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WITHDRAWN: Bisphenol S triggers the malignancy of hemangioma cells via regulation of basic fibroblast growth factor
Journal Pre-proof Bisphenol S triggers the malignancy of hemangioma cells via regulation of basic fibroblast growth factor Dahai Liu, Yubo Hui, Junrong Wang, Cong Ye, Jianshi Du PII:
S0009-2797(19)31596-0
DOI:
https://doi.org/10.1016/j.cbi.2019.108866
Reference:
CBI 108866
To appear in:
Chemico-Biological Interactions
Received Date: 21 September 2019 Revised Date:
15 October 2019
Accepted Date: 21 October 2019
Please cite this article as: D. Liu, Y. Hui, J. Wang, C. Ye, J. Du, Bisphenol S triggers the malignancy of hemangioma cells via regulation of basic fibroblast growth factor, Chemico-Biological Interactions (2019), doi: https://doi.org/10.1016/j.cbi.2019.108866. 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 B.V.
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Bisphenol S triggers the malignancy of hemangioma cells via
2
regulation of basic fibroblast growth factor
3 Dahai Liu2, Yubo Hui3, Junrong Wang1, Cong Ye1*, Jianshi Du2*
4 5 6
1
Department of Obstetrics and Gynecology, China-Japan Union Hospital of Jilin University,
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Changchun, Jilin 130033, China; 2
8 9 10 11
Lymph and Vascular Surgery Department, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, China;
3
Department of Anesthesiology, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, China
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* Co-corresponding authors:
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Dr Jianshi Du, Email: [email protected] ,
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Lymph and Vascular Surgery Department, China-Japan Union Hospital of Jilin
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University, Changchun, Jilin 130033, China
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Dr Cong Ye, Email: [email protected] ,
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Department of Obstetrics and Gynecology, China-Japan Union Hospital of Jilin
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University, Changchun, Jilin 130033, China.
20 21 22 1
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ABSTRACT: Hemangioma (IHA) is the most common benign tumor in infants caused by
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hyperproliferation of mesoblastic vascular tissues. Studies indicated that estrogenic signals
25
have positive roles in its progression, while potential roles of endocrine disrupting compounds
26
(EDCs) on its development are largely unknown. Our present study found that bisphenol S
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(BPS), the “safety” analog of bisphenol A (BPA), can trigger proliferation of HA cells and
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induce G1 to S transition of cell cycle at nanomolar concentrations. Further, BPS increased
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the expression of basic fibroblast growth factor (bFGF) in HA cells. Knockdown of bFGF can
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attenuate BPS-induced proliferation of HA cells, suggesting the essential role of bFGF in
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BPS-induced HA development. BPS can increase the transcription and mRNA stability of
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bFGF, while had no effect on its mRNA export or protein stability. Mechanistically, BPS can
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activate p65 to initiate bFGF transcription and decrease miR-155-5p to upregulate the mRNA
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stability of bFGF. Collectively, our study revealed that BPS can trigger the malignancy of HA
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cells via induction of cell proliferation and cell cycle transition. This is due to that BPS can
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increase the expression of bFGF via activation of p65 and down regulation of miR-155-5p.
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Keywords: IL-6; hemangioma; bFGF; proliferation; cell cycle;
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Running title: IL-6 promotes malignancy of HA cells via bFGF
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2
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1. INTRODUCTION
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Infantile hemangioma (IHA), caused by hyperproliferation of mesoblastic vascular
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tissues, is the most common benign tumor in infants at present (Mabeta and Pepper, 2011).
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HA can cause a heavy physical and mental burden to patients, particularly for patients with
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disfigured skin lesions (Chibbaro et al., 2018). Pharmacotherapy and surgery are major
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approaches for HA therapy (Chen et al., 2018). However, there are still 25% HA patients
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undergoing resection had persistence of symptoms (Haggstrom et al., 2007). The
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understanding about initiation and risk factors for HA is warranted for discovery of novel
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treatment approaches and prevent of HA.
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It has been reported that about 75%–80% of HA patients are females (Chibbaro et al.,
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2018). The detailed cause of the female preponderance is not yet understood. It has been
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reported that level of estradiol (E2) in healthy children was significantly lower than that in
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HA patients (Sasaki et al., 1984). The serum levels of E2 in proliferating HA tissues are
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greater than that in involuting phase (Yu et al., 2006). Laboratory studies indicated that
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estrogen can promote the in vitro proliferation of HA cells, which may depend on certain
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growth factors (GFs) and be inhibited by tamoxifen (Xiao et al., 1999). Further, estrogen and
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VEGF had synergistic effects on proliferation of HA cells (Xiao et al., 2004). All these data
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suggested the positive roles of estrogenic signals on HA progression.
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Endocrine disrupting compounds (EDCs) are environmental compounds which have
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similar characteristics with E2 (Rubin, 2011). They can influence multiple endocrine related
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pathways and then disrupt hormone functions (Henley and Korach, 2006). Bisphenol A (BPA)
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is a typical EDC banned from many human consumer products due to the negative effects on 3
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human health (Lu et al., 2013). Its analog bisphenol S (BPS) are widely used as substitutes for
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industrial application particularly in many commercial products labeled “BPA-free”
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(Rochester and Bolden, 2015). Recently, numerous studies indicated that BPS can also
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accumulate in human body and is an urgent issue for public health (Rochester and Bolden,
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2015). For example, BPS can reduce the steroid hormone synthesis and down regulate
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steroidogenic gene transcripts (Feng et al., 2016). BPS also has a comparable estrogenic
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activity as BPA (Kuruto-Niwa et al., 2005). It can induce epithelial‐mesenchymal transition
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(EMT) in HA cells via induction of Snail (Zhai et al., 2016). While the potential effects of
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BPS on the progression of HA are not investigated.
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The cytokines such as vascular endothelial growth factor (VEGF) and basic fibroblast
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growth factor (bFGF) are critical for HA progression (Fu et al., 2017; Przewratil et al., 2010).
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For example, VEGFA can facilitate the growth of HA-derived endothelial cells (HemECs)
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(Jinnin et al., 2008). While targeted inhibition of VEGF can inhibit the proliferation of HA
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cells (Pan et al., 2015). As to bFGF, its expression was associated with incidence of HA
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(Bielenberg et al., 1999) and proliferation of HA cells (Przewratil et al., 2010). However, the
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regulators about bFGF and other cytokines in HA were not well illustrated.
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Our present study found that nanomolar BPS can trigger the proliferation of HA cells via
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increasing the expression of bFGF. While the neutralization antibody of bFGF can abolish
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BPS-induced proliferation of HA cells. Mechanistically, BPS increased the transcription of
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bFGF via activation of p65 and stabilized the mRNA of bFGF via decreasing miR-155-5p.
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2. MATERIALS AND METHODS 4
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2.1 Cell culture and treatment
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The primary HA derived endothelial cell (HDEC) and CRL 2586 cells were
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maintained in our laboratory and cultured in DMEM supplied with FBS to a final
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concentration of 10% and streptomycin sulfate and penicillin (Life Technologies, Inc,
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Gaithersburg, Maryland) at 37°C under 5% CO2. Bisphenol S (99%, 4,4’-sulfonyldiphenol)
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was bought from Molecular Probes (USA) and dissolved in DMSO to get a stock solution of
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100 mM. Medium contains the same amount (less than 0.5%) of DMSO, which had no toxic
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effect on cells, was used as control.
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2.2 Cell proliferation assay
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The effects of BPS on proliferation of HA cells were tested by use of CCK-8 kit
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according to the previous study (Pang et al., 2019). Briefly, cells (2 × 103 cells/well) seeded in
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the 96-well plates were treated with different concentrations of BPS for the indicated time
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periods. At the end of experiments, 10 µL of CCK-8 solution was added to each well and
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incubated with 2 h at 37 °C. The absorbance at 450 nm was measured using a microplate
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reader (PerkinElmer, USA).
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2.3 Colony formation assay
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Cells (2 × 102 cells/well) seeded in six-well plates were treated with or without BPS as
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the indicated conditions. Then cells were allowed to grow for 5–14 days with media changed
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every 5 days. The formation of colony was stained and counted according to the previous
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study (Kalailingam et al., 2019). 5
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2.4 Cell cycle analysis
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Cells were treated with or without BPS before cell cycle analysis. After treatment, cells
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were fixed with cold 70% ethanol for 4 h, stained with propidium iodide (PI), and
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analyzed by flow cytometry using a FACSort Flow Cytometer (San Jose, CA)
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equipped with CellQuest Software according to the previous study (Yu et al., 2019).
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For each assay, 10,000 single cells were collected for analysis by use of ModFit
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software to determine each phase of cell cycle and are reported as percentage of
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G0/G1, S, and G2/M for each sample.
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2.5 Real-time RT-PCR analysis
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After treatment, cells were harvested with TransZolUp (TransGen Biotech, Beijing,
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China) and extracted with an RNA Purification Kit (Qiagen, Valencia, CA). Then, 1000 ng of
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total RNA was used to generate cDNA by use of reverse transcription reagent kit (Takara
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Biotechnology, Kusatsu, Shiga, Japan) for mRNA and miRNA reverse transcription kit
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(Promega Corporation, Madison, WI, USA) for miRNAs, respectively. The qRT-PCR was
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conducted by use of an iCycler (Bio-rad, Hercules, USA) with the primers as follow: VEGFA,
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5′- AGGGCAGAATCATCACGAAGT -3′ and 5′- AGGGTCTCGATTGGATGGCA -3′;
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aFGF, 5′- CTCCCGAAGGATTAAACGACG -3′ and 5′- GTCAGTGCTGCCTGAATGCT
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-3′;
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CGGTTAGCACACACTCCTTTG -3′; FGF3, 5′- GGCGTCTACGAGCACCTTG -3′ and 5′-
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CCACTGCCGTTATCTCCAAAA-3′;
bFGF,
5′-AGAAGAGCGACCCTCACATCA-3′
and
5′-
IGF1, 5′- GCTCTTCAGTTCGTGTGTGGA -3′ and 6
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5′- GCCTCCTTAGATCACAGCTCC-3′; HGF, 5′- GCTATCGGGGTAAAGACCTACA -3′
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and 5′- CGTAGCGTACCTCTGGATTGC -3′; TGFB1, 5′- GGCCAGATCCTGTCCAAGC
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-3′ and 5′- GTGGGTTTCCACCATTAGCAC -3′; GAPDH, forward 5′-GCA CCG TCA
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AGG CTG AGA AC-3′ and reverse 5′-TGG TGA AGA CGC CAG TGG A-3′. GAPDH and
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U6 were used to normalize the level of mRNAs and miRNAs, respectively. The results were
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based on the ∆∆Ct method and expressed as relative changes as compared to untreated
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control groups.
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2.6 Western blot analysis
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The protein expression was checked by western blot analysis according to the previous
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study (Song et al., 2019). The primary antibodies were listed as follows: bFGF (ab126861,
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Abcam, 1:500); GAPDH (ab181603, Abcam, 1:500); p-p65 (ab76302, Abcam, 1:500); p65
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(ab16502, Abcam, 1:500); p-c-fos (ab27793, Abcam, 1:500); c-fos (ab208942, Abcam, 1:500);
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p-c-Jun (ab32385, Abcam, 1:500); c-Jun (ab32137, Abcam, 1:500). GAPDH was used as an
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internal reference.
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2.7 Enzyme-linked immunosorbent assay (ELISA)
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The levels of bFGF in medium of cells treated with or without BPS were measured by
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use of ELISA kit according to the manufacturer's protocol (USCN Business Co. Ltd., Wuhan,
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China).
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2.8 Promoter activity assay 7
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The promoter (1kb upstream of TSS) of bFGF was cloned into the pGL-Basic plasmid
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to generate pGL-bFGF-Basic plasmid. Effects of BPS on the promoter activity of bFGF were
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tested by luciferase assay according to previously described protocol (Jiang et al., 2013).
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Briefly, cells were transfected with pGL-bFGF-basic and pRL-TK for 12 h and then further
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treated with or without BPS for the indicated time periods. The luciferase was measured by
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Dual-Glo Luciferase Assay system (Promega). Renilla Luciferase (R-luc) was used to
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normalize firefly luciferase (F-luc) activity to evaluate reporter translation efficiency.
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2.9 Statistical analysis
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All values are expressed as the mean ± standard deviation (SD). Data were analyzed by
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use of SPSS 14.0 software (SPSS, Inc., Chicago, IL, USA). The student t test was used to
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assess the difference between two groups. P ≤ 0.05 was considered as statistically significant.
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3. RESULTS
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3.1 BPS triggers the proliferation and cell cycle transition of HA cells
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To evaluate the potential functions of BPS on progression of HA, we treated cells with
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increasing concentration of BPS for 48 h. CCK-8 analysis showed that 10 nM BPS can
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significantly trigger the proliferation of HDEC cells (Figure 1 A), however, BPS with the
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concentrations greater than 1 mM can suppress proliferation of HDEC cells. Similarly,
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nanomolar concentrations of BPS can also trigger the proliferation of CRL-2586 cells (Figure
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1 B). Considering that nanomolar was more closely to concentration of BPS observed in
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human body, we only focused on the potential roles of nanomolar BPS in next studies. 8
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Colony formation analysis showed that 10 nM BPS can also significantly trigger the
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colonization of both HDEC and CRL-2586 cells (Figure 1 C). Cell cycle analysis showed that
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population of the cells in G0/G1 was decreased in the groups treated with BPS. The
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distribution of S phase was increased by treatment with BPS (Sun et al., 2018). It indicated
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that BPS can trigger the proliferation of HA cells via inducing cell cycle transition.
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3.2 BPS increases the expression of bFGF in HA cells
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Growth factors (GFs) such as EGF and hepatocyte growth factor (HGF) are suggested
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to play important roles in epithelial cell proliferation (Han et al., 2011; Zelenka and Arpitha,
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2008). We then tested the potential effects of BPS on the expression of various GFs including
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acidic fibroblast growth factor (aFGF), bFGF, FGF3, Insulin-like growth factor-1 (IGF-1),
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HGF, VEGF, and transforming growth factor beta (TGF-β). Our data showed that BPS can
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increase the expression of bFGF and VEGF in HDEC cells (Figure 2 A). However, BPS can
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only increase the expression of bFGF in CRL-2586 cells (Figure 2 B). Consistently, BPS can
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increase the expression of bFGF in HDEC cells via both time (Figure 2 C) and concentration
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(Figure 2 D) manners. ELISA confirmed that BPS increased expression of bFGF in both
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HDEC and CRL-2586 cells (Figure 2 E). All these results suggested that BPS increased the
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expression of bFGF in HA cells.
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3.3 bFGF is involved in BPS induced proliferation of HA cells
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The potential roles of bFGF were measured in BPS induced proliferation of HA cells.
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We found that neutralization antibody of bFGF can attenuate BPS induced proliferation of 9
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HDEC cells (Figure 3 A). However, the neutralization antibody for VEGF, which was also
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upregulated by BPS, had no significant effect on BPS induced proliferation of HDEC cells
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(Figure 3 B).
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cells (Figure 3 C). In addition, anti-bFGF also partially attenuated BPS decreased proportions
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of G0/G1 phase (Figure 3 D) while increased proportions of S phase (Figure 3 E) in HDEC
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cells. All these data suggested that bFGF was involved in BPS induced proliferation of HA
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cells.
Consistently, anti-bFGF also reversed BPS induced proliferation of CRL-2586
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3.4 BPS can increase the transcription and mRNA stability of bFGF
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We then further investigated the mechanisms responsible for upregulation of bFGF.
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Our data showed that BPS treatment can increase the mRNA of bFGF since treatment for 4 h.
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We then analyzed the promoter activity of bFGF in BPS treated HA cells by use of promoter
209
luciferase assay. Our data showed that BPS can increase the promoter activity of bFGF in
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both HDEC and CRL-2586 cells (Figure 4 A). Further, BPS can increase the half-life of
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bFGF mRNA in HDEC cells (Figure 4 B). However, BPS had no effect on mRNA
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distribution in cellular fractions such as cytoplasm and nucleus in HDEC cells (Figure 4 C).
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Further, BPS had no effect on protein stability of bFGF in HDEC cells (Figure 4 D). These
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data suggested that bFGF can increase the transcription and mRNA stability of bFGF.
215 216 217 218
3.5 NF-κB is involved in BPS induced expression of bFGF in HA cells Previous studies indicated that AP-1 and NF-κB can regulate the transcription of bFGF (Wu et al., 2016). We therefore investigated whether AP-1 or NF-κB was involved in BPS 10
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induced transcription of bFGF. We found that BPS can increase the phosphorylation of p65,
220
while not c-Fos or c-Jun, in HDEC cells (Figure 5 A). Consistently, BPS also increased the
221
phosphorylation of p65 in CRL-2586 cells (Figure 5 B). Further, BAY, the inhibitor of NF-κB,
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can reverse BPS induced upregulation of bFGF in HDEC cells (Figure 5 C). Consistently,
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BAY also partially attenuated BPS induced proliferation of HDEC cells (Figure 5 D). These
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data suggested that NF-κB was involved in BPS induced expression of bFGF in HA cells.
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3.6 miR-155-5p is involved in BPS increased mRNA stability of bFGF
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miRNAs can target the 3’UTR of mRNA and then lead to degradation of its target
228
(Mohajeri et al., 2018). Based on the prediction results of miRTarBase (Chou et al., 2016),
229
miRecords (Xiao et al., 2009) and starBase version 2.0 (Li et al., 2014), miR-16-5p,
230
miR-140-5p, miR-503-5p, and miR-155-5p can directly target the 3’UTR of bFGF to regulate
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its expression. Our data showed that BPS can significantly inhibit the expression of
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miR-155-5p, while not others, in HDEC cells (Figure 6 A). Consistently, our data showed that
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BPS can also decrease the expression of miR-155-5p in CRL-2586 cells (Figure 6 B). The
234
mimic of miR-155-5p can attenuate BPS induced upregulation of bFGF in HDEC cells
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(Figure 6 C). Consistently, mimic of miR-155-5p can also partially attenuate BPS induced
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proliferation of HDEC cells (Figure 6 D). All these data indicated that miR-155-5p is
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involved in BPS increased mRNA stability of bFGF.
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4. DISCUSSION
11
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Recent studies indicated that BPA can increase the progression of HA via induction of
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EMT and upregulation of Snail (Zhai et al., 2016). Our present study found that BPS, the
242
“safety” analog of BPA, can trigger the proliferation of HA cells and its cell cycle transition
243
via upregulation of bFGF. Further, BPS can increase the mRNA stability of bFGF via
244
decreasing the expression of miR-155-5p. As well, BPS can increase the transcription of
245
bFGF via activation of NF-κB.
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BPS have existed estrogenic and cellular functions in many recent studies (Chin et al.,
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2018; Kinch et al., 2015; LaPlante et al., 2017). As to cell proliferation, BPS significantly
248
promoted the proliferation of ERα positive MCF-7 cells, but failed to promote the
249
proliferation of ERα negative MDA-MB-231 and SK-BR-3 cells (Lin et al., 2019), which
250
was consistent with our present study that BPS at nanomolar can significantly promote
251
proliferation of HA cells. Our results also observed that BPA can increase S phase and
252
decrease G0/G1 phase of HA cell, which was also consistent with previous data that EDCs
253
such as BPA(Wu et al., 2012), Perfluorooctanoic acid (PFOA) (Pierozan et al., 2018), and
254
polybrominated diphenyl ethers (Li et al., 2012) can reduce the percentage of cells at G0/G1
255
phase and increase percentage of cells at S phase to trigger the cell cycle transition and cell
256
proliferation. In MCF-7 cells, BPS treatment also resulted in an acceleration of G1-S phase
257
transition (Lin et al., 2019). Since BPS existed comparable estrogenic potency to E2 (Vinas
258
and Watson, 2013) and HA cells can be exposed to BPS via blood circulation, the potential
259
effects of BPS on HA progression need further study.
260
We found the upregulation of bFGF was involved in BPS induced proliferation of HA
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cells. The expression of bFGF and its receptor are closely associated with proliferation of 12
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infantile cutaneous hemangioma (Przewratil et al., 2010). In situ hybridization and
263
immunohistochemical analysis confirmed that the expression of bFGF is closely correlated
264
with incidence of hemangioma (Bielenberg et al., 1999). The bFGF is an effective stimulator
265
of breast epithelial cells proliferation and differentiation (Korah et al., 2000). Our present
266
study found that the neutralization antibody against bFGF can suppress the proliferation of
267
HA cells and block promotion effect of BPS on cells proliferation, which further confirmed
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the essential roles of bFGF in HA progression.
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We found that activation of p65 and down regulation of miR-155-5p were responsible
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for BPS induced transcription and upregulation mRNA stability of bFGF, respectively, in HA
271
cells. In bovine mammary epithelial cells (BMEC), the expression of bFGF is dependent on
272
the NF-κB and AP-1 signaling pathways (Wu et al., 2018). While BPS had no significant
273
effect on the phosphorylation of AP1 in HA cells. Further, activation of p65 was also
274
involved in BPA induced migration of cervical cancer cells (Ma et al., 2015). In male
275
zebrafish, BPS exposure can change the expression of 14 miRNAs involved in hematopoiesis,
276
lymphoid organ development, and immune system development (Lee et al., 2018). In
277
pheochromocytoma PC12 cells, BPS can regulate the expression of miR-10b to inhibit the
278
expression of KLF4 and induce cell migration (Jia et al., 2018). It has been reported that
279
miR-15a (Zhu et al., 2017), miR-146a (Liu et al., 2016), and miR-195 (Wang et al., 2017)
280
can also regulate the mRNA stability and expression of bFGF. Whether these miRNAs are
281
involved in BPS regulated expression of bFGF needs further study.
282
In conclusion, our results demonstrated that nanomolar BPS can induce the proliferation
283
of HA cells via induction of bFGF through p65 induced transcription and miR-155 regulated 13
284
mRNA stabilization. It indicated the potential risks about BPS on HA progression and
285
hormone related diseases need more attention.
286 287
Declarations
288
Ethics approval and consent to participate
289
No human/animal study was included in the present study.
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Consent for publication
291
All authors give the consent for the publish of this study.
292
Availability of data and material
293
All data and material are available.
294
Disclosure of potential conflicts of interest
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The authors declare no conflict of interest.
296
Funding
297
No funding information
298
Authors' contributions
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Data collecting: Dahai Liu, Yubo Hui, Junrong Wang
300
Writing: Junrong Wang, Jianshi Du, Cong Ye
301
Data analysis: Dahai Liu, Yubo Hui, Cong Ye
302
Design: Dahai Liu, Yubo Hui, Junrong Wang, Jianshi Du,
303
Acknowledgements
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No applicable
305 14
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impaired angiogenic capacity of BM-MSCs in high fat diet-fed mice. Mol Med Rep 16, 7609-7616.
439 440
18
441
Figure legends
442
Figure 1 BPS triggers the proliferation and cell cycle transition of HA cells. HDEC (A) or
443
CRL-2586 (B) cells were treated with increasing concentrations of BPS for 48 h, cell
444
proliferation was tested by CCK-8 kit; (C) Cells (2 × l05) treated with or without 100 nM
445
BPS were cultured in 6-well plates for two weeks before colonies were counted; (D)
446
HDEC cells were treated with or without 100 nM BPS for 24 h, the cell cycle was
447
detected using flow cytometry. Data are shown as means ± SD. **p < 0.01 compared to
448
control.
449
Figure 2 BPS increases the expression of bFGF in HA cells. HDEC (A) or CRL-2586 (B)
450
cells were treated with or without 100 nM BPS for 24 h, the expression of GFs was
451
measured by qRT-PCR; HDEC cells were treated with 100 nM BPS for the increased
452
time periods (C) or increasing concentrations of BPS for 24 h (D), the expression of
453
bFGF was checked by qRT-PCR; (E) Cells were treated with or without 100 nM BPS for
454
24 h, the expression of bFGF was checked by ELISA. Data are shown as means ± SD.
455
**p < 0.01 compared to control.
456
Figure 3 bFGF is involved in BPS induced proliferation of HA cells. HDEC cells were
457
pre-treated with anti-bFGF (A) or anti-VEGF (B) (100 ng/ml) for 2 h and then further
458
treated with or without 100 nM BPS for 24 h, the cell proliferation was tested by CCK-8
459
kit; (C) CRL-2586 cells were pre-treated with or without anti-bFGF for 2 h and then
460
further treated with or without 100 nM BPS for 24 h, the cell proliferation was tested by
461
CCK-8 kit; HDEC cells were pre-treated with or without anti-bFGF and then further
462
treated with or without 100 nM BPS for 24 h, the cell cycle of G0/G1 (D) or S (E) phase 19
463
of cells were detected using flow cytometry. Data are shown as means ± SD. **p < 0.01
464
compared to control.
465
Figure 4 bFGF can increase the transcription and mRNA stability of bFGF. (A) Cells were
466
treated with or without 100 nM BPS for 24 h, the luciferase activities of bFGF promoter
467
were measured by dual-luciferase assay; (B) HDEC cells pretreated with or without 100
468
nM BPS for 24 h were further treated with or without transcriptional inhibitor Act-D for
469
the indicated times, the mRNA of bFGF was checked by qRT-PCR; (C) After treated
470
with or without 100 nM BPS for 24 h, the mRNA of bFGF in cytoplasm and nucleus of
471
HDEC cells was checked by qRT-PCR; (D) HDEC cells were pre-treated with or without
472
100 nM BPS for 24 h and further treated with CHX (100 µg/ml) for increasing time
473
periods, the expression of bFGF was recorded (left) and quantitatively analyzed (right).
474
Data are shown as means ± SD. **p < 0.01 compared to control.
475
Figure 5 NF-κB is involved in BPS induced expression of bFGF in HA cells. HDEC (A) or
476
CRL-2586 (B) cells were treated with or without 100 nM BPS for 15 min, the
477
phosphorylation and total expression of p65, c-fos and c-Jun was measured by western
478
blot analysis; HDEC cells were pretreated with or without BAY, the inhibitor of NF-κB,
479
for 30 min, and then further treated with or without 100 nM BPS for 24 h, the mRNA
480
expression of bFGF was tested by qRT-PCR (C), and cell proliferation was tested by
481
CCK-8 kit (D). Data are shown as means ± SD. **p < 0.01 compared to control.
482
Figure 6 miR-155-5p is involved in BPS increased mRNA stability of bFGF. HDEC (A) or
483
CRL-2586 (B) cells were treated with or without 100 nM BPS for 15 min, the expression
484
of miRNAs was checked by qRT-PCR; HDEC cells pretreated with scramble control or 20
485
miR-155 mimic and then further treated with or without 100 nM BPS for 24 h, the
486
mRNA expression of bFGF was checked by qRT-PCR (C), the cell proliferation was
487
detected by CCK-8 kit (D).
488
control.
Data are shown as means ± SD. **p < 0.01 compared to
489 490
21
491
Figure 1
492
493 494 495
22
C
498 0 0
200
4
300
D
**
** **
100
8 12 24 h Relative mRNA expression (%)
200
100
0
300 **
200
0 ** **
100
0 10 20 50100 nM
F FG F3 IG F1 H G F VE G TG F Fβ
F
**
G
**
ELISA (pg/ml)
G
B Relative mRNA expression (%)
HDEC
bF
aF
F1 H G F VE G TG F Fβ
F3
F
F
300
IG
FG
bF G
aF G
Relative mRNA expression (%)
A
Relative mRNA expression (%)
496 Figure 2
497
400 CRL-2586
300 **
E
Con BPS
200
100 0
600 **
0 HDEC
Con BPS
400
200 **
100 CRL
499
500
23
501
Figure 3
B HDEC 300
Relative cell proliferation (%)
Relative cell proliferation (%)
A **
200 NS 100
0
HDEC 300
** 200
100
0
Con BPS Con BPS
Relative cell phase (%)
D
80
Anti-bFGF
G0/G1 ** NS
60
40
20
Con
E
50
Anti-VEGF
CRL-2586 300
**
200 NS 100
0 Con BPS Con BPS Con
Anti-bFGF
S **
40 NS 30 20 10 0
Con BPS Con BPS
503
Con BPS Con BPS
Relative cell phase (%)
Con
**
C
Relative cell proliferation (%)
502
Con
Anti-bFGF
Con BPS Con BPS Con
Anti-bFGF
504 505
24
506
Figure 4
507
508 509 510
25
511
Figure 5
512
A
Con
B
BPS
Con
p-p65
p-p65
p65
p65
p-c-fos
GAPDH
BPS
CRL-2586
c-fos
C
D
p-c-Jun c-Jun GAPDH HDEC
513 514 515
26
Figure 6
150
100
m
m
iR
03 -5
HDEC
40 -1 iR m
Con
55 -1
**
BPS
iR
B
D
Relative miRNA expression (%) Relative cell proliferation (%)
50
6 -1
**
BPS
0
iR m
**
150
100
50
0
iR m
300
6 -1 m
**
03 -5 iR
CRL-2586
m
Con
55
**
BPS
-1 iR m
miR-155
**
40 -1 iR
BPS
A
C 300 200
100
0 Con
NC
BPS
200
Con
miR-155
Con
100
0
NC
Con
516 517
518 519 520
BPS
Relative miRNA expression (%)
Relative mRNA expression (%)
27
Highlights 1. BPS triggers the proliferation of HA cells; 2. bFGF is involved in BPS induced proliferation of HA cells; 3. BPS increases the mRNA stability of bFGF via decreasing miR-155-5p; 4. BPS activates p65 to initiate bFGF transcription
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0009-2797(19)31596-0
DOI:
https://doi.org/10.1016/j.cbi.2019.108866
Reference:
CBI 108866
To appear in:
Chemico-Biological Interactions
Received Date: 21 September 2019 Revised Date:
15 October 2019
Accepted Date: 21 October 2019
Please cite this article as: D. Liu, Y. Hui, J. Wang, C. Ye, J. Du, Bisphenol S triggers the malignancy of hemangioma cells via regulation of basic fibroblast growth factor, Chemico-Biological Interactions (2019), doi: https://doi.org/10.1016/j.cbi.2019.108866. 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 B.V.
1
Bisphenol S triggers the malignancy of hemangioma cells via
2
regulation of basic fibroblast growth factor
3 Dahai Liu2, Yubo Hui3, Junrong Wang1, Cong Ye1*, Jianshi Du2*
4 5 6
1
Department of Obstetrics and Gynecology, China-Japan Union Hospital of Jilin University,
7
Changchun, Jilin 130033, China; 2
8 9 10 11
Lymph and Vascular Surgery Department, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, China;
3
Department of Anesthesiology, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, China
12 13
* Co-corresponding authors:
14
Dr Jianshi Du, Email: [email protected] ,
15
Lymph and Vascular Surgery Department, China-Japan Union Hospital of Jilin
16
University, Changchun, Jilin 130033, China
17
Dr Cong Ye, Email: [email protected] ,
18
Department of Obstetrics and Gynecology, China-Japan Union Hospital of Jilin
19
University, Changchun, Jilin 130033, China.
20 21 22 1
23
ABSTRACT: Hemangioma (IHA) is the most common benign tumor in infants caused by
24
hyperproliferation of mesoblastic vascular tissues. Studies indicated that estrogenic signals
25
have positive roles in its progression, while potential roles of endocrine disrupting compounds
26
(EDCs) on its development are largely unknown. Our present study found that bisphenol S
27
(BPS), the “safety” analog of bisphenol A (BPA), can trigger proliferation of HA cells and
28
induce G1 to S transition of cell cycle at nanomolar concentrations. Further, BPS increased
29
the expression of basic fibroblast growth factor (bFGF) in HA cells. Knockdown of bFGF can
30
attenuate BPS-induced proliferation of HA cells, suggesting the essential role of bFGF in
31
BPS-induced HA development. BPS can increase the transcription and mRNA stability of
32
bFGF, while had no effect on its mRNA export or protein stability. Mechanistically, BPS can
33
activate p65 to initiate bFGF transcription and decrease miR-155-5p to upregulate the mRNA
34
stability of bFGF. Collectively, our study revealed that BPS can trigger the malignancy of HA
35
cells via induction of cell proliferation and cell cycle transition. This is due to that BPS can
36
increase the expression of bFGF via activation of p65 and down regulation of miR-155-5p.
37 38
Keywords: IL-6; hemangioma; bFGF; proliferation; cell cycle;
39 40
Running title: IL-6 promotes malignancy of HA cells via bFGF
41 42
2
43
1. INTRODUCTION
44
Infantile hemangioma (IHA), caused by hyperproliferation of mesoblastic vascular
45
tissues, is the most common benign tumor in infants at present (Mabeta and Pepper, 2011).
46
HA can cause a heavy physical and mental burden to patients, particularly for patients with
47
disfigured skin lesions (Chibbaro et al., 2018). Pharmacotherapy and surgery are major
48
approaches for HA therapy (Chen et al., 2018). However, there are still 25% HA patients
49
undergoing resection had persistence of symptoms (Haggstrom et al., 2007). The
50
understanding about initiation and risk factors for HA is warranted for discovery of novel
51
treatment approaches and prevent of HA.
52
It has been reported that about 75%–80% of HA patients are females (Chibbaro et al.,
53
2018). The detailed cause of the female preponderance is not yet understood. It has been
54
reported that level of estradiol (E2) in healthy children was significantly lower than that in
55
HA patients (Sasaki et al., 1984). The serum levels of E2 in proliferating HA tissues are
56
greater than that in involuting phase (Yu et al., 2006). Laboratory studies indicated that
57
estrogen can promote the in vitro proliferation of HA cells, which may depend on certain
58
growth factors (GFs) and be inhibited by tamoxifen (Xiao et al., 1999). Further, estrogen and
59
VEGF had synergistic effects on proliferation of HA cells (Xiao et al., 2004). All these data
60
suggested the positive roles of estrogenic signals on HA progression.
61
Endocrine disrupting compounds (EDCs) are environmental compounds which have
62
similar characteristics with E2 (Rubin, 2011). They can influence multiple endocrine related
63
pathways and then disrupt hormone functions (Henley and Korach, 2006). Bisphenol A (BPA)
64
is a typical EDC banned from many human consumer products due to the negative effects on 3
65
human health (Lu et al., 2013). Its analog bisphenol S (BPS) are widely used as substitutes for
66
industrial application particularly in many commercial products labeled “BPA-free”
67
(Rochester and Bolden, 2015). Recently, numerous studies indicated that BPS can also
68
accumulate in human body and is an urgent issue for public health (Rochester and Bolden,
69
2015). For example, BPS can reduce the steroid hormone synthesis and down regulate
70
steroidogenic gene transcripts (Feng et al., 2016). BPS also has a comparable estrogenic
71
activity as BPA (Kuruto-Niwa et al., 2005). It can induce epithelial‐mesenchymal transition
72
(EMT) in HA cells via induction of Snail (Zhai et al., 2016). While the potential effects of
73
BPS on the progression of HA are not investigated.
74
The cytokines such as vascular endothelial growth factor (VEGF) and basic fibroblast
75
growth factor (bFGF) are critical for HA progression (Fu et al., 2017; Przewratil et al., 2010).
76
For example, VEGFA can facilitate the growth of HA-derived endothelial cells (HemECs)
77
(Jinnin et al., 2008). While targeted inhibition of VEGF can inhibit the proliferation of HA
78
cells (Pan et al., 2015). As to bFGF, its expression was associated with incidence of HA
79
(Bielenberg et al., 1999) and proliferation of HA cells (Przewratil et al., 2010). However, the
80
regulators about bFGF and other cytokines in HA were not well illustrated.
81
Our present study found that nanomolar BPS can trigger the proliferation of HA cells via
82
increasing the expression of bFGF. While the neutralization antibody of bFGF can abolish
83
BPS-induced proliferation of HA cells. Mechanistically, BPS increased the transcription of
84
bFGF via activation of p65 and stabilized the mRNA of bFGF via decreasing miR-155-5p.
85 86
2. MATERIALS AND METHODS 4
87
2.1 Cell culture and treatment
88
The primary HA derived endothelial cell (HDEC) and CRL 2586 cells were
89
maintained in our laboratory and cultured in DMEM supplied with FBS to a final
90
concentration of 10% and streptomycin sulfate and penicillin (Life Technologies, Inc,
91
Gaithersburg, Maryland) at 37°C under 5% CO2. Bisphenol S (99%, 4,4’-sulfonyldiphenol)
92
was bought from Molecular Probes (USA) and dissolved in DMSO to get a stock solution of
93
100 mM. Medium contains the same amount (less than 0.5%) of DMSO, which had no toxic
94
effect on cells, was used as control.
95 96
2.2 Cell proliferation assay
97
The effects of BPS on proliferation of HA cells were tested by use of CCK-8 kit
98
according to the previous study (Pang et al., 2019). Briefly, cells (2 × 103 cells/well) seeded in
99
the 96-well plates were treated with different concentrations of BPS for the indicated time
100
periods. At the end of experiments, 10 µL of CCK-8 solution was added to each well and
101
incubated with 2 h at 37 °C. The absorbance at 450 nm was measured using a microplate
102
reader (PerkinElmer, USA).
103 104
2.3 Colony formation assay
105
Cells (2 × 102 cells/well) seeded in six-well plates were treated with or without BPS as
106
the indicated conditions. Then cells were allowed to grow for 5–14 days with media changed
107
every 5 days. The formation of colony was stained and counted according to the previous
108
study (Kalailingam et al., 2019). 5
109 110
2.4 Cell cycle analysis
111
Cells were treated with or without BPS before cell cycle analysis. After treatment, cells
112
were fixed with cold 70% ethanol for 4 h, stained with propidium iodide (PI), and
113
analyzed by flow cytometry using a FACSort Flow Cytometer (San Jose, CA)
114
equipped with CellQuest Software according to the previous study (Yu et al., 2019).
115
For each assay, 10,000 single cells were collected for analysis by use of ModFit
116
software to determine each phase of cell cycle and are reported as percentage of
117
G0/G1, S, and G2/M for each sample.
118 119
2.5 Real-time RT-PCR analysis
120
After treatment, cells were harvested with TransZolUp (TransGen Biotech, Beijing,
121
China) and extracted with an RNA Purification Kit (Qiagen, Valencia, CA). Then, 1000 ng of
122
total RNA was used to generate cDNA by use of reverse transcription reagent kit (Takara
123
Biotechnology, Kusatsu, Shiga, Japan) for mRNA and miRNA reverse transcription kit
124
(Promega Corporation, Madison, WI, USA) for miRNAs, respectively. The qRT-PCR was
125
conducted by use of an iCycler (Bio-rad, Hercules, USA) with the primers as follow: VEGFA,
126
5′- AGGGCAGAATCATCACGAAGT -3′ and 5′- AGGGTCTCGATTGGATGGCA -3′;
127
aFGF, 5′- CTCCCGAAGGATTAAACGACG -3′ and 5′- GTCAGTGCTGCCTGAATGCT
128
-3′;
129
CGGTTAGCACACACTCCTTTG -3′; FGF3, 5′- GGCGTCTACGAGCACCTTG -3′ and 5′-
130
CCACTGCCGTTATCTCCAAAA-3′;
bFGF,
5′-AGAAGAGCGACCCTCACATCA-3′
and
5′-
IGF1, 5′- GCTCTTCAGTTCGTGTGTGGA -3′ and 6
131
5′- GCCTCCTTAGATCACAGCTCC-3′; HGF, 5′- GCTATCGGGGTAAAGACCTACA -3′
132
and 5′- CGTAGCGTACCTCTGGATTGC -3′; TGFB1, 5′- GGCCAGATCCTGTCCAAGC
133
-3′ and 5′- GTGGGTTTCCACCATTAGCAC -3′; GAPDH, forward 5′-GCA CCG TCA
134
AGG CTG AGA AC-3′ and reverse 5′-TGG TGA AGA CGC CAG TGG A-3′. GAPDH and
135
U6 were used to normalize the level of mRNAs and miRNAs, respectively. The results were
136
based on the ∆∆Ct method and expressed as relative changes as compared to untreated
137
control groups.
138 139
2.6 Western blot analysis
140
The protein expression was checked by western blot analysis according to the previous
141
study (Song et al., 2019). The primary antibodies were listed as follows: bFGF (ab126861,
142
Abcam, 1:500); GAPDH (ab181603, Abcam, 1:500); p-p65 (ab76302, Abcam, 1:500); p65
143
(ab16502, Abcam, 1:500); p-c-fos (ab27793, Abcam, 1:500); c-fos (ab208942, Abcam, 1:500);
144
p-c-Jun (ab32385, Abcam, 1:500); c-Jun (ab32137, Abcam, 1:500). GAPDH was used as an
145
internal reference.
146 147
2.7 Enzyme-linked immunosorbent assay (ELISA)
148
The levels of bFGF in medium of cells treated with or without BPS were measured by
149
use of ELISA kit according to the manufacturer's protocol (USCN Business Co. Ltd., Wuhan,
150
China).
151 152
2.8 Promoter activity assay 7
153
The promoter (1kb upstream of TSS) of bFGF was cloned into the pGL-Basic plasmid
154
to generate pGL-bFGF-Basic plasmid. Effects of BPS on the promoter activity of bFGF were
155
tested by luciferase assay according to previously described protocol (Jiang et al., 2013).
156
Briefly, cells were transfected with pGL-bFGF-basic and pRL-TK for 12 h and then further
157
treated with or without BPS for the indicated time periods. The luciferase was measured by
158
Dual-Glo Luciferase Assay system (Promega). Renilla Luciferase (R-luc) was used to
159
normalize firefly luciferase (F-luc) activity to evaluate reporter translation efficiency.
160 161
2.9 Statistical analysis
162
All values are expressed as the mean ± standard deviation (SD). Data were analyzed by
163
use of SPSS 14.0 software (SPSS, Inc., Chicago, IL, USA). The student t test was used to
164
assess the difference between two groups. P ≤ 0.05 was considered as statistically significant.
165 166
3. RESULTS
167
3.1 BPS triggers the proliferation and cell cycle transition of HA cells
168
To evaluate the potential functions of BPS on progression of HA, we treated cells with
169
increasing concentration of BPS for 48 h. CCK-8 analysis showed that 10 nM BPS can
170
significantly trigger the proliferation of HDEC cells (Figure 1 A), however, BPS with the
171
concentrations greater than 1 mM can suppress proliferation of HDEC cells. Similarly,
172
nanomolar concentrations of BPS can also trigger the proliferation of CRL-2586 cells (Figure
173
1 B). Considering that nanomolar was more closely to concentration of BPS observed in
174
human body, we only focused on the potential roles of nanomolar BPS in next studies. 8
175
Colony formation analysis showed that 10 nM BPS can also significantly trigger the
176
colonization of both HDEC and CRL-2586 cells (Figure 1 C). Cell cycle analysis showed that
177
population of the cells in G0/G1 was decreased in the groups treated with BPS. The
178
distribution of S phase was increased by treatment with BPS (Sun et al., 2018). It indicated
179
that BPS can trigger the proliferation of HA cells via inducing cell cycle transition.
180 181
3.2 BPS increases the expression of bFGF in HA cells
182
Growth factors (GFs) such as EGF and hepatocyte growth factor (HGF) are suggested
183
to play important roles in epithelial cell proliferation (Han et al., 2011; Zelenka and Arpitha,
184
2008). We then tested the potential effects of BPS on the expression of various GFs including
185
acidic fibroblast growth factor (aFGF), bFGF, FGF3, Insulin-like growth factor-1 (IGF-1),
186
HGF, VEGF, and transforming growth factor beta (TGF-β). Our data showed that BPS can
187
increase the expression of bFGF and VEGF in HDEC cells (Figure 2 A). However, BPS can
188
only increase the expression of bFGF in CRL-2586 cells (Figure 2 B). Consistently, BPS can
189
increase the expression of bFGF in HDEC cells via both time (Figure 2 C) and concentration
190
(Figure 2 D) manners. ELISA confirmed that BPS increased expression of bFGF in both
191
HDEC and CRL-2586 cells (Figure 2 E). All these results suggested that BPS increased the
192
expression of bFGF in HA cells.
193 194
3.3 bFGF is involved in BPS induced proliferation of HA cells
195
The potential roles of bFGF were measured in BPS induced proliferation of HA cells.
196
We found that neutralization antibody of bFGF can attenuate BPS induced proliferation of 9
197
HDEC cells (Figure 3 A). However, the neutralization antibody for VEGF, which was also
198
upregulated by BPS, had no significant effect on BPS induced proliferation of HDEC cells
199
(Figure 3 B).
200
cells (Figure 3 C). In addition, anti-bFGF also partially attenuated BPS decreased proportions
201
of G0/G1 phase (Figure 3 D) while increased proportions of S phase (Figure 3 E) in HDEC
202
cells. All these data suggested that bFGF was involved in BPS induced proliferation of HA
203
cells.
Consistently, anti-bFGF also reversed BPS induced proliferation of CRL-2586
204 205
3.4 BPS can increase the transcription and mRNA stability of bFGF
206
We then further investigated the mechanisms responsible for upregulation of bFGF.
207
Our data showed that BPS treatment can increase the mRNA of bFGF since treatment for 4 h.
208
We then analyzed the promoter activity of bFGF in BPS treated HA cells by use of promoter
209
luciferase assay. Our data showed that BPS can increase the promoter activity of bFGF in
210
both HDEC and CRL-2586 cells (Figure 4 A). Further, BPS can increase the half-life of
211
bFGF mRNA in HDEC cells (Figure 4 B). However, BPS had no effect on mRNA
212
distribution in cellular fractions such as cytoplasm and nucleus in HDEC cells (Figure 4 C).
213
Further, BPS had no effect on protein stability of bFGF in HDEC cells (Figure 4 D). These
214
data suggested that bFGF can increase the transcription and mRNA stability of bFGF.
215 216 217 218
3.5 NF-κB is involved in BPS induced expression of bFGF in HA cells Previous studies indicated that AP-1 and NF-κB can regulate the transcription of bFGF (Wu et al., 2016). We therefore investigated whether AP-1 or NF-κB was involved in BPS 10
219
induced transcription of bFGF. We found that BPS can increase the phosphorylation of p65,
220
while not c-Fos or c-Jun, in HDEC cells (Figure 5 A). Consistently, BPS also increased the
221
phosphorylation of p65 in CRL-2586 cells (Figure 5 B). Further, BAY, the inhibitor of NF-κB,
222
can reverse BPS induced upregulation of bFGF in HDEC cells (Figure 5 C). Consistently,
223
BAY also partially attenuated BPS induced proliferation of HDEC cells (Figure 5 D). These
224
data suggested that NF-κB was involved in BPS induced expression of bFGF in HA cells.
225 226
3.6 miR-155-5p is involved in BPS increased mRNA stability of bFGF
227
miRNAs can target the 3’UTR of mRNA and then lead to degradation of its target
228
(Mohajeri et al., 2018). Based on the prediction results of miRTarBase (Chou et al., 2016),
229
miRecords (Xiao et al., 2009) and starBase version 2.0 (Li et al., 2014), miR-16-5p,
230
miR-140-5p, miR-503-5p, and miR-155-5p can directly target the 3’UTR of bFGF to regulate
231
its expression. Our data showed that BPS can significantly inhibit the expression of
232
miR-155-5p, while not others, in HDEC cells (Figure 6 A). Consistently, our data showed that
233
BPS can also decrease the expression of miR-155-5p in CRL-2586 cells (Figure 6 B). The
234
mimic of miR-155-5p can attenuate BPS induced upregulation of bFGF in HDEC cells
235
(Figure 6 C). Consistently, mimic of miR-155-5p can also partially attenuate BPS induced
236
proliferation of HDEC cells (Figure 6 D). All these data indicated that miR-155-5p is
237
involved in BPS increased mRNA stability of bFGF.
238 239
4. DISCUSSION
11
240
Recent studies indicated that BPA can increase the progression of HA via induction of
241
EMT and upregulation of Snail (Zhai et al., 2016). Our present study found that BPS, the
242
“safety” analog of BPA, can trigger the proliferation of HA cells and its cell cycle transition
243
via upregulation of bFGF. Further, BPS can increase the mRNA stability of bFGF via
244
decreasing the expression of miR-155-5p. As well, BPS can increase the transcription of
245
bFGF via activation of NF-κB.
246
BPS have existed estrogenic and cellular functions in many recent studies (Chin et al.,
247
2018; Kinch et al., 2015; LaPlante et al., 2017). As to cell proliferation, BPS significantly
248
promoted the proliferation of ERα positive MCF-7 cells, but failed to promote the
249
proliferation of ERα negative MDA-MB-231 and SK-BR-3 cells (Lin et al., 2019), which
250
was consistent with our present study that BPS at nanomolar can significantly promote
251
proliferation of HA cells. Our results also observed that BPA can increase S phase and
252
decrease G0/G1 phase of HA cell, which was also consistent with previous data that EDCs
253
such as BPA(Wu et al., 2012), Perfluorooctanoic acid (PFOA) (Pierozan et al., 2018), and
254
polybrominated diphenyl ethers (Li et al., 2012) can reduce the percentage of cells at G0/G1
255
phase and increase percentage of cells at S phase to trigger the cell cycle transition and cell
256
proliferation. In MCF-7 cells, BPS treatment also resulted in an acceleration of G1-S phase
257
transition (Lin et al., 2019). Since BPS existed comparable estrogenic potency to E2 (Vinas
258
and Watson, 2013) and HA cells can be exposed to BPS via blood circulation, the potential
259
effects of BPS on HA progression need further study.
260
We found the upregulation of bFGF was involved in BPS induced proliferation of HA
261
cells. The expression of bFGF and its receptor are closely associated with proliferation of 12
262
infantile cutaneous hemangioma (Przewratil et al., 2010). In situ hybridization and
263
immunohistochemical analysis confirmed that the expression of bFGF is closely correlated
264
with incidence of hemangioma (Bielenberg et al., 1999). The bFGF is an effective stimulator
265
of breast epithelial cells proliferation and differentiation (Korah et al., 2000). Our present
266
study found that the neutralization antibody against bFGF can suppress the proliferation of
267
HA cells and block promotion effect of BPS on cells proliferation, which further confirmed
268
the essential roles of bFGF in HA progression.
269
We found that activation of p65 and down regulation of miR-155-5p were responsible
270
for BPS induced transcription and upregulation mRNA stability of bFGF, respectively, in HA
271
cells. In bovine mammary epithelial cells (BMEC), the expression of bFGF is dependent on
272
the NF-κB and AP-1 signaling pathways (Wu et al., 2018). While BPS had no significant
273
effect on the phosphorylation of AP1 in HA cells. Further, activation of p65 was also
274
involved in BPA induced migration of cervical cancer cells (Ma et al., 2015). In male
275
zebrafish, BPS exposure can change the expression of 14 miRNAs involved in hematopoiesis,
276
lymphoid organ development, and immune system development (Lee et al., 2018). In
277
pheochromocytoma PC12 cells, BPS can regulate the expression of miR-10b to inhibit the
278
expression of KLF4 and induce cell migration (Jia et al., 2018). It has been reported that
279
miR-15a (Zhu et al., 2017), miR-146a (Liu et al., 2016), and miR-195 (Wang et al., 2017)
280
can also regulate the mRNA stability and expression of bFGF. Whether these miRNAs are
281
involved in BPS regulated expression of bFGF needs further study.
282
In conclusion, our results demonstrated that nanomolar BPS can induce the proliferation
283
of HA cells via induction of bFGF through p65 induced transcription and miR-155 regulated 13
284
mRNA stabilization. It indicated the potential risks about BPS on HA progression and
285
hormone related diseases need more attention.
286 287
Declarations
288
Ethics approval and consent to participate
289
No human/animal study was included in the present study.
290
Consent for publication
291
All authors give the consent for the publish of this study.
292
Availability of data and material
293
All data and material are available.
294
Disclosure of potential conflicts of interest
295
The authors declare no conflict of interest.
296
Funding
297
No funding information
298
Authors' contributions
299
Data collecting: Dahai Liu, Yubo Hui, Junrong Wang
300
Writing: Junrong Wang, Jianshi Du, Cong Ye
301
Data analysis: Dahai Liu, Yubo Hui, Cong Ye
302
Design: Dahai Liu, Yubo Hui, Junrong Wang, Jianshi Du,
303
Acknowledgements
304
No applicable
305 14
306
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Figure legends
442
Figure 1 BPS triggers the proliferation and cell cycle transition of HA cells. HDEC (A) or
443
CRL-2586 (B) cells were treated with increasing concentrations of BPS for 48 h, cell
444
proliferation was tested by CCK-8 kit; (C) Cells (2 × l05) treated with or without 100 nM
445
BPS were cultured in 6-well plates for two weeks before colonies were counted; (D)
446
HDEC cells were treated with or without 100 nM BPS for 24 h, the cell cycle was
447
detected using flow cytometry. Data are shown as means ± SD. **p < 0.01 compared to
448
control.
449
Figure 2 BPS increases the expression of bFGF in HA cells. HDEC (A) or CRL-2586 (B)
450
cells were treated with or without 100 nM BPS for 24 h, the expression of GFs was
451
measured by qRT-PCR; HDEC cells were treated with 100 nM BPS for the increased
452
time periods (C) or increasing concentrations of BPS for 24 h (D), the expression of
453
bFGF was checked by qRT-PCR; (E) Cells were treated with or without 100 nM BPS for
454
24 h, the expression of bFGF was checked by ELISA. Data are shown as means ± SD.
455
**p < 0.01 compared to control.
456
Figure 3 bFGF is involved in BPS induced proliferation of HA cells. HDEC cells were
457
pre-treated with anti-bFGF (A) or anti-VEGF (B) (100 ng/ml) for 2 h and then further
458
treated with or without 100 nM BPS for 24 h, the cell proliferation was tested by CCK-8
459
kit; (C) CRL-2586 cells were pre-treated with or without anti-bFGF for 2 h and then
460
further treated with or without 100 nM BPS for 24 h, the cell proliferation was tested by
461
CCK-8 kit; HDEC cells were pre-treated with or without anti-bFGF and then further
462
treated with or without 100 nM BPS for 24 h, the cell cycle of G0/G1 (D) or S (E) phase 19
463
of cells were detected using flow cytometry. Data are shown as means ± SD. **p < 0.01
464
compared to control.
465
Figure 4 bFGF can increase the transcription and mRNA stability of bFGF. (A) Cells were
466
treated with or without 100 nM BPS for 24 h, the luciferase activities of bFGF promoter
467
were measured by dual-luciferase assay; (B) HDEC cells pretreated with or without 100
468
nM BPS for 24 h were further treated with or without transcriptional inhibitor Act-D for
469
the indicated times, the mRNA of bFGF was checked by qRT-PCR; (C) After treated
470
with or without 100 nM BPS for 24 h, the mRNA of bFGF in cytoplasm and nucleus of
471
HDEC cells was checked by qRT-PCR; (D) HDEC cells were pre-treated with or without
472
100 nM BPS for 24 h and further treated with CHX (100 µg/ml) for increasing time
473
periods, the expression of bFGF was recorded (left) and quantitatively analyzed (right).
474
Data are shown as means ± SD. **p < 0.01 compared to control.
475
Figure 5 NF-κB is involved in BPS induced expression of bFGF in HA cells. HDEC (A) or
476
CRL-2586 (B) cells were treated with or without 100 nM BPS for 15 min, the
477
phosphorylation and total expression of p65, c-fos and c-Jun was measured by western
478
blot analysis; HDEC cells were pretreated with or without BAY, the inhibitor of NF-κB,
479
for 30 min, and then further treated with or without 100 nM BPS for 24 h, the mRNA
480
expression of bFGF was tested by qRT-PCR (C), and cell proliferation was tested by
481
CCK-8 kit (D). Data are shown as means ± SD. **p < 0.01 compared to control.
482
Figure 6 miR-155-5p is involved in BPS increased mRNA stability of bFGF. HDEC (A) or
483
CRL-2586 (B) cells were treated with or without 100 nM BPS for 15 min, the expression
484
of miRNAs was checked by qRT-PCR; HDEC cells pretreated with scramble control or 20
485
miR-155 mimic and then further treated with or without 100 nM BPS for 24 h, the
486
mRNA expression of bFGF was checked by qRT-PCR (C), the cell proliferation was
487
detected by CCK-8 kit (D).
488
control.
Data are shown as means ± SD. **p < 0.01 compared to
489 490
21
491
Figure 1
492
493 494 495
22
C
498 0 0
200
4
300
D
**
** **
100
8 12 24 h Relative mRNA expression (%)
200
100
0
300 **
200
0 ** **
100
0 10 20 50100 nM
F FG F3 IG F1 H G F VE G TG F Fβ
F
**
G
**
ELISA (pg/ml)
G
B Relative mRNA expression (%)
HDEC
bF
aF
F1 H G F VE G TG F Fβ
F3
F
F
300
IG
FG
bF G
aF G
Relative mRNA expression (%)
A
Relative mRNA expression (%)
496 Figure 2
497
400 CRL-2586
300 **
E
Con BPS
200
100 0
600 **
0 HDEC
Con BPS
400
200 **
100 CRL
499
500
23
501
Figure 3
B HDEC 300
Relative cell proliferation (%)
Relative cell proliferation (%)
A **
200 NS 100
0
HDEC 300
** 200
100
0
Con BPS Con BPS
Relative cell phase (%)
D
80
Anti-bFGF
G0/G1 ** NS
60
40
20
Con
E
50
Anti-VEGF
CRL-2586 300
**
200 NS 100
0 Con BPS Con BPS Con
Anti-bFGF
S **
40 NS 30 20 10 0
Con BPS Con BPS
503
Con BPS Con BPS
Relative cell phase (%)
Con
**
C
Relative cell proliferation (%)
502
Con
Anti-bFGF
Con BPS Con BPS Con
Anti-bFGF
504 505
24
506
Figure 4
507
508 509 510
25
511
Figure 5
512
A
Con
B
BPS
Con
p-p65
p-p65
p65
p65
p-c-fos
GAPDH
BPS
CRL-2586
c-fos
C
D
p-c-Jun c-Jun GAPDH HDEC
513 514 515
26
Figure 6
150
100
m
m
iR
03 -5
HDEC
40 -1 iR m
Con
55 -1
**
BPS
iR
B
D
Relative miRNA expression (%) Relative cell proliferation (%)
50
6 -1
**
BPS
0
iR m
**
150
100
50
0
iR m
300
6 -1 m
**
03 -5 iR
CRL-2586
m
Con
55
**
BPS
-1 iR m
miR-155
**
40 -1 iR
BPS
A
C 300 200
100
0 Con
NC
BPS
200
Con
miR-155
Con
100
0
NC
Con
516 517
518 519 520
BPS
Relative miRNA expression (%)
Relative mRNA expression (%)
27
Highlights 1. BPS triggers the proliferation of HA cells; 2. bFGF is involved in BPS induced proliferation of HA cells; 3. BPS increases the mRNA stability of bFGF via decreasing miR-155-5p; 4. BPS activates p65 to initiate bFGF transcription
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: