MiR-20a-5p suppressed TGF-β1-triggered apoptosis of human bronchial epithelial BEAS-2B cells by targeting STAT3

Journal Pre-proof MiR-20a-5p suppressed TGF-β1-triggered apoptosis of human bronchial epithelial BEAS-2B cells by targeting STAT3 Xiuyun Lv, Lihong Wang, Tianji Zhu PII:

S0890-8508(19)30489-X

DOI:

https://doi.org/10.1016/j.mcp.2019.101499

Reference:

YMCPR 101499

To appear in:

Molecular and Cellular Probes

Received Date: 16 December 2019 Accepted Date: 24 December 2019

Please cite this article as: Lv X, Wang L, Zhu T, MiR-20a-5p suppressed TGF-β1-triggered apoptosis of human bronchial epithelial BEAS-2B cells by targeting STAT3, Molecular and Cellular Probes (2020), doi: https://doi.org/10.1016/j.mcp.2019.101499. 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.

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MiR-20a-5p suppressed TGF-β1-triggered apoptosis of human bronchial epithelial

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BEAS-2B cells by targeting STAT3

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Xiuyun Lv, Lihong Wang*, Tianji Zhu

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Department of Respiratory and Critical Care Medicine, The Affiliated Hospital of Inner

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Mongolia Medical University, Hohhot, 010050, P. R. China

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* Corresponding author: Lihong Wang

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Department of Respiratory and Critical Care Medicine, The Affiliated Hospital of Inner

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Mongolia Medical University, No. 1 Tongdao North Street, Hohhot, 010050, P. R. China

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Tel & Fax: 0471-3451168

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E-mail: [email protected]

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Running title: MiR-20a-5p inhibits apoptosis of BEAS-2B cells

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1

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Abstract

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Apoptosis of bronchial epithelial cells contributes to lung diseases, including asthma.

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Although miR-20a-5p is reportedly downregulated in the bronchial epithelia of asthmatic

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patients, its function and mechanism still need to be explored. Here, we explored how

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miR-20a-5p affects human bronchial epithelial cells stimulated with transforming growth

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factor (TGF)-β1. Using qRT-PCR, we observed downregulated miR-20a-5p levels in these

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cells. After transfecting miR-20a-5p mimics or inhibitors into human bronchial epithelium

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BEAS-2B cells, a Cell Counting Kit-8 assay and flow cytometry analysis showed that the

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mimics mitigated suppression of cell viability and acceleration of apoptosis that was triggered

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by TGF-β1, whereas the inhibitors exerted the opposite effects. TGF-β1 induced a decrease in

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expression of Bcl-2 and an increase in expression of Bax, both of which were inhibited by

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miR-20a-5p mimics and further enhanced by miR-20a-5p inhibitors. Further study verified

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that miR-20a-5p targeted the signal transducer and activator of transcription 3 (STAT3) and

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the STAT3 level was inversely related to the miR-20a-5p level. Furthermore, STAT3

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overexpression partly counteracted the miR-20a-5p-induced anti-apoptotic effect in

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TGF-β1-treated BEAS-2B cells. In summary, this study suggested that miR-20a-5p restrained

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apoptosis in TGF-β1-stimulated BEAS-2B cells by targeting STAT3. MiR-20a-5p thus may

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be a novel therapeutic target for asthma treatment.

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Key words: miR-20a-5p; asthma; proliferation; apoptosis; BEAS-2B cells; STAT3

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Introduction

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Bronchial asthma is a common and frequent respiratory disorder and among the most

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important chronic respiratory diseases endangering public health worldwide. Airway

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epithelial cells are essential as a first line of defense against harmful stimuli and external

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damage in the respiratory tract. These cells therefore serve as the initial link to and

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pathological basis of many respiratory diseases, including asthma [1]. A series of studies have

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associated the pathogenesis and clinical manifestations of asthma with changes in the physical

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and biological barriers of airway epithelial cells [2]. Excessive apoptosis of airway epithelial

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cells has been observed in severe asthma [3], suggesting an important mechanism leading to

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airway injury. Therefore, protecting these cells from abnormal apoptosis may prevent the

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development and progression of asthma.

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Transforming growth factor (TGF)-β1 participates in cell growth, proliferation, migration,

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and apoptosis. TGF-β1 promotes apoptosis in podocytes [4], human mesangial cells [5],

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human gingival epithelial cells [6], and airway epithelial cells [7, 8]. Increased TGF-β1 levels

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in the lung tissue have been observed in asthmatic rats [9] and in patients with asthma [10].

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Gagliardo et al. show how TGF-β1 contributes to airway inflammation in childhood asthma

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[11]. Establishing innovative therapeutic tactics to target TGF-β1-enhanced airway epithelial

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cell apoptosis thus is vital for treating asthma.

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Numerous studies have shown that microRNAs (miRNAs) help regulate many biological

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processes, including cell growth and apoptosis [12]. Because MiRNAs have roles in asthma

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progression, they may represent potential therapeutic targets for this condition [13-15].

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Accumulating reports reveal abnormal expression of many miRNAs in the airway epithelial

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cells of asthmatic patients [16, 17]. MiRNAs also regulate the biological function of bronchial

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epithelial cells. For example, in a study of patients with severe asthma, Haj-Salem et al. reveal

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that miR-19a is upregulated in bronchial epithelial cells and that it accelerates cell

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proliferation by targeting TGFβR2 [18]. Huo et al. show that decreased miR-181b-5p in

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epithelial cells is associated with inflammation of airway eosinophils in asthma [19].

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MiR-221 participates in bronchial epithelial cell injury in asthma by targeting SIRT1 [20]. 3

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MiR-20a-5p is decreased in the bronchial epithelia of patients with severe asthma [21]. Yet,

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the potential influence of miR-20a-5p in asthma remains unclear. Thus, this study aimed to

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investigate the effects of miR-20a-5p on TGF-β1-triggered apoptosis of human bronchial

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epithelial cells and to explore its mechanisms, thus providing a theoretical and experimental

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basis for effective asthma prevention strategies.

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Materials and methods

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Cell culture

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Human embryonic kidney (HEK) 293T cells and human bronchial epithelial BEAS-2B cells

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(ATCC, Rockville, MD, USA) were grown in Dulbecco's Modified Eagle's medium

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(Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum and maintained in a 5% CO2

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atmosphere at 37 °C.

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TGF-β1 treatment

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Upon 90% confluence, TGF-β1 (10 ng/mL; Sigma, St. Louis, MO, USA) was added to the

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BEAS-2B cell medium and incubated for 24 hours [8, 22].

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Transfection

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Before transfection, BEAS-2B cells were seeded into 96-well plates. The miR-20a-5p mimics,

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miR-20a-5p inhibitors, and their respective negative controls (GenePharma, Shanghai, China)

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each were transfected into the BEAS-2B cells using Lipofectamine™ 2000 (Invitrogen). After

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24 hours, transfection efficacy was assessed using qRT-PCR. To construct the signal

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transducer and activator of transcription 3 (STAT3) overexpression vectors (pcDNA/STAT3),

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the pcDNA 3.1 vectors containing the full sequence of STAT3 were synthesized by

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GenePharma. The pcDNA/STAT3 then was transfected into BEAS-2B cells using

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Lipofectamine 2000.

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Cell Counting Kit-8 assay

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BEAS-2B cells were seeded into 96-well plates (2×103 cells per well) and incubated for 48

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hours. Then, 10 µL of Cell Counting Kit-8 solution (Beyotime Technology, Jiangsu, China)

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was added to each well. After 2 hours, absorbance at 450 nm was tested. 4

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Flow cytometry assay

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BEAS-2B cells (1×106/mL) were reacted with 5 µL of propidium iodine and 5 µL of

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FITC-linked Annexin-V, both from BD Biosciences (Bedford, MA, USA). Following 15

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minutes of incubation in the dark, apoptotic cells were detected using a flow cytometer (BD

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Biosciences, San Jose, CA, USA).

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Quantitative real-time PCR

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The total RNA of BEAS-2B cells was extracted using TRIzol (Invitrogen). For miR-20a-5p

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quantification, cDNA synthesis was performed using an M-MLV reverse transcriptase kit

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(Promega, Madison, WI, USA). MiRNA was quantified with SYBR Premix ExTaq (Takara,

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Dalian,

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5’-UAAAGUGCUUAUAGUGCAGGUAG-3’

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5’-CUACCUGCACUAUAAGCACUUUA-3’

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5’-TGCGGGTGCTCGCTTCGGCAGC-3’ and reverse 5’-CCAGTGCAGGGTCCGAGGT-3’.

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For STAT3 mRNA quantification, RNA samples were reverse-transcribed into template

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cDNA and then quantified using a SYBR Green Master Mix Kit (Qiagen, Hilden, Germany).

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The

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5’-ATCACGCCTTCTACAGACTGC-3’

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5’-CATCCTGGAGATTCTCTACCACT-3’

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5’-CAGGGCTGCTTTTAACTCTGGTAA-3’

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5’-ACTTGATTTTGGAGGGATCTCGCT-3’. Relative gene expression was calculated using

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the 2-∆∆Ct method.

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Western blot

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Proteins were extracted from BEAS-2B cells and quantified using the BCA method. First, 20

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µg of protein were separated onto SDS-PAGE gels (Invitrogen), transferred to a

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polyvinylidene difluoride membrane (Millipore, Schwalbach, Germany), and then blocked in

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5% fat-free milk. After 1 hour, the membrane was incubated overnight at 4 ºC with the

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primary antibodies STAT3 (1:1000), Bcl-2 (1:1000), Bax (1:1000), and β-actin (1:2000), all

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from Abcam (Cambridge, UK), followed by incubation for 1 hour with secondary antibodies

China).

PCR

primer

The

primers

sequence

were

as

follows:

miR-20a-5p

and and

sequences

were

as

reverse U6

follows: and

and

forward

STAT3

forward reverse

GAPDH and

5

forward

forward reverse

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(1:4000), all from Abcam. Enhanced chemiluminescence (Amersham, Little Chalfont, UK)

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was used to detect the blots, and Image Quant software was used to quantify the blots.

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Dual-luciferase reporter assay

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The wild or mutant sequence of the 3’ untranslated region (UTR) of STAT3 was amplified and

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cloned into the pGL3 luciferase reporter vector (Promega) to create the wide-type plasmid

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(Wt STAT3) or mutant-type plasmid (Mut STAT3), respectively. HEK293T cells were plated

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into 96-well plates, and Wt STAT3 or Mut STAT3 was co-transfected with miR-20a-5p

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mimics or mimic controls into HEK293 cells using Lipofectamine 2000 (Invitrogen). A

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dual-luciferase reporter assay kit (Promega) was used to detect luciferase activity after 48

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hours of transfection.

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Statistical analysis

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All statistical analyses were repeated at least three times for each experiment to collect valid

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data. Data are represented as the mean ± standard deviation and were analyzed using SPSS

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22.0 software (SPSS Inc., Chicago, IL, USA). Differences were determined via one-way

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ANOVA or t test. P<0.05 was considered statistically significant.

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Results

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MiR-20a-5p was downregulated in TGF-β1-treated human bronchial epithelial cells

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MiR-20a-5p expression was 36% lower in BEAS-2B cells stimulated with TGF-β1, compared

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to unstimulated cells. (P<0.05; Fig. 1A), implying that miR-20a-5p might have a crucial role

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in asthma. After transfecting the miR-20a-5p mimics or miR-20a-5p inhibitors into

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TGF-β1-treated BEAS-2B cells (P<0.05; Fig. 1B), we observed that the mimics significantly

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inhibited downregulation of miR-20a-5p and that the inhibitors further downregulated

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miR-20a-5p in these cells (P<0.05; Fig. 1C).

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MiR-20a-5p mediated anti-apoptotic effects in TGF-β1-stimulated BEAS-2B cells

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To elucidate the contribution of miR-20a-5p in BEAS-2B cells, cell viability and apoptosis

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were detected. TGF-β1 significantly decreased cell viability, which was mitigated by the

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miR-20a-5p mimics and aggravated by the miR-20a-5p inhibitors (P<0.05; Fig. 2A). The 6

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mimics suppressed apoptosis of TGF-β1-treated BEAS-2B cells, whereas the inhibitors

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markedly enhanced this effect (P<0.05; Fig. 2B). MiR-20a-5p mimics restrained the

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TGF-β1-induced reduction in Bcl-2 and increase in Bax in BEAS-2B cells (P<0.05; Fig. 2C),

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and the miR-20a-5p inhibitors displayed the opposite effects (Fig. 2C). Our data suggested

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that miR-20a-5p mediated the anti-apoptotic effects in TGF-β1-treated bronchial epithelial

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cells.

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MiR-20a-5p targeted the STAT3 gene

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To identify potential targets of miR-20a-5p for the functional studies in asthma, we performed

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bioinformatic analyses and found binding sites of miR-20a-5p in the 3’-UTR of STAT3 (Fig.

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3A). Co-transfection with the Wt 3’-UTR of STAT3 and miR-20a-5p mimics led to a

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reduction in relative luciferase activity, whereas luciferase activity was unaffected in cells that

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were co-transfected with the Mut 3’-UTR of STAT3 and miR-20a-5p mimics (Fig. 3B).

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Moreover, the miR-20a-5p mimics downregulated STAT3 expression, whereas transfection

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with the miR-20a-5p inhibitors upregulated STAT3 expression in BEAS-2B cells under

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TGF-β1 exposure (Fig. 3C and 3D). As shown in Fig. 3E, miR-20a-5p negatively regulated

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STAT3 protein expression in these cells. Together, these data suggest that miR-20a-5p directly

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modulated STAT3 expression in BEAS-2B cells under TGF-β1 stimulation.

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STAT3 was essential for miR-20a-5p to modulate apoptosis in TGF-β1-treated BEAS-2B

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cells

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To assess whether miR-20a-5p derives its function in TGF-β1-induced apoptosis via STAT3,

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BEAS-2B cells were transfected with the STAT3 overexpression vector and either the

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miR-20a-5p mimics or mimic control. Notably, transfection with the STAT3 overexpression

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vector alleviated the downregulation of STAT3 that was induced by the miR-20a-5p mimics

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(P<0.05, Fig. 4A). STAT3 overexpression also markedly reversed the function of the

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miR-20a-5p mimics on cell viability (Fig. 4B) and apoptosis (Fig. 4C) in TGF-β1-treated

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BEAS-2B cells (P<0.05). Similarly, STAT3 overexpression also counteracted the levels of

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Bax and Bcl-2 that were regulated by the miR-20a-5p mimics (P<0.05; Fig. 4D), indicating

7

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that miR-20a-5p regulated the biofunction of TGF-β1-treated BEAS-2B cells by targeting

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STAT3.

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Discussion

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In this study, we found downregulation of miR-20a-5p in the bronchial epithelial cells of

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patients with asthma and in TGF-β1-stimulated BEAS-2B cells. MiR-20a-5p increased cell

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viability and decreased apoptosis in these cells. Subsequently, our study corroborated that

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miR-20a-5p directly regulated its target gene, STAT3. As a recent study has revealed that

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hsa-miR-20a-5p attenuates allergic inflammation [23], it is worth investigating miR-20a-5p as

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a novel gene target for asthma treatment.

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Asthma is a common chronic illness of the respiratory system. Apoptosis of bronchial

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epithelial cells is a main cause of airway epithelial injury in asthma [24, 25]. Abundant

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evidence implicates miRNAs, which are epigenetic regulators, in the pathophysiology of

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asthma [15, 26, 27]. Thus, targeting specific miRNAs may have therapeutic potential in

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asthma treatment [14].

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Abnormal changes in miRNA expression have been found in airway epithelial cells of

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asthmatic patients [16]. Downregulated miR-20a-5p occurs in the bronchial epithelia of

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patients with severe asthma [21]. TGF-β1, a pro-apoptosis factor, can induce apoptosis in

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human bronchial epithelial cells [7]. Our study analyzed miR-20a-5p expression in

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TGF-β1-stimulated BEAS-2B cells and found that it was markedly decreased, implying a

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crucial role of miR-20a-5p in asthma. Moreover, TGF-β1 triggered apoptosis in BEAS-2B

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cells, which is consistent with Sun et al. [28].

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The Bcl-2 gene family has an essential role in the signal transduction pathway of

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apoptosis, among which Bcl-2 and Bax are the most representative genes that inhibit and

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promote apoptosis in airway epithelial cells [29, 30]. Our study showed that miR-20a-5p

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overexpression increased cell viability and suppressed TGF-β1-triggered apoptosis in

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BEAS-2B cells, accompanied by upregulation of Bcl-2 and reductions in Bax. MiR-20a-5p

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inhibition showed the opposite effects. 8

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To understand the molecular mechanism responsible for the effects of miR-20a-5p in

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TGF-β1-triggered apoptosis, bioinformatic analyses were conducted that identified STAT3 as

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a potential target of miR-20a-5p. Consistent with this prediction, miR-20a-5p was confirmed

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to directly target the STAT3 3’-UTR. STAT3 participates in various cellular processes, such as

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growth and apoptosis [31, 32]. STAT3 also is reported to be a crucial regulator in the

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development and pathogenesis of asthma. STAT3 expression in airway epithelia contributes to

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allergic inflammation in asthmatic mice [33]. Gavino et al. reveal that STAT3 repression

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decreases lung inflammation, suppresses airway remodeling, and reduces Th2/Th17-type cell

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accumulation in a mouse model of asthma [34].

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In this research, the expression of STAT3 was elevated in BEAS-2B cells after TGF-β1

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stimulation, and miR-20a-5p negatively regulated its expression. To verify whether

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miR-20a-5p exerts its effects in BEAS-2B cells by targeting STAT3, a STAT3 overexpression

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vector was transfected into cells. STAT3 overexpression markedly abrogated the regulatory

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function of miR-20a-5p on TGF-β1-triggered apoptosis in BEAS-2B cells. These data suggest

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that miR-20a-5p inhibited TGF-β1-induced apoptosis by downregulating STAT3.

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Our data confirmed miR-20a-5p as an important regulator of TGF-β1-initiated apoptosis

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in human bronchial epithelial cells. Overexpression of miR-20a-5p markedly restrained

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TGF-β1-triggered apoptosis, downregulated Bax levels, and upregulated Bcl-2 levels.

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Furthermore, miR-20a-5p alleviated TGF-β1-triggered apoptosis in BEAS-2B cells by

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targeting STAT3. Limitations of our study included the use of only one cell line and the use of

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an in vitro experiment to explore the role of miR-20a-5p. It is necessary to investigate these

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effects in an in vivo asthma model to support our conclusions. Nevertheless, our results

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indicate that miR-20a-5p might be a novel target for treating asthma.

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Author’s Contributions

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Xiuyun Lv and Lihong Wang disigned the study; Xiuyun Lv conducted experiments and wrote

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the manuscript; Tianji Zhu analyzed experiments results; Lihong Wang revised the paper. All

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authors contributed to and approved the final version of the manuscript.

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Funding

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This work was supported by the Natural science foundation of Inner Mongolia autonomous

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region (grant numbers 2014MS08114).

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Acknowledgments

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None.

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Declaration of interests

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None.

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Figure legends

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Figure 1. MiR-20a-5p level in TGF-β1-stimulated BEAS-2B cells. (A) MiR-20a-5p level in

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BEAS-2B cells after TGF-β1 exposure. After exposure to TGF-β1 (10 ng/ml) for 24 hours, the

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miR-20a-5p level in BEAS-2B cells was detected. N=3, #P<0.05 vs. control. (B) MiR-20a-5p

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expression after transfecting BEAS-2B cells with miR-20a-5p mimics or miR-20a-5p

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inhibitors. N=3, #P<0.05 vs. control. (C) Expression of miR-20a-5p in TGF-β1-stimulated

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BEAS-2B cells after transfection with miR-20a-5p mimics or miR-20a-5p inhibitors. N=3,

350

#

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Figure 2. MiR-20a-5p inhibited TGF-β1-induced apoptosis of BEAS-2B cells. (A) Cell

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viability was measured using a Cell Counting Kit-8 assay. (B) Apoptosis was assessed using

353

flow cytometry. (C) The protein levels of Bax and Bcl-2 were detected using western blot.

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N=3, #P<0.05 vs. control; *P<0.05 vs. TGF-β1.

355

Figure 3. MiR-20a-5p directly targeted the 3’ untranslated region (UTR) of STAT3. (A)

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Predicted interaction between miR-20a-5p and the STAT3 3’-UTR. (B) A dual-luciferase

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reporter assay identified STAT3 as a direct target of miR-20a-5p. #P<0.05 vs. mimic control.

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(C) STAT3 relative mRNA level. N=3, #P<0.05 vs. control. (D) STAT3 relative protein level.

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N=3, #P<0.05 vs. control. (E) Effect of miR-20a-5p on the protein level of STAT3 in

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TGF-β1-induced BEAS-2B cells. N=3, #P<0.05 vs. control; *P<0.05 vs. TGF-β1.

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Figure 4. Restoration of STAT3 expression in BEAS-2B cells counteracted the repression

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of miR-20a-5p on TGF-β1-induced apoptosis. (A) Relative protein level of STAT3. Cells

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were co-transfected with a STAT3 overexpression vector and miR-20a-5p mimics. (B) Cell

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viability in TGF-β1-treated cells. (C) Apoptosis in TGF-β1-treated cells. (D) The protein levels

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of Bax and Bcl-2. N=3, #P<0.05 vs. TGF-β1; *P<0.05 vs. TGF-β1+miR-20a-5p mimics.

P<0.05 vs. control; *P<0.05 vs. TGF-β1.

366 367

15

Highlights 1.MiR-20a-5p is downregulated in in TGF-β1-treated BEAS-2B cells 2.MiR-20a-5p mediates anti-apoptotic effects in BEAS-2B cells 3.MiR-20a-5p targetes STAT3 to modulate apoptosis