, miRNA-380 has very limited reports on its function except for its high expression in acute myocardial infraction [10]. Furthermore, miR-380-5p has been reported to regulate cell viability via suppressing p53, as well as associated with poor prognosis
in mycn-amplified neuroblastoma [11]. However, research about the function of miR380 in COPD was limited. In our research, miR-380 has been confirmed to be highly expressed in COPD patients and cells. Moreover, miR-380 suppressed cells proliferation and promoted cells apoptosis and inflammation response. Cholinergic Receptor Nicotinic Alpha 4 subunit (CHRNA4), which was lower expressed in COPD patients, was affirmed as a target of miR-380 and negatively regulated by miR-380. Depletion of CHRNA4 blocked the protective effects of miR-380 inhibitor on cells damage caused by CSE, indicating that miR-380/CHRNA4 might be potential therapeutic targets for COPD treatment. 2. Materials and methods 2.1 Data acquisition Differentially expression levels of miR-380 and CHRNA4 were determined by analyzing GEO database with accession number GSE61741 (including 94 normal and 47 COPD) and GSE106986 (containing 5 normal and 14 COPD). 2.2 Preparation of cigarette smoke extract (CSE) One cigarette (Yuxi, China) was smoked by a syringe and 500 mL smoke was dissolved into 30 mL serum-free RPMI 1640 medium (Hyclone, Tauranga, New Zealand). The PH was adjusted to 7.4 and the obtained CSE was filtered by a 0.22mm filter as the initial solution for CSE. CSE was prepared freshly before each assay and diluted to 20% as working concentration. 2.3 Cell culture and transfection
Chinese Academy of Science Cell Bank (Shanghai, China) supplied Human Bronchial Epithelial (HBE) cells. RPMI-1640 medium (Hyclone, Tauranga, New Zealand) was applied to culture cells accompanied by 10% fetal bovine serum (Gibco, ThermoFisher Scientific, USA), 100 mg/mL streptomycin, and 100 U/mL penicillin (Sigma, USA) at 37 °C incubator with 5% CO2. After 24 h of culture, the cells were exposed to 20% cigarette smoke extract (CSE) condition for 24 h to simulate the cells in COPD. GenePharma Co. (Shanghai, China) provided all small fragments including miR380
mimic/inhibitor,
negative
GCTCATCGAGTCCATGCATAA-3’),
control and
(NC),
si-CHRNA4 si-con
(5’(5’-
CGAACUCACUGGUCUGACC-3’). All small fragments were transfected into cells by Lipofectamine 2000 reagent (Invitrogen, USA) to alter the expression of miR-380 and CHRNA4. 2.4 RNA extraction and Quantitative Real-time PCR (qRT-PCR) Entire RNA was extracted by TRIzol (Invitrogen, USA), and PrimeScript RT Reagent Kit (Takara, Japan) was applied for reverse transcription of mRNA into cDNA. Then, real-time PCR was carried out on the 7900HT real-time PCR system using SYBR Premix Ex Taq II (TaKaRa, Japan). GAPDH was regarded as an internal standard for mRNA detection. MiScript Reverse Transcription kit (Qiagen) was utilized to inverse transcribe miRNA into cDNA, and MiScript SYBR-Green PCR kit (Qiagen) was used for real-time PCR to detect miRNA expression. U6 was considered as an internal control for miRNA analysis. The relative expression of miR-380 and CHRNA4 were calculated by 2-∆∆Ct method. The sequences of primers synthesized by GenePharma were attached below:
miR-380: F: 5’-GGTTGACCATAGAACATG-3’, R: 5’-GAACATGTCTGCGTATCTC-3’; U6: F: 5’-CTCGCTTCGGCAGCA CATATACT-3’, R: 5’-ACGCTTCACGAATTTGCGTGTC-3’; CHRNA4: F: 5’-CTCCGAGCTCATCTGGCG-3’, R: 5’-GGAGCCGAATTTCATGGTGC-3’; GAPDH: F: 5’-TGAAGGTCGGAGTCAACGGATTTGGT-3’, R: 5’-CATGTGGGCCATGAGGTCCACCAC-3’. 2.5 Cell proliferation detection Cell proliferation was analyzed by cell counting kit 8 (CCK8, Dojindo, Japan). The treated cells were implanted into 96-well plates at the density of 1000 cells/well, and cultured in CO2 incubators. The cell activity was measured at 0 h, 24 h, 48 h, and 72 h. Before each detection, the cells were added into 10 µL of CCK-8 reagent and cultured for 1.5 h. Finally, the optical density (OD) values were measured by microplate reader at the 450 nm wavelength, and the proliferation curve was drawn with GraphPad Prism 5.0.
2.6 Cell apoptosis detection Cell apoptosis was measured by Annexin V/Propidium iodide (PI) double staining. Cells were collected into a centrifuge tube, which was centrifuged at 1000 rpm for 5 min. Pre-cooled PBS was added to suspend the cells, and the cells were centrifuged again. Suspended the cells with 1× binding buffer, and adjusted the cell density to 1-5 × 106 cells/mL. Put 100 µL of cell suspension and 5 µL of Annexin V/FITC (Invitrogen, USA) into 5 mL flow tube, and the mixture was cultured in the dark at room temperature for 5 min. Before detection, 10 µL of PI (Invitrogen, USA) and 400 µL of PBS were added. The results were analyzed by Flowjo software. 2.7 Luciferase reporter assay
The downstream target of miR-380 was predicted by bioinformatics software, and further confirmed by dual luciferase assay. Firstly, wild-type (WT) 3’ UTR of CHRNA4 or mutant (MUT) 3’ UTR of CHRNA4 was cloned into luciferase vector pGL3. Subsequently, miR-380 mimic or miR-380 mimic NC and WT-CHRNA4 or MUT-CHRNA4 were transfected into cells by Lipofectamine 2000 reagent (Invitrogen, USA). The transfected cells were implanted in 96-well plates at a density of 1 × 104 cells/well, and cultured for another 48 h. Finally, collected and lysed the cells, and the fluorescence intensity was measured through dual-luciferase report analysis (Promega, Madison, WI, USA). 2.8 Western blot RIPA lysate including protease inhibitor was used to lyse and extract protein. BCA method was applied to determine protein concentration. 20 µg protein was added into each well of the vertical electrophoresis tank, separated by SDS-polyacrylamide gel
electrophoresis, and transferred to PVDF membrane. Then, the membranes were blocked with 5% non-fat milk for 1 h, and incubated with primary antibodies (Abcam, UK) including CHRNA4 (ab41172, 1:800), IL-10 (ab133575, 1:1000), TNF-α (ab6671, 1:1000), IL-6 (ab178013, 1:1000) at 4 °C over 16 h. The membranes were washed and incubated with secondary antibody for 1 h at room temperature. Eventually, after washing, the membranes were used to develop the signals with the help of ECL (BioVision, USA). GAPDH was regarded as an internal control. QUANTITY ONE software was used to scan the protein bands. The relative expression of each protein was calculated by target protein/internal reference. 2.9 Statistical analysis The experimental data, which were obtained by repeating the experiment at least three times, was processed with SPSS 19.0 and GraphPad Prism 5.0. The difference between two groups was analyzed with student’s t test, and ANOVA was applied to analyze the comparison among multiple groups with post hoc test Dunnett and Bonferroni. P value < 0.05 was regarded as statistically significant. 3. Results 3.1 High expression of miR-380 was observed in COPD patients and CESinduced cells To gain insight into the function of miRNA in the development of COPD, microarray-based analysis was carried out according to the samples from GEO database including 47 COPD patients and 94 normal people. Among all detected miRNAs, miR-380 was selected due to its high expression and limited research in COPD. As shown in Figure 1A, miR-380 exhibited a high expression trend in COPD patients compared with the normal. Subsequently, HBE cells were applied to simulate
the cells in COPD by exposure to 20% CSE condition. The data from Figure 1B indicated that miR-380 was significantly up-regulated in CSE condition. High expression of miR-380 in COPD patients and CSE-induced cells suggested that miR380 has an important relationship with the development of COPD. 3.2 Depletion of miR-380 attenuated cells damage caused by CSE In our study, miR-380 inhibitor and mimic were used to regulate the expression of miR-380. The results from Figure 2A exhibited that the expression of miR-380 could be repressed or promoted by miR-380 inhibitor or mimic transfection. Subsequently, CCK-8, flow cytometry, and western blot were performed to explore functions of miR-380 on CSE-induced cells. After CSE treatment, cells OD values were reduced about 40%, cells apoptosis rate was elevated nearly tripled, as well as the inflammatory response was enhanced accompanied by the increase of TNF-α and IL-6 and decrease of IL-10. These phenomena indicated that CSE treatment can induce cells damage. And overexpression of miR-380 promoted cells damage caused by CSE, while inhibition of miR-380 alleviated cells damage induced by CSE (Figure 2B-F). All data suggested that inhibition of miR-380 attenuated cells damage caused by CSE, manifesting the importance of miR-380 in COPD. 3.3 CHRNA4 functioned as a direct target of miR-380 and was down-regulated in COPD patients Using prediction websites (miRanda, miRWalk, TargetScan, miRDB) to analyze the possible target genes of miR-380, a total of 2128 genes were obtained. According to GEO dataset, 400 differentially expressed genes in COPD patients were acquired, including 222 up-regulated genes and 178 down-regulated genes. The predicted target genes of miR-380 were intersected with down-regulated genes in COPD patients, and
8 genes were obtained. Based on literature review, CHRNA4 was selected as a target of miR-380 for further exploration. Subsequently, dual luciferase assay was performed to detect the relationship between miR-380 and CHRNA4. The binding sites between miR-380 and CHRNA4 were presented in Figure 3A. And the data in Figure 3B exhibited that overexpression of miR-380 reduced the luciferase activity in WT-CHRNA4 group, but has no impact on MUT-CHRNA4 group. Moreover, the data from GEO database indicated that CHRNA4 was significantly down-regulated in COPD patients (n=14) compared with the normal (n=5, Figure 3C). Subsequently, the expression of CHRNA4 at mRNA and protein levels were analyzed after different treatment. The interference efficiency of si-CHRNA4 was presented in Figure 3D. Compared with the control and si-con, the expression of CHRNA4 was profoundly decreased after si-CHRNA4 treatment. In CSE condition, the expression of CHRNA4 was significantly reduced compared with control. However, after CSE+miR-380 inhibitor treatment, the expression of CHRNA4 was remarkably increased compared with the cells in CSE. While, after CSE+miR-380 inhibitor+si-CHRNA4 treatment, the expression of CHRNA4 was declined sharply in contrast with the CSE+miR-380 inhibitor group (Figure 3E-G). All data indicated that CHRNA4, which was lower expressed in COPD patients, was a target of miR-380 and negatively modulated by miR-380, providing the basis for the following experiments. 3.4 Knockdown of CHRNA4 attenuated the remission effects of miR-380 inhibitor on cells damage caused by CSE To explore functions of miR-380/CHRNA4 on COPD, CCK-8 and flow cytometry assays were employed after co-transfected miR-380 inhibitor and siCHRNA4 into CSE-induced cells. The data from Figure 4A showed that the OD values were significantly elevated after knockdown of miR-380 in CSE condition,
however, depletion of miR-380 and CHRNA4 attenuated the cells OD values under same condition. Whilst, the data from flow cytometry indicated that the apoptosis rate of cells reduced from 21.45% to 10.79% after silencing of miR-380 in CSE condition, but the apoptosis rate of cells increased from 10.79 to 20.68 after knockdown of miR380 and CHRNA4 under same condition (Figure 4B-C). The results suggested that knockdown of CHRNA4 inhibited cells proliferation and promoted cells apoptosis, suggesting that silencing of CHRNA4 suppressed the alleviation effects of miR-380 inhibitor on cells damage caused by CSE. 4. Discussion COPD, as a multifactorial disease, is determined by both environment and genetic factors [12]. Currently, few research has successfully uncovered the pathogenetic mechanisms of COPD [13]. In the study, we discovered that miR-380 was significantly up-regulated in COPD patients. Subsequently, HBE cells were exposed to CSE condition to simulate the cells in COPD. Similarly, high expression of miR-380 was observed in CSE-induced cells. And the cells in CSE condition showed decreased proliferation and increased apoptosis. Moreover, overexpression of miR-380 promoted the influences on cells behaviors induced by CSE, and knockdown of miR-380 suppressed the impacts on cells behaviors caused by CSE. Through bioinformatics software, CHRNA4 was predicted as a target of miR-380. Then, the prediction was affirmed by dual luciferase assay. CHRNA4 showed a lower expression level in COPD patients and was negatively modulated by miR-380. Further experiments indicated that silencing of CHRNA4 suppressed the mitigation effects of miR-380 inhibitor on cells behaviors induced by CSE.
As a novel gene expression regulator, miRNA plays a central role in different pathophysiological processes, including COPD [14]. At present, several research indicated that multiple miRNAs were involved in COPD patients [15]. For example, miR-186 has been illustrated to conduce to the pathogenesis of COPD by regulating HIF-1α [16]. Moreover, research from Jiang et al. indicated that miR-190a-5p might regard as a biomarker of diagnosis and prognosis in COPD patients(2018). In our research, miR-380 was picked out for exploration in COPD according to bioinformatics analysis. Meanwhile, miR-380 showed a high expression level in both COPD patients and CSE-induced cells. In addition, overexpression of miR-380 suppressed cells proliferation, and promoted cells apoptosis and inflammation response. While, inhibition of miR-380 accelerated cells growth, and limited cells apoptosis and inflammation response. These findings underscored the importance of miR-380 in COPD. Differently, in neuroblastoma, miR-380 acted as an oncogene by promoting the growth of tumor [11]. Generally speaking, miRNAs regulated gene expression and protein coding through base pairing with the 3’UTR of target mRNAs [17]. On the basis of bioinformatics software prediction and literature analysis, CHRNA4 was selected as a target of miR-380. CHRNA4, which encodes a nicotinic acetylcholine receptor, has been reported to be associated with smoking [18], alcoholism [19], and family epilepsy [20]. In our study, CHRNA4 was affirmed as a target of miR-380, and significantly down-regulated in COPD patients and CES-induced cells. Furthermore, CHRNA4 was illustrated to be negatively regulated by miR-380. And depletion of CHRNA4 suppressed the protective effects of miR-380 inhibitor on cells damage caused by CSE. These data indicated that depletion of miR-380 could attenuate cells damage through targeting CHRNA4.
COPD, as an inflammatory disease, is commonly associated with an abnormal inflammatory response of lungs [21]. Recent studies have exhibited increase in different pro-inflammatory cytokines containing TNF-α and IL-6 [22-24], and decrease in anti-inflammatory IL10 in COPD patients [25]. Importantly, these makers of systemic inflammation were changed in our research. After being treated with CSE, the expression of TNF-α and IL-6 increased, while IL-10 decreased. Whilst, overexpression of miR-380 could strengthen the effects of TNF-α, IL-6, and IL-10 expression, which were caused by CSE. While, knockdown of miR-380 has an opposite result. All data indicated that depletion of miR-380 attenuated CSE-induced cells damage through limiting inflammation response. In summary, depletion of miR-380 alleviated the cells damage caused by CES through targeting CHRNA4, suggesting that miR-380/CHRNA4 might serve as potential molecular targets for the diagnosis and treatment of COPD. Acknowledgments None Declaration of interest statement Author states that there is no conflict of interest.
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Figure legends Figure 1. miR-380 was over expressed in COPD patients and CSE-induced cells. A. Data from GEO database showed that the expression of miR-380 was up-regulated in COPD patients compared with the normal, P < 0.0001. B. The expression of miR380 was increased in HBE cells after being treated with CSE, **P < 0.01. Figure 2. Cells damage induced by CSE was alleviated by miR-380 inhibitor. A. The expression of miR-380 was decreased or increased after transfected with miR380 inhibitor or mimic,
**
P < 0.01 vs. miR-380 mimic NC,
##
P < 0.01 vs. miR-380
inhibitor NC. B. Cells activity was reduced after exposed to CSE condition, and the descending trend of cells activity was promoted or inhibited by miR-380 mimic or inhibitor,
**
P < 0.01 vs. control,
##
P < 0.01 vs. CSE. C. The apoptosis of cells was
increased in CSE condition, and the rising trend of apoptosis was increased or decreased after transfected with miR-320 mimic or inhibitor. D. The apoptosis rate was calculated,
**
P < 0.01 vs. control,
##
P < 0.01 vs. CSE. E. The expression of
inflammatory factors were changed after being treated with miR-380 mimic or inhibitor. F. The statistics of gray value of protein bands, **P < 0.01 vs. control, ##P < 0.01 vs. CSE. Figure 3. CHRNA4 was as a target of miR-380 and down-regulated in COPD patients. A. The sequences of WT-CHRNA4 including miR-380 binding site, MUTCHRNA4 excluding miR-380 binding site and miR-380 were exhibited. B. The luciferase activity was detected after transfected with miR-380 mimic, **P < 0.01 vs. mimic NC. C. The expression of CHRNA4 in COPD patients was analyzed on the basis of GEO database, P = 0.0103. D. The expression of CHRNA4 was significantly decreased after si-CHRNA4 treatment,
**
P < 0.01 vs. control,
##
P < 0.01 vs. si-con.
The mRNA (E) and protein (F) expression of CHRNA4 were evaluated after different treatment, G was the statistic of F, **P < 0.01 vs. control, ##P < 0.01 vs. CSE,
&&
P<
0.01 vs. CSE+miR-380 inhibitor. Figure 4. Silencing of CHRNA4 reduced the protective effects of miR-380 inhibitor on cells damage caused by CSE. A. Cells proliferation was measured after transfected with miR-380 inhibitor and si-CHRNA4. B. Cells apoptosis was detected by flow cytometry after being treated with miR-380 inhibitor and si-CHRNA4. C. The apoptosis rate was calculated, **P < 0.01 vs. control, ##P < 0.01 vs. CSE, vs. CSE+miR-380 inhibitor..
&&
P < 0.01
1. miR-380 was overexpressed in COPD patients and CES-induced cells. 2. Depletion of miR-380 attenuated cell damage caused by CSE. 3. CHRNA4, as a direct target of miR-380, was down-regulated in COPD patients. 4. Knockdown of CHRNA4 promoted the protective effect of miR-380 inhibition on cell injury caused by CSE.