Tumor Biol. DOI 10.1007/s13277-013-1418-3

RESEARCH ARTICLE

NOV promoted the growth and migration of pancreatic cancer cells Lei Cui & Rong Xie & Shenchun Dang & Qing Zhang & Shengfa Mao & Jixiang Chen & Jianguo Qu & Jianxin Zhang

Received: 3 October 2013 / Accepted: 12 November 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract NOV, a member of the CCN (Cyr61, CTGF and NOV) family, is involved in diverse biological processes, such as cell adhesion, proliferation and angiogenesis. However, its function in pancreatic cancer remains poorly understood. Here, we found that the expression of NOV was upregulated in pancreatic cancer tissues. Moreover, overexpression of NOV in pancreatic cancer cells promoted cell proliferation and migration, while knock down the expression of NOV impaired the tumorigenecity of pancreatic cancer cells in vitro and in vivo. Mechanistically, NOV induced epithelial–mesenchymal transition (EMT) and regulated the expression of multiple EMT marker. Taken together, our study suggested the important role of NOV in pancreatic cancer and NOV might be an important therapeutic target. Keywords NOV . Pancreatic cancer . Cancer metastasis . EMT

Introduction Pancreatic cancer is one of the most common malignancies and its poor response to chemotherapy makes the management of pancreatic cancer a complicated challenge [1]. Pancreatic cancer is an invasive and metastatic malignancy, so an approach decreasing its invasion and metastasis may facilitate the therapy. L. Cui : R. Xie : S. Dang : Q. Zhang : S. Mao : J. Chen : J. Qu : J. Zhang General Surgery Department, Affiliated hospital, Jiangsu University, Zhenjiang, Jiangsu, China J. Zhang (*) Affiliated Hospital, Jiangsu University, 438 Jie-Fang Road, Zhenjiang 212003, China e-mail: [email protected]

Tumor metastatic cascade consists of multiple successive steps [2]. To facilitate cell motility, invading cells turn to epithelial–mesenchymal transition (EMT), change the cell– cell adhesion properties and rearrange the extracellular matrix (ECM) environment [3]. The concept of EMT, which was initially developed in the field of embryology, has been extended to explain cancer progression and metastasis. Mesenchymal–epithelial transition (MET) is the reverse process of EMT, involving the stabilization of distant metastasis by allowing cancer cells to regain epithelial properties. One of the hallmarks of EMT in cancer is the down-regulation of Ecadherin, which is thought to be a repressor of invasion and metastasis [4–8]. The process of EMT was regulated by multiple inducer or repressor, such as the secreted protein and microRNA [9]. NOV belongs to the CCN (Cyr61, CTGF and NOV) family [10]. Recent studies have shown that the CCN protein family members play important roles in both physiological and pathological processes, such as tumorigenesis [11]. However, the functions of NOV protein in the tumorigenesis might be very different depend on the tissues types. For example, in Wilm's tumor, chondrosarcomas and rhabdomyosarcomas, NOV was originally found to inhibit the growth of cancer cells [12]. However, more recent study correlated NOV with increased proliferation of 3 T3 fibroblast and tissue samples of prostate and renal carcinomas [13]. Up to date, the function of NOV in the tumorigenesis of pancreatic cancer was poorly understood. In this study, the function of NOV in pancreatic cancer were studied. It showed that the expression of NOV was elevated in the clinical pancreatic cancer samples. The in vitro study revealed that over-expression of NOV in pancreatic cancer cells promoted cell growth and migration, while silencing the expression of NOV inhibited the growth and migration of pancreatic cancer cells. Moreover, downregulation of NOV impaired the metastasis of pancreatic cancer cells in vivo. Mechanically, it showed that NOV induced

Tumor Biol.

EMT of pancreatic cancer cells. Taken together, our study revealed the oncogenic role of NOV in pancreatic cancer and suggested that NOV might be a potential target for the treatment.

Materials and methods Primary pancreatic cancer samples Primary tissues were collected from patients who received surgery for pancreatic cancer at Affiliated Hospital of Jiangsu University. All patients have given informed consent. Dissected samples were frozen immediately after surgery and stored at −80 °C until needed. Cell culture and transfection Pancreatic cancer cell lines (MIAPaca2, BXPC3 and Suit2) were obtained from the American Type Culture Collection and cultured in DMEM medium (Invitrogen) containing 10 % fetal bovine serum, 10 units/ml penicillin-G and 10 mg/ml streptomycin. Cells were incubated at 37 °C in humidified air containing 5 % CO2. To generate the NOV expression vector, the open reading frame of human NOV cDNA was cloned into the eukaryotic expression vector pcDNA3.1myc. The NOV expression vector and empty pcDNA3.1myc vector were transfected into MIAPaca2 and BXPC3 cells using Lipofectamine 2000 reagent (Invitrogen). The transfected cells were selected in the presence of 600 μg/ ml G418, and the resistant cells were pooled and used for the further study. Immunohistochemistry Paraffin-embedded pancreatic cancer tissues and paired normal tissues were obtained from Affiliated Hospital of Jiangsu University. First, 5-μm-thick consecutive sections were cut and mounted on glass slides. After deparaffinizing, rehydrateing, antigen retrieval and blocking endogenous peroxidases, the sections were washed thrice in 0.01 mol/l PBS (8 mmol/l Na2HPO4, 2 mmol/l NaH2PO4 and 150 mmol/l NaCl) for 5 min each, blocked for 1 h in 0.01 mol/l PBS supplemented with 0.3 % Triton X-100 and 5 % normal goat serum, followed by addition of anti-NOV antibody (1:200; Santa Cruz) at 4 °C overnight. After brief washes in 0.01 mol/l PBS, sections were exposed for 2 h to 0.01 mol/l PBS containing horseradish peroxidase-conjugated mouse anti-goat IgG (1:500), followed by development with 0.3 % H2O2 and 0.03 % 3,30-diaminobenzidine in 0.05 mol/l Tris–HCl (pH 7.6). Immunohistochemistry was performed at least three times, and the sections were counterstained with hematoxylin.

Immunoblotting Western blot analysis was performed according to the standard protocols. Primary antibodies to NOV, N-cadherin, Vimentin, Fibronectin, E-cadherin and c-Myc (also for myc tag) were purchased from Santa Cruz Biotechnology and antibody to GAPDH was purchased from Sigma. Secondary antibodies: rabbit anti-mouse IgG (Sigma) and goat anti-rabbit IgG (Cell Signaling Technology) were used at a dilution of 1:1500. Primary antibodies were diluted in TBST containing 1 % bovine serum albumin (BSA). The immunoreactive protein bands were visualized by ECL kit (Pierce). Immunostaining Cells (MIAPaca2/pcDNA3.1 or MIAPaca2/myc-NOV) were digested with trypsin and plated on the cover slides. The next day, the cells were washed thrice with PBS at room temperature and fixed in methanol at −20 °C for 10 min. Cells were blocked with 2 % BSA at room temperature for 1 h. Primary antibody incubations were performed overnight at 4 °C (E-cadherin, 1:300; N-cadherin, 1:100). After extensive washing with PBS, secondary antibody (1:1,000) incubations were performed at room temperature for 1 h. Samples were washed again with PBS and then mounted in Mowiol 488 (Sigma-Aldrich, St. Louis, MO, USA). Fluorescence was monitored by inverted confocal laser microscopy (Carl Zeiss, Thornwood, NY, USA). RT-PCR analysis TRIzol reagent (Invitrogen) was used to isolate total RNA from tissues. Two micrograms of total RNA with high quality was processed directly to cDNA with the reverse transcription kit (Promega, Madison, WI, USA), following the manufacturer's instructions, in a total volume of 25 μl. The primer pair used for amplification of the human NOV gene was as follows: forward primer, 5′-ATGCAGAGTGTGCAGAGCA-3′ and reverse primer, 5′-CGTGCAGATGCCAGTCTGGT-3′. As an internal standard, a fragment of human beta-actin was amplified by PCR using the following primers: forward primer, 5′-GATCATTGCTCCTCCTGAGC-3′, and reverse primer, 5′-ACTCCT GCTTGCTGATCCAC-3′. Amplification reactions were performed in a 20-μl volume of the LightCyclerDNA Master SYBR Green I mixture from Roche Applied Science as follows: with 10 pmol of primer, 2 mM MgCl2, 200 μM dNTP mixture, 0.5 units of Taq DNA polymerase and universal buffer. All of the reactions were performed in triplicate in an iCycler iQ System (Bio-Rad), and the thermal cycling conditions were as follows: 95 °C for 3 min; 40 cycles of 95 °C for 30 s, 58 °C for 20 s, and 72 °C for 30 s; 72 °C for 10 min. To confirm the specificity of amplification, the PCR products from each primer pair were subjected to a melting curve analysis and electrophoresis in 2 % agarose gel.

Tumor Biol. Fig. 1 The expression of NOV was up-regulated in pancreatic cancer. a Relative mRNA level of NOV in human pancreatic cancer samples and paired normal tissues. Semi-quantitative RT-PCR was performed on 61 pancreatic cancer samples and 80 normal tissues. The NOV expression was normalized to that of beta-actin. Data was calculated from triplicates. P

NOV promoted the growth and migration of pancreatic cancer cells.

NOV, a member of the CCN (Cyr61, CTGF and NOV) family, is involved in diverse biological processes, such as cell adhesion, proliferation and angiogene...
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