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Regulation of Pancreatic Stellate Cell Activation by Notch3 Haiyan Song and Yuxiang Zhang*

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Regulation of Pancreatic Stellate Cell Activation by Notch3 Haiyan Song and Yuxiang Zhang* Song and Zhang BMC Cancer (2018) 18:36 DOI 10.1186/s12885-017-3957-2 RESEARCHARTICLE Open Access Regulation of pancreatic stellate cell activation by Notch3 Haiyan Song and Yuxiang Zhang* Abstract Background: Activated pancreatic stellate cells (PaSCs) are the key cellular source of cancer-associated fibroblasts in the pancreatic stroma of patients with pancreatic ductal adenocarcinoma (PDAC), however, the activation mechanism of PaSCs is not yet known. The Notch signaling pathway, components of which are expressed in stromal cells, is involved in the fibrosis of several organs, including the lung and liver. In the current study, we investigated whether Notch signal transduction is involved in PaSC activation in PDAC. Methods: The expression of Notch signaling pathway components in human PDAC was examined via immunohistochemical staining and assessed in mouse PaSCs using RT-qPCR and western blotting. Notch3 expression in both PDAC stromal cells and activated mouse PaSCs was evaluated using immunofluorescence, RT-qPCR and western blotting. The impact of siRNA-mediated Notch3 knockdown on PaSC activation was detected with RT-qPCR and western blotting, and the impact on PaSC proliferation and migration was detected using CCK-8 assays and scratch experiments. The effect of conditioned medium from PaSCs activated with Notch3 siRNA on pancreatic cancer (LTPA) cells was also detected with CCK-8 assays and scratch experiments. The data were analyzed for statistical significance using Student’s t-test. Results: Notch3 was overexpressed in both human PDAC stromal cells and activated mouse PaSCs, and Notch3 knockdown with Notch3 siRNA decreased the proliferation and migration of mouse PaSCs. The levels of markers related to PaSC activation, such as α-smooth muscle actin (α-SMA), collagen I and fibronectin, decreased in response to Notch3 knockdown, indicating that Notch3 plays an important role in PaSC activation. Furthermore, we confirmed that inhibition of PaSC activation via Notch3 siRNA reduced the proliferation and migration of PaSC-induced mouse pancreatic cancer (LTPA) cells. Conclusions: Notch3 inhibition in PaSCs can inhibit the activation, proliferation and migration of PaSCs and reduce the PaSC-induced pro-tumorigenic effect. Therefore, Notch3 silencing in PaSCs is a potential novel therapeutic option for patients with PDAC. Keywords: Pancreatic ductal adenocarcinoma, Pancreatic stellate cells, Notch3, Activation Background constitute 4–7% of all pancreatic cells and are quiescent Pancreatic stellate cells (PaSCs) are myofibroblast-like [5–7], however, PaSCs can switch between quiescent and cells found in exocrine areas of the pancreas, and they activated phenotypes. In their quiescent state they have play an important role in the pathogenesis of pancrea- abundant vitamin-A-containing lipid droplets in their titis and pancreatic cancer [1–3]. Fibrosis is a major fea- cytoplasm and express specific markers, such as desmin ture of chronic pancreatitis and desmoplasia, a stromal and glial fibrillary acidic protein (GFAP) [6]. When the reaction characteristic of pancreatic ductal carcinoma pancreatic cells are injured, PaSCs transform into their cancer (PDAC) [4]. In a normal pancreas, PaSCs active state, which is characterized by loss of the cytoplasmic vitamin-A-containing lipid droplets and * Correspondence: [email protected] upregulated expression of the cytoskeletal protein Department of Biochemistry and Molecular Biology, Cancer Institute, Beijing α-smooth muscle actin (α-SMA) [6, 7]. Activated PaSCs Key Laboratory for Cancer Invasion and Metastasis Research, Capital Medical University, No. 10 Xitoutiao, You An Men, Fengtai District, Beijing 100069, subsequently synthesize excessive extracellular matrix People’s Republic of China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Song and Zhang BMC Cancer (2018) 18:36 Page 2 of 12 (ECM) proteins, such as collagen, fibronectin and Methods laminin, and the proliferation and migration of PaSCs Pancreatic tissues and animals increases [8]. Human pancreatic cancer tissue microarrays were Recently, attention has been focused on the desmo- purchased from Xi’an Alena Biotechnology Co., Ltd. of plastic reaction in pancreatic cancer, specifically how it China. For this study, male C57BL/6 J wild-type mice regulates cancer progression. This desmoplastic reaction (6 weeks old, weight range 20–25 g) were supplied by the occurs because activated PaSCs secrete large quantities of Laboratory Animal Services Center of Capital Medical ECM proteins, including collagen types I, III, and IV, into University. Mouse LTPA cells (ATCC Number: CRL- the tumor microenvironment [7, 8]. There is strong evi- 2389™)wereobtainedfromAmericanTypeCulture dence of a correlation between activated PaSCs and PDAC Collection (ATCC). [9–11]. Thus, elucidation of the mechanism underlying PaSC transformation from a quiescent to an activated Cell isolation and culture conditions phenotype has many important implications. We isolated normal mouse PaSCs from the pancreas using Several signaling pathways and molecules that mediate the outgrowth method described by Apte and Bachem [5, PaSC activation have been identified, including Sonic 6]. PaSCs were cultured in DMEM/F12 (Gibco, New York, hedgehog [12, 13], mitogen-activated protein kinases USA) containing 20% fetal bovine serum (FBS) (Gibco, [14], peroxisome proliferator activated receptor γ [15, New York, USA) and antibiotics (1% penicillin and 16], the Janus kinase/signal transducer and activator of streptomycin) (Beyotime, Haimen, China). PaSCs were transcription pathway, and the transcription factor confirmed by their fibroblast-like morphology and nuclear factor-kappa B [17–20]. immunocytochemical positivity for PaSC markers such as However, more research is required to understand the α-SMA, collagen I and fibronectin. details of PaSC activation. The Notch signaling family is an evolutionarily highly conserved signaling pathway. Immunohistochemical staining Notch activation plays critical roles in embryonic Xylene and a graded alcohol series (ZSGB-BIO, Beijing, development, cell differentiation, cell proliferation and China) were used for dewaxing and rehydration. Subse- apoptosis [21, 22]. The canonical Notch signaling quently, sections were treated with citrate salt buffer pathway is known as the CSL-dependent pathway. Notch (pH 6.0) in the microwave for 15 min for antigen re- receptor proteins can be activated by interacting with a trieval, followed by 3% hydrogen peroxide (ZSGB-BIO, family of ligands on adjacent cells. Upon activation, the Beijing, China) for 15 min to block endogenous peroxid- Notch receptor is cleaved, and the intracellular domain ase activity. Then, the samples were blocked with 5% of the Notch receptor (NICD) is released from the mem- donkey blood serum (Jackson, West Grove, USA) in brane into the cytoplasm and translocates into the nu- phosphate-buffered saline (PBS) for 1 h at room cleus. NICD in the nucleus binds with CSL (CBF1/ temperature. The primary antibodies used in our experi- Su(H)/LAG-1, also known as RBP-Jκ) and forms a tran- ments are listed in Table 1. The samples were then incu- scriptional activation complex that acts as a potent bated with primary antibodies (against Notch1, Notch2, transcriptional activator of CSL target genes, such as Notch3, Notch4, Jagged1, Jagged2, Delta1, Delta3 and Hes1, and thus promotes downstream gene expression Delta4) at 4 °C overnight, followed by incubation with [23]. Notch signaling pathway components are highly secondary horseradish peroxidase (HRP)-conjugated expressed in PDAC [24–26], and inhibition of the Notch antibodies (ZSGB-BIO, Beijing, China) for 1 h at room signaling pathway inhibits PDAC progression [27, 28]. temperature. Next, diaminobenzidine (DAB) and These results indicate that the Notch signaling pathway hematoxylin (ZSGB-BIO, Beijing, China) were applied plays an important role in PDAC occurrence and for staining and counterstaining. After dehydration with progression. In addition, the Notch pathway is involved a graded alcohol series and xylene, the samples were in stromal cell activation during lung and hepatic sealed with coverslips and neutral gum. fibrosis [29–32], however, the role of the Notch pathway in PaSC activation remains undefined. We hypothesized Immunofluorescence that components of the Notch pathway are present in Following dewaxing, rehydration and antigen retrieval, PaSCs and that Notch signaling regulates the activation the samples were immunostained with mouse monoclo- of these cells. To date, four Notch receptors have been nal anti-α-SMA (1:100) and rabbit polyclonal anti- identified in mammals, and the presence of multiple Notch3 (1:50) antibodies at 4 °C overnight. The sections Notch receptors and ligands suggests that different were then incubated with Alexa Fluor 594-conjugated receptors play different roles in PaSC activation. In the donkey anti-rabbit IgG (Invitrogen, Chicago, USA) present
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