Lung myofibroblast transition and fibrosis is regulated by circ0044226

Journal Pre-proof Lung myofibroblast transition and fibrosis is regulated by circ0044226 Lijuan Zhang, Xiaowen Chi, Wen Luo, Shihuan Yu, Jiawen Zhang, Yuening Guo, Qiu Ren, Wei Zhang

PII:

S1357-2725(19)30237-7

DOI:

https://doi.org/10.1016/j.biocel.2019.105660

Reference:

BC 105660

To appear in:

International Journal of Biochemistry and Cell Biology

Received Date:

20 September 2019

Revised Date:

26 November 2019

Accepted Date:

27 November 2019

Please cite this article as: Zhang L, Chi X, Luo W, Yu S, Zhang J, Guo Y, Ren Q, Zhang W, Lung myofibroblast transition and fibrosis is regulated by circ0044226, International Journal of Biochemistry and Cell Biology (2019), doi: https://doi.org/10.1016/j.biocel.2019.105660

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.

Lung myofibroblast transition and fibrosis is regulated by circ0044226

Running title Circ0044226 regulates lung FMT through miR-7

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Lijuan Zhang1, Xiaowen Chi2, Wen Luo1, Shihuan Yu1, Jiawen Zhang1, Yuening Guo1,

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Qiu Ren3, Wei Zhang1, *

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

Pediatrics, The First Affiliated Hospital of Harbin Medical University, Harbin,

Heilongjiang, 150036, China.

Department of Respiratory and Critical Care Medicine, Heilongjiang Hospital Affiliated

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Harbin Medical University, Harbin, Heilongjiang, 150001, China.

*

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to Harbin Institute of Technology, Harbin, Heilongjiang, 150001, China.

Corresponding author: Wei Zhang, MD. Department of Respiratory and Critical Care

Medicine, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, 150001, China.

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Address: NO.23 youzheng street, Nangang District, Harbin, Heilongjiang, 150001, China. TEL: +86 13030052121 E-mail: [email protected]

Abstract Background and purpose: Idiopathic pulmonary fibrosis (IPF) is a life-threatening progressive disease characterized by aberrant fibroblast activation. This study aims to explore the role of the circ0044226 on fibroblast-to-myofibroblast transition (FMT).

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Methods: Bleomycin and TGF-β1 were respectively used to induce the IPF mice model and human lung fibroblasts to myofibroblast differentiation. The mRNA and protein

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levels were examined by qRT-PCR and western blot. Localization of α-SMA was

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evaluated by immunofluorescence staining. Cell viability and proliferation were evaluated by CCK8 and EDU test. Dual-luciferase reporter assay was used to analyze the

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interaction between miR-7 and circ0044226 or sp1. Fluorescence in situ hybridization analysis (FISH) assay was used for the identification of sub-location of circ0044226 and

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miR-7 in cells. The IPF mice model received intratracheal injection of AAV-sh-NC and AAV-sh- circ0044226, and lung fibrosis was detected by HE staining, Masson staining

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and immunohistochemistry assay.

Results: The circ0044226 was upregulated while miR-7 was downregulated in IPF mice model and FMT-derived myofibroblasts. miR-7 was a target of circ0044226 and sp1 was

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a target of miR-7. circ0044226 was distributed mostly in the cytoplasm and functioned as a miR-7 sponge to positively regulate the expression of sp1. Intervention of circ0044226 could ameliorate FMT and suppress fibroblast viability and proliferation by functioning as an endogenous miR-7 sponge. Conclusion: Circ0044226 knockdown alleviates fibroblast proliferation and FMT by functioning as a competing endogenous RNA, which may represent a promising therapy

for pulmonary fibrosis.

Keywords: Circ0044226; fibroblast-to-myofibroblast transition; miR-7; pulmonary

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fibrosis.

1. Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease characterized by the aberrant accumulation of fibrotic tissue in the lungs parenchyma. Fibroblasts are tissue mesenchymal cells committed to re-establish a normal and wellstructured extracellular matrix (ECM) in wound healing repair process. During IPF

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pathogenesis, both lung and fibrocytes-derived fibroblasts are persistently exposed to profibrotic mediators secreted by activated fibroblasts, leading to ECM production and

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trans-differentiation to myofibroblasts. The presence of myofibroblasts in fibroblast foci,

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which is one of the histopathological hallmarks of pulmonary fibrosis, can be induced by inflammatory cytokines and chemokines such as transforming growth factor-β1 (TGF-β1),

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which is a key mediator of pulmonary fibrosis and other fibrotic diseases. This process, known as TGF-β1-induced fibroblast-to-myofibroblast transition (FMT), results in

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abnormal hyperplasia within collagen in lung interstitial tissues (Sgalla, 2018, Zhang Q, 2019). FMT is a complex pathophysiological process whereby fibroblasts lose their

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specific markers and acquire myofibroblastic phenotype and express mesenchymal cell products such as α-smooth muscle actin (α-SMA) and collagen (Piera-Velazquez S, 2016). Circular RNA (circRNA), a novel class of endogenous non-coding RNA, is

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characterized by their covalently closed-loop structures without a 5’cap or a 3’Poly A tail. Emerging evidence has shown that circRNA is related to fibrosis activity and can potentially be a monitoring factor for fibrosis or, more excitingly, can be a target for treatment (Yao J, 2018). Recent research has shown that circRNAs may function as potential molecular markers for pulmonary fibrosis diagnosis and treatment (Li R, 2018, Yang L, 2019), and play an important role in the initiation and progression of human

pulmonary fibrosis, such as circRNA-012091 (Cheng Y, 2019), CDR1as (Yao W, 2018), circHECTD1 (Fang S, 2018) and circHIPK3 (Zhang JX, 2019). It has been reported that circ0044226 (named circRNA_102100) was significantly upregulated in IPF patients (Li R, 2018). However, whether circ0044226 participates in mediating the biological function of fibroblast remains unclear. miRNA is a small non-coding RNA molecule that is involved in regulating gene

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expression by binding to the 3’UTR of target mRNAs. Yao et al (Yao W, 2018) have

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reported that miR-7 was decreased in the lung tissues of silica-induced mouse pulmonary fibrosis, indicating that miR-7 may function as a regulator of pulmonary fibrosis.

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Bioinformatics prediction using Circular RNA Interactome database shows that miR-7 is

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a common miRNA that potentially binds to circ0044226. Sp1, an important profibrogenic mediator (Grzegorzewska AP, 2017) and a transcription factor for TGF-β1 (Si-Si, 2015),

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is found to contain potential binding sites of miR-7. Thus, we speculated that circ0044226 might promote progression of human pulmonary fibrosis via miR-7/sp1 axis.

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In this study, we utilized a bleomycin (BLM)-induced IPF mice model and TGF-β1treated human lung fibroblast as in vitro model of pulmonary fibrosis, and characterized the expression of circ0044226 and investigated for the first time its role in fibroblast proliferation and pulmonary FMT. We revealed that silencing circ0044226 could result in

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inhibition of fibroblast proliferation and differentiation into myofibroblasts in vivo and in vitro. Intervention of circ0044226 may represent a promising therapy for pulmonary fibrosis.

2. Materials and methods

2.1 Mice pulmonary fibrosis model and circ0044226 shRNA intratracheal injection C57BL/6 mice with a mean weight of 20 g were divided into two groups: saline group (control) and bleomycin (BLM) group. Model mice under anesthesia were injected with BLM sulfate (3 mg/kg) intratracheally. Control mice were treated with saline. For AAV production, shRNA3 or scrambled sequences were inserted into AAV vector. C57BL/6 mice under anesthesia were intratracheally administered about 30 μL (2.34 ×

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1013 viral particles/mL) AAV6 containing circ0044226 shRNA or scrambled shRNA.

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After 3 weeks, mice were challenged with BLM for pulmonary fibrosis experiments. On Day 28 after BLM treatments, the mice were sacrificed for experiments. Parts of lung

subsequent

quantitative

real-time

PCR

(qRT-PCR),

western

blot

and

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for

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lobes were fixed in 4% paraformaldehyde for histopathologic analyses, parts were frozen

immunofluorescent studies. Lung microsections were stained with Masson staining to

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visualize fibrotic lesions. The animal experiments were performed in accordance with the Animal Ethics Committee of The First Affiliated Hospital of Harbin Medical University.

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2.2 In vitro model of pulmonary fibrosis

FMT-derived myofibroblasts are the major source of ECM proteins during pathological matrix remodeling in pulmonary fibrosis. Importantly, persistent TGF-β signaling is the primary mechanism of FMT and fibrosis. Also, TGF-β1 is the most potent

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profibrotic cytokine known to mediate the process of pulmonary fibrosis by accumulation of fibroblast and FMT. The human lung fibroblasts (WI-38 cells) were incubated with 10 ng/mL TGF-β1 for 48 h to simulate an in vitro model of pulmonary fibrosis. 2.3 qRT-PCR analysis Total RNA obtained from lung tissues or cells was extracted by Trizol reagent. The

reverse transcription kit (Takara, Nanjing, China ) was used to reverse-transcribe RNA to cDNA. The SYBR Green PCR kit was used for circ0044226, miR-7 and sp1 amplification. qRT-PCR analysis was performed with ABI 7900HT Real-Time PCR system (Applied Biosystems). The expression levels of target genes were normalized to GAPDH expression levels by using the 2-ΔΔCT method. The primers were used as follows: circ0044226 forward, 5’-TCTATTAGGGCATGAGTTTGTCTT-3’ and reverse, 5’-

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TCCTTGGTTGTGGAGCTGTC-3’; miR-7 forward, 5’-TGGAAGACTAGTGATTTTG-

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3’ and reverse, 5’- GAACATGTCTGCGTATCTC-3’; Sp1 forward, 5’-TTGAAAAAG GAGTTGGTGGC-3’; reverse, 5’-TGCTGGTTCTGTAAGTTGGG-3’; GAPDH forward, and

reverse,

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5’-GCACCGTCAAGGCTGAGAAC-3’

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TGGTGAAGACGCCAGTGGA-3’.

5’-

2.4 Western blot analysis

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Total proteins were extracted from lung tissues or cells using RIPA buffer, separated by 10% SDS-PAGE and PVDF membranes (Millipore, Bedford, MA, USA). After

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blocking the nonspecific binding sites with 5% skim milk, the membranes were incubated with primary antibody against α-SMA (1:500 dilution), Collagen type I (COL-1, 1:500 dilution), sp1 (1:1000 dilution) and GAPDH (1:1000 dilution; Abcam) overnight at 4°C, followed by another 1h of incubation with HRP-conjugated secondary antibody. Protein

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quantification was examined by using an ECL reagent (Beckman Coulter, Brea, CA, USA).

2.5 Immunofluorescence assay Lung microsections or cells were fixed with 4% carbinol, washed with PBST, and blocked with BSA (Phygene, USA). Then, they were incubated overnight at 4°C with

anti-α-SMA antibody (Abcam, ab32575, 1:500). At last, after washed with PBST, the lung microsections were incubated with anti-rabbit antibody labeled with Cy3 or Alexa 488 for α-SMA staining and DAPI (Beyotime, Shanghai, China, 1:200) for nuclear staining. Cell imaging was acquired through Fluoview 300 confocal laser scanning microscopy (Olympus, Tokyo, Japan). 2.6 Cell proliferation

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Cell proliferation was performed using CCK-8 assay and 5-Ethynyl-2’-

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Deoxyuridine (EDU) (Ribibio, Guangzhou, China) assay. After treatment, 10 μL of CCK8 was added to the WI-38 cells, and the optical density was measured at 490 nm. For

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EDU assay, the WI-38 cells were incubated with 50 nM EDU for 2 h. The proliferating

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cells were fixed with 4% paraformaldehyde and incubated with Apollo Dye Solution. Cell nuclei were stained with DAPI. We observed the cells using a fluorescence

2.7 Luciferase assay

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microscope.

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The 3’-UTR or mutant 3’-UTR of sp1, and the wide type or mutant circ0044226 were separately inserted into the downstream of the luciferase gene in the pmirGLOcontrol vector. The HEK-293T cells were co-transfected with circ0044226/sp1 plasmids, miR-7 mimics, and negative control mimics at appropriate concentration. The luciferase

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activity was detected using a dual-luciferase reporter assay system. For comparison, the Firefly luciferase activity was normalized to Renilla luciferase activity. 2.8 Fluorescence in situ hybridization analysis (FISH) FITC-labeled RNA probes to circ0044226, Cy3-labelled miR-7 were designed and synthesized by GenePharma (Shanghai, China). Signals were detected by Fluorescent in

situ Hybridization Kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. The images were acquired under a laser confocal microscope (Leica TCS SP8, Germany). 2.9 Hematoxylin and eosin (HE) and Masson staining The lung tissues were dehydrated in gradient alcohol and treated with xylene. The tissues were embedded with paraffin and sliced (4 μm). For HE staining, after treated on

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40°C platform to remove paraffin, the slices were stained with hematoxylin for few

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minutes. The 70% and 90% ethanol were used to dehydrate. In the end, the slices were treated with eosin for several minutes and examined under a light microscope. For

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Masson staining, the paraffin slices were dewaxed and washed. The slices were stained

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using hematoxylin. After being washed, the sliced were re-stained by Masson ponceaux. The slices were treated with 2% glacial acetic acid and 1% phosphomolybdic acid. Then,

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2.10 Statistical analysis

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aniline blue was used to stain and dehydrated. In the end, the slices were stained with

Values were expressed as the mean ± standard deviation. Student's t‑test was used for comparison between two groups. P < 0.05 was considered as significantly different.

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3. Results

3.1 Circ0044226 was upregulated in pulmonary fibrosis mice model and FMTderived myofibroblasts. The pulmonary fibrosis

mouse model was

established by intratracheal

administration of BLM. Compared to control mice, circ0044226 was significantly higher

in the lung of BLM-induced mice (Figure 1A). As expected, BLM caused increased myofibroblast accumulation, such as the increased markers of myofibroblast including αSMA and COL-1 (Figure 1B and 1C). WI-38 cells treatment with 10 ng/mL TGF-β1 for 48 h were used as an in vitro pulmonary fibrosis model. We found that circ0044226 was also significantly upregulated in TGF-β1-treated cell (Figure 1D), and TGF-β1 caused increased expression of α-SMA and COL-1(Figure 1E and 1F), and induced WI-38 cell

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viability and proliferation (Figure 1G and 1H).

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In addition, the miR-7 expression level was downregulated in both BLM-induced mice and TGF-β1-treated WI-38 cells (Figure 2A and 2B). These data revealed that the

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model and FMT-derived myofibroblasts.

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expression trend of miR-7 was opposite with circ0044226 in pulmonary fibrosis mice

3.2 Circ0044226 regulated FMT and fibroblast proliferation in vitro.

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To investigate the effect of circ0044226 on FMT and fibroblast proliferation, a siRNA targeting the sequence of circ0044226 was designed. As shown in Figure 2C, si-

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circ0044226 transfection significantly downregulated circ0044226 expression. We also found that circ0044226 silencing increased TGF-β1-inhibited miR-7 expression and attenuated TGF-β1-induced expression of α-SMA and COL-1 (Figure 2D, 2E and 2F). CCK-8 assays revealed that circ0044226 silencing significantly decreased the viability of

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WI-38 cells under TGF-β1 treatment (Figure 2G). EDU assays revealed that circ0044226 silencing significantly reduced TGF-β1-induced fibroblast proliferation (Figure 2H). These results suggested that circ0044226 was essential for the activation of TGF-β1induced FMT and fibroblast proliferation. 3.3 Circ0044226 functioned as a miR-7 sponge

Bioinformatics prediction using Circular RNA Interactome database shows that miR-7 is a common miRNA that potentially binds to circ0044226. circ0044226 cDNA was cloned into the downstream of luciferase gene (Luc-circ0044226) and co-transfected into HEK-293T cells with miR-7 mimics or negative control mimic (mimic NC). The activity of Luc-circ0044226 was significantly reduced by miR-7 mimic, but not by mimic NC (Figure 3A). miR-7 mimic transfection had no effect on the activity of Luc-Mut.

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FISH assays revealed that circ0044226 and miR-7 were co-localized in the cytoplasm of

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TGF-β1-treated WI-38 cells (Figure 3B). These results implied that circ0044226 may function as a sponge of miR-7.

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Sp1, an important transcription factor for TGF-β1, was found to contain potential

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binding sites of miR-7. Then a luciferase reporter plasmid containing 3’UTR region of sp1 mRNA was constructed for the luciferase assay. The results showed that the

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translational activity of sp1 3’UTR was significantly suppressed by miR-7 mimic. However, the mutant sp1 plasmid was not affected (Figure 3C). These results implied that

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sp1 was a target of miR-7.

3.4 Circ0044226 regulated FMT and fibroblast proliferation by acting as a miR-7 sponge

Since miR-7 was sponged by circ0044226, we then determined the role of miR-7 in

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vitro. miR-7 mimic transfection could repress TGF-β1-induced protein expression of αSMA and COL-1, whereas the repression was abrogated by circ0044226 overexpression (Figure 4A and 4B). These results indicated circ0044226/miR-7 interaction was involved in regulating TGF-β1-induced FMT. As we have proved that sp1 was a target of miR-7, the sp1 mRNA and protein

expression were detected by qRT-PCR and western blot. miR-7 mimic transfection could repress TGF-β1-induced expression of sp1, whereas the repression was abrogated by circ0044226 overexpression (Figure 4C and 4D). Moreover, we found that miR-7 mimic transfection could repress TGF-β1-induced fibroblast viability and proliferation, whereas the repression was abrogated by circ0044226 overexpression (Figure 4E and 4F). These results revealed that circ0044226/miR-7/sp1 network was involved in regulating FMT

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and fibroblast proliferation.

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3.5 Circ0044226 silencing attenuated BLM-induced pulmonary fibrosis in vivo.

To further explore that effect of circ0044226 in pulmonary fibrosis, the adeno-

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associated viral shRNA for circ0044226 silencing was designed and intratracheally

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injected into BLM-induced pulmonary fibrosis mice model. BLM significantly induced TGF-β1 expression, but circ0044226 shRNA inhibited it (Figure 5A). Also, circ0044226

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shRNA significantly reduced lung circ0044226 expression (Figure 5B) and sp1 expression (Figure 5D and 5E). In contrast, injection of circ0044226 shRNA significantly

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increased the BLM-inhibited miR-7 expression (Figure 5C). The HE staining was used to evaluate histopathological changes of lung tissue. As shown in Figure 5F, the control group showed clear alveolar space structure and alveolar interval thickness, while the alveolar interval thickness and alveolar structure was significantly damaged in BLM

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group, suggesting a successful pulmonary fibrosis model. Compared with model group, the alveolar space structure, alveolar interval thickness and inflammatory cell infiltration were significantly improved in BLM + sh-circ0044226, suggesting that circ0044226 interference reduced the degree of pulmonary interstitial fibrosis induced by BLM in mice. Meanwhile, circ0044226 shRNA attenuate collagen deposition, as demonstrated by

Masson’s staining (Figure 5G and 5I). As initially expected, circ0044226 shRNA also decreased myofibroblast accumulation, as α-SMA and COL-1 (Figure 5H and 5J). Collectively, these results indicated that circ0044226 silencing attenuated BLM-induced pulmonary fibrosis in vivo. 4. Discussion Pathological injury of pulmonary fibrosis is characterized by alveolar interstitial

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inflammatory cell infiltration, fibroblast proliferation and alveolar interstitial fibrous

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connective tissues deposition. This process eventually results in the loss of lung elasticity and alveolar surface area, leading to the impairment of gas exchange and lung function.

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Myofibroblasts are the main effector cells in IPF (Salton F, 2019). Myofibroblasts have

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the characteristics of both fibroblasts and smooth muscle cells, which could synthesize and secrete extracellular matrix. Pulmonary myofibroblasts may be derived from a

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variety of sources, including fibroblasts, epithelial cells, and bone marrow-derived circulating fibroblasts, among which fibroblasts are considered to be the main source of

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myofibroblasts. Therefore, interfering with myofibroblast generation by regulating fibroblasts is an important direction for the treatment of IPF. Several studies have shown that circRNAs are aberrantly expressed in IPF patients and implicated in IPF development. For instance, circRNA-012091, CDR1as,

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circHECTD1 and circHIPK3 (Cheng Y, 2019, Fang S, 2018, Yao W, 2018, Zhang JX, 2019). Li et al (Li R, 2018) have reported that circ0044226 was significantly upregulated in IPF patients compared with the healthy controls. In this study, circ0044226 was found to be significantly increased in BLM-induced IPF mice and TGF-β1-induced WI-38 cell, and the further experiments showed that circ0044226 silencing significantly reduced

TGF-β1-induced FMT and fibroblast proliferation, which indicated that circ0044226 may play an important role in the initiation and progression of human pulmonary fibrosis. Next, the mechanism of circ0044226 on regulating FMT and fibroblast proliferation was studied in this paper. circRNA functions mainly as the miRNA sponge, competitively binding to miRNAs to regulate target gene expressions. As confirmed by the luciferase reporter gene assay and FISH assay, we found that circ0044226 could function as a miR-

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7 sponge. The miR-7 expression was reported to be decreased in the lung tissues of silica-

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induced mouse pulmonary fibrosis (Yao W, 2018), and we also observed the downregulated miR-7 in BLM-induced IPF mice and TGF-β1-induced WI-38 cell, which

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was consistent with the previous study. The sp1 is a transcription factor involved in

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processes of pulmonary fibrosis, and TGF-β1 can be activated by the transcriptional factor Sp1(Grzegorzewska AP, 2017, Martin-Gallausiaux C, 2018). Here, utilizing the

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luciferase reporter gene assay, we found that the translational activity of sp1 3’UTR was significantly suppressed by miR-7 mimic, demonstrating sp1 was a target of miR-7. And

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miR-7 mimic transfection could repress TGF-β1-induced expression of sp1, whereas the repression was abrogated by circ0044226 overexpression, implying that circ0044226 can act as molecular sponge of miR-7 to regulate sp1 expression. In addition, miR-7 mimic transfection could repress TGF-β1-induced FMT and fibroblast proliferation, whereas the

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repression was abrogated by circ0044226 overexpression, suggesting circ0044226/miR7/sp1 network was involved in regulating FMT and fibroblast proliferation. Finally, the BLM-induced pulmonary fibrosis mice were treated by circ0044226 shRNA, and the results showed that circ0044226 silencing attenuated BLM-induced pulmonary fibrosis. Epithelial mesenchymal transition (EMT) appearing on the airway epithelial cell

plays an essential role in myofibroblast accumulation and the formation and development of IPF (Lekkerkerker et al. , 2012, Xiong et al. , 2017). miR-7 has been shown to negatively regulate EMT of human bronchial epithelial cells (Yao et al. , 2018). In contrast, sp1 was shown to contribute to EMT in the epithelial cells (Chen et al. , 2017, Yu et al. , 2014). Thus, we may speculate that, the anti-fibrotic efficacy of circ0044226 knockdown could also be attributed to other possible mechanisms such as EMT, which

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might be mediated by miR-7 and sp1.

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In conclusion, our study demonstrates circ0044226 is upregulated in BLM-induced pulmonary fibrosis mice model and FMT-derived myofibroblasts. circ0044226 silencing

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can ameliorate FMT and suppress fibroblast proliferation in vivo and in vitro.

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circ0044226 regulates FMT by functioning as an endogenous miR-7 sponge to increase

Authors' contributions

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sp1 expression.

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WZ conceived and designed the analysis. LZ, XC, WL, SY, JZ, YG, QR and WZ collected the data. LZ, XC, WL and SY contributed data or analysis tools. JZ and YG performed the analysis. LZ wrote the paper. All authors read and approved the

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manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Competing interests All the authors declare no conflict of interest.

Reference

of

Chen SJ, Wu P, Sun LJ, Zhou B, Niu W, Liu S, et al. miR-204 regulates epithelial-

ro

mesenchymal transition by targeting SP1 in the tubular epithelial cells after acute kidney injury induced by ischemia-reperfusion. Oncology reports. 2017;37:1148-58.

-p

Cheng Y LW, Li Z,Cao M,Zhu Z,Han C,Dai X,Zhang W,Wang J,Yao H,Chao J.

re

CircRNA-012091/PPP1R13B-Mediated Lung Fibrotic Response in Silicosis via ER Stress and Autophagy. American journal of respiratory cell and molecular biology.

lP

2019;undefined:undefined.

Fang S GH, Cheng Y,Zhou Z,Zhang W,Han B,Luo W,Wang J,Xie W,Chao J.

ur na

circHECTD1 promotes the silica-induced pulmonary endothelial-mesenchymal transition via HECTD1. Cell death & disease. 2018;9:396. Grzegorzewska AP SF, Han R,Czajka CA,Makino K,Stawski L,Isenberg JS,Browning JL,Trojanowska M. Dimethyl Fumarate ameliorates pulmonary arterial hypertension and

Jo

lung fibrosis by targeting multiple pathways. Scientific reports. 2017;7:41605. Lekkerkerker AN, Aarbiou J, van Es T, Janssen RA. Cellular players in lung fibrosis. Current pharmaceutical design. 2012;18:4093-102. Li R WY, Song X,Sun W,Zhang J,Liu Y,Li H,Meng C,Zhang J,Zheng Q,Lv C. Potential regulatory role of circular RNA in idiopathic pulmonary fibrosis. International journal of molecular medicine. 2018;42:3256-68.

Martin-Gallausiaux C B-CF, Marinelli L,Jamet A,Ledue F,Blottière HM,Lapaque N. Butyrate produced by gut commensal bacteria activates TGF-beta1 expression through the transcription factor SP1 in human intestinal epithelial cells. Scientific reports. 2018;8:9742. Piera-Velazquez S MF, Jimenez SA. Endothelial to Mesenchymal Transition (EndoMT) in the Pathogenesis of Human Fibrotic Diseases. Journal of clinical medicine.

of

2016;5:undefined.

ro

Salton F VM, Confalonieri M. Epithelial⁻ Mesenchymal Transition in the Pathogenesis of Idiopathic Pulmonary Fibrosis. Medicina (Kaunas, Lithuania). 2019;55:undefined. G,

Iovene,

Bruno,Calvello,

Mariarosaria,Ori,

-p

Sgalla

Margherita,Varone,

Respiratory Research. 2018;19:32.

re

Francesco,Richeldi, Luca. Idiopathic pulmonary fibrosis: pathogenesis and management.

lP

Si-Si Y, Jiang-Lin, Tan,Dai-Song, Liu,Fabrizio, Loreni,Xu, Peng,Qing-Qing, Yang,WeiFeng, He,Zhi-Hui, Yao,Xiao-Rong, Zhang,Ilaria, Dal Prà. Eukaryotic initiation factor 6

ur na

modulates myofibroblast differentiation at transforming growth factor-β1 transcription level via H2A.Z occupancy and Sp1 recruitment. Journal of Cell Science. 2015;128:3977-89.

Xiong Y, Zhang J, Shi L, Ning Y, Zhu Y, Chen S, et al. NOGO-B promotes EMT in lung

Jo

fibrosis via MMP14 mediates free TGF-beta1 formation. Oncotarget. 2017;8:71024-37. Yang L LX, Zhang N,Chen L,Xu J,Tang W. Investigation of circular RNAs and related genes in pulmonary fibrosis based on bioinformatics analysis. Journal of cellular biochemistry. 2019;undefined:undefined. Yao J DQ, Liu Z,Zhou L,Xu J. Circular RNAs in Organ Fibrosis. Advances in

experimental medicine and biology. 2018;1087:259-73. Yao W, Li Y, Han L, Ji X, Pan H, Liu Y, et al. The CDR1as/miR-7/TGFBR2 Axis Modulates EMT in Silica-Induced Pulmonary Fibrosis. Toxicological sciences : an official journal of the Society of Toxicology. 2018;166:465-78. Yao W LY, Han L,Ji X,Pan H,Liu Y,Yuan J,Yan W,Ni C. The CDR1as/miR-7/TGFBR2

official journal of the Society of Toxicology. 2018;166:465-78.

of

Axis Modulates EMT in Silica-Induced Pulmonary Fibrosis. Toxicological sciences : an

ro

Yu JW, Duan WJ, Huang XR, Meng XM, Yu XQ, Lan HY. MicroRNA-29b inhibits peritoneal fibrosis in a mouse model of peritoneal dialysis. Laboratory investigation; a

-p

journal of technical methods and pathology. 2014;94:978-90.

re

Zhang JX LJ, Xie H,Wang DP,Ni HE,Zhu Y,Ren LH,Meng XX,Wang RL. circHIPK3 regulates lung fibroblast-to-myofibroblast transition by functioning as a competing

lP

endogenous RNA. Cell death & disease. 2019;10:182.

Zhang Q WY, Qu D, Yu J, Yang J. The Possible Pathogenesis of Idiopathic Pulmonary

Jo

ur na

Fibrosis considering. BioMed research international. 2019;2019:9712464.

Figure legends Figure 1. circ0044226 is upregulated in mice model of pulmonary and FMT-derived myofibroblasts. (A) qRT-PCR was performed to detect the expression of circ0044226 in the lungs of normal and bleomycin (BLM)-treated mice. (B) Lung tissue sections from normal and BLM-treated mice were stained with myofibroblast marker α-SMA. Red, α-SMA; blue,

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nuclei. Scale bar, 50 μm. (C) Myofibroblast markers, COL-1 and α-SMA in mouse lungs

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were determined by western bolt. (D) WI-38 cells were treated with 10 ng/mL TGF-β1 for 48 h or left untreated (control). qRT-PCR was performed to detect the expression of

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circ0044226. (E) Immunofluorescence assays were performed to detect the myofibroblast

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marker α-SMA. Green, α-SMA; blue, nuclei. Scale bar, 25 μm. (F) Myofibroblast markers, COL-1 and α-SMA were determined by western bolt. (G) Cell viability were

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detected by CCK-8 assay. (H) DNA synthesis was assessed using EDU assay. Red, EDU; blue, nuclei. Scale bar, 25 μm. **P<0.01,***P<0.001 vs. Control.

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Figure 2. circ0044226 regulates FMT and fibroblast proliferation in vitro. (A) qRT-PCR was performed to detect the expression of miR-7 in the lungs of normal and BLM-treated mice. (B) WI-38 cells were treated with 10 ng/mL TGF-β1 for 48 h or left untreated (control). qRT-PCR was performed to detect the expression of miR-7. (C)

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The circ0044226 gene silencing efficiency in WI-38 cells was detected by qRT-PCR. (D) WI-38 cells were treated with circ0044226 siRNA (Si-circ0044226), scrambled siRNA (Si-NC) and then incubated with 10 ng/mL TGF-β1 for 48 h. qRT-PCR was performed to detect the expression of miR-7 in cells. (E) Myofibroblast markers, COL-1 and α-SMA were determined by western bolt. (F) Immunofluorescence assays were performed to

detect the myofibroblast marker α-SMA. green, α-SMA; blue, nuclei. Scale bar, 25 μm. (G) Cell viability were detected by CCK-8 assay. (H) DNA synthesis was assessed using EDU assay. Red, EDU; blue, nuclei. Scale bar, 25 μm. **P<0.01,***P<0.001 vs. Control or si-NC or TGF-β1+si-NC. Figure 3. circ0044226 function as a miR-7 sponge and miR-7 represses sp1 mRNA via pairing to its 3’UTR.

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(A) Schematic of putative binding sites of miR-7 on circ0044226. HEK-293T cells were

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co-transfected with LUC-circ0044226 or LUC-circ0044226 mutant with miR-7 mimics or negative control mimics. Luciferase activity was detected 48 h after transfection. (B)

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RNA-FISH assay was conducted to detect the expression distribution of circ0044226 and

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miR-7 in TGF-β1-treated WI-38 cells. Green, circ0044226; Red, miR-7; blue, nuclei. Scale bar, 10 μm. (C) Schematic of putative binding sites of miR-7 on 3’UTR of SP1.

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HEK-293T cells were co-transfected with LUC-sp1 or LUC-sp1 mutant with miR-7 mimics or negative control mimics. Luciferase activity was detected 48 h after

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transfection. **P<0.01,***P<0.001 vs. mimic NC. Figure 4.circ0044226/miR-7/sp1 network is involved in regulating FMT (A) WI-38 cells were transfected with miR-7 mimics or negative control mimics or left untreated plus LV-NC (vector) or LV-circ0044226, and then treated with TGF-β1 for 48 h.

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Immunofluorescence assays were performed to detect the myofibroblast marker α-SMA. Green, α-SMA; blue, nuclei. Scale bar, 25 μm. (B) Myofibroblast markers, COL-1 and αSMA were determined by western bolt. (C-D) The sp1 mRNA and protein expression were detected by qRT-PCR and western blot, respectively. (E) Cell viability were detected by CCK-8 assay. (F) DNA synthesis was assessed using EDU assay. Red, EDU;

blue, nuclei. Scale bar, 25 μm. ***P<0.001 vs. Control; ###P<0.001 vs. TGF-β1+mimic NC; &&&P<0.001 vs. TGF-β1+miR-7 mimic+vector. Figure 5. circ0044226 silencing attenuates BLM-induced pulmonary fibrosis in vivo. The mice were received an intratracheal injection of sh-circ0044226, sh-NC or left untreated. Three weeks later, mice were received a single intratracheal administration of BLM or saline. (A) The TGF-β1 level in lung tissue was detected by ELISA. (B) The

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circ0044226 expression in lung tissues was detected by qRT-PCR. (C) The miR-7

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expression in lung tissues was detected by qRT-PCR. (D-E) The sp1 mRNA and protein levels in lung tissues were detected by qRT-PCR and western blot, respectively. (F) Lung

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fibrosis was determined by HE staining. Scale bar, 50 μm. (G) Lung fibrosis was

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determined by Masson’s staining. Scale bar, 50 μm. (H) Immunofluorescence assays were performed to detect the myofibroblast marker α-SMA. Red, α-SMA; blue, nuclei. Scale

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bar, 50 μm. (I) Quantification of positive staining area was measured and statistically analyzed. (J) Myofibroblast markers, COL-1 and α-SMA were determined by western

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bolt. **P<0.01,***P<0.001 vs. Control; #P<0.05,##P<0.01,###P<0.001 vs. BLM+sh-NC.