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CPNE1 Silencing Inhibits the Proliferation, Invasion and Migration of Human Osteosarcoma Cells

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CPNE1 Silencing Inhibits the Proliferation, Invasion and Migration of Human Osteosarcoma Cells ONCOLOGY REPORTS 39: 643-650, 2018 CPNE1 silencing inhibits the proliferation, invasion and migration of human osteosarcoma cells ZHENHUAN JIANG1*, JIANNONG JIANG1*, BIZENG ZHAO2*, HUILIN YANG3*, YUNLIANG WANG4*, SHANG GUO2*, YOUPING DENG5, DEYI LU6, TIELIANG MA7, HONGWEI WANG8 and JINZHI WANG9 1Department of Orthopaedics, Affiliated Yixing Hospital of Jiangsu University, Yixing, Jiangsu 214200; 2Department of Orthopedics, Affiliated Sixth People's Hospital of Shanghai JiaoTong University, Shanghai 20023; 3Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215200; 4Department of Neurology, The 148 Central Hospital of PLA, Zibo, Shangdong 255000, P.R. China; 5Bioinformatics Core, Department of Complementary and Integrative Medicine, University of Hawaii John A. Burns School of Medicine, Honolulu, HI 96813; 6Department of Bioengineering, University of Illinois at Chicago, Chicago, IL 60607, USA; 7Central Laboratory, Affiliated Yixing Hospital of Jiangsu University, Yixing, Jiangsu 214200; 8Shanghai Realgen Biotech Inc., Pudong New District, Shanghai 203215; 9Department of Cell Biology, School of Medicine, Soochow University, Suzhou, Jiangsu 215007, P.R. China Received June 12, 2017; Accepted November 13, 2017 DOI: 10.3892/or.2017.6128 Abstract. Osteosarcoma (OS) is the most common primary The present study provides deep insight into the clinical use of malignancy of the bone affecting children and adolescents. lentiviral-mediated CPNE1 silencing for osteosarcoma therapy. Copine 1 (CPNE1) is a highly conserved calcium-dependent phospholipid-binding protein and may function in regulating Introduction signal transduction and membrane trafficking. In the present study, we investigated CPNE1 expression in osteosarcoma Osteosarcoma is one of the most common primary malignant tissues and cells, and studied the effects of small interfering bone tumors in children, young adults and adolescents (1,2). RNA (siRNA)-targeting CPNE1 on proliferation, metastasis Despite newly developed multi-agent chemotherapy and and chemosensitivity of the osteosarcoma cells. The results gradually improving surgical techniques, the prognosis for demonstrated that CPNE1 was highly expressed in the patients with metastatic osteosarcoma is still poor (3). It is osteosarcoma tissues and cell lines. Moreover, functional important to investigate the complex molecular mechanisms investigations confirmed that CPNE1 knockdown significantly and identify novel biomarkers for the treatment, diagnosis inhibited cell proliferation, colony formation, invasion and and prognosis of osteosarcoma. The copines are a widely metastasis in Saos-2 and HOS cells. Western blot analysis indi- distributed class of calcium-dependent phospholipid-binding cated that CPNE1 silencing downregulated the expression of proteins that are evolutionally conserved from Arabidopsis many proteins associated with tumorigenesis and development, to Homo sapiens (4,5). Copine 1 (CPNE1), a soluble including Ras, MEK-1/2, WNT1, β-catenin, cyclin A1, IRAK2 calcium-dependent membrane-binding protein, is ubiquitously and cIAP2. In addition, CPNE1 downregulation enhanced the expressed in various tissues and organs. CPNE1 is located sensitivity of Saos-2 cells towards cisplatin and adriamycin. on chromosome 20q11.21 region in humans and has several alternative splicing forms coding for the same 537-amino acid protein (6). CPNE1 has a pair of C2 domains (C2A and C2B) at the N-terminus and a Von Willebrand factor A (VWA) domain Correspondence to: Dr Jinzhi Wang, Department of Cell Biology, (A domain) at the C-terminus (7). It was previously reported School of Medicine, Soochow University, Suzhou, Jiangsu 215007, that the C2 domains of CPNE1 may function in cell signaling P. R. Ch i na and/or membrane trafficking pathways, and the A domain E-mail: [email protected] facilitates the binding of CPNE1 with various intracellular Dr Hongwei Wang, Shanghai Realgen Biotech Inc., Pudong New proteins (8,9). Lentivirus-based vectors with small hairpin District, Shanghai 203215, P.R. China RNA (shRNA) have been used as a successful tool for silencing E-mail: [email protected] target gene expression, particularly in cancer cells, with high specificity, stability and efficiency in vitro and in vivo (10,11). * Contributed equally However, no valid evidence concerning the biological function of CPNE1 in osteosarcoma exists to date. In the present study, Key words: lentivirus, RNA interference, CPNE1, osteosarcoma we successfully silenced CPNE1 expression in Saos-2 and cells, chemosensitivity HOS cells using RNA interference (RNAi) technology and investigated the biological role of CPNE1 in osteosarcoma. 644 jiang et al: Suppression of CPNE1 inhibits the malignant phenotype of osteosarcoma cells Materials and methods Invitrogen, was 5'-CACACAACTGGTCTCATACTT-3'. The non-silencing (NS) sequence, 5'-TTCTCCGAACGTGTCA Main reagents. Rabbit anti-human CPNE1 polyclonal antibody CGT-3', was used as a scrambled control (12). The following (cat. no. AB155675) was purchased from Abcam (Cambridge, oligonucleotides were synthesized, annealed and ligated into UK); mouse anti-human antibodies to Ras (cat. no. YT2960), the pGCSIL-GFP plasmid vector between the AgeI and EcoRI MEK-1/2 (cat. no. YT2715), cyclin A1 (cat. no. YT1168) and sites. Then, these plasmids were amplified in DH5α-competent IRAK2 (cat. no. YT2392) were purchased from ImmunoWay Escherichia coli cells and purified using the Qiagen plasmid. Biotechnology Company (Plano, TX, USA); mouse anti- Recombinant lentiviruses were produced in 293T cells by human antibodies to WNT1 (cat. no. YM0649), β-catenin co-transfection of the recombinant pGCSIL-GFP vector, along (cat. no. YM3065) and cIAP2 (cat. no. Ym1343) were also with packaging plasmids, pHelper1.0 and pHelper2.0, using purchased from ImmunoWay Biotechnology Company; Lipofectamine™ 2000. For lentivirus transduction, the Saos-2 Lipofectamine™ 2000, TRIzol reagent and Opti-MEM and HOS cells were subcultured at a density of 5x104 cells/well were purchased from Invitrogen Corporation (Carlsbad, into 6-well culture plates. After growing to 30% confluence, CA, USA); AgeI, EcoRI and SYBR-Green Master Mix kits the recombinant lentiviruses were transfected into cells at a were purchased from New England Biolabs (NEB; Beijing, multiplicity of infection (MOI) of 20. Each cell line was China); Taq DNA polymerase was purchased from Takara divided into the following groups: the scr-siRNA (cells Biotechnology Co., Ltd. (Dalian, China). Cisplatin (DDP) infected with Lv-si-CTRL) and the CPNE1-siRNA group was purchased from Qilu Pharmaceutical (Hainan) Co., Ltd. (cells infected with Lv-si-CPNE1). At 48 h post infection, the (Haikou, China). Adriamycin (ADR) was purchased from the infection efficiencies were determined using a fluorescence Zhejiang Haizheng Pharmaceutical Co., Ltd. (Taizhou, China). microscope (Leica Microsystems, Wetzlar, Germany). CPNE1- knockdown efficiency was evaluated by quantitative reverse Cell culture and tissue collection. The human osteosarcoma transcription-polymerase chain reaction (RT-qPCR) and cell lines (Saos-2 and HOS) were purchased from the Cell western blot analysis. Bank of the Chinese Academy of Sciences (Shanghai, China) and were identified by short tandem repeat (STR) method in RNA extraction and RT-PCR. Total RNA was extracted from 2015. Dulbecco's modified Eagle's medium (DMEM; HyClone, the cells after 5 days of infection using TRIzol reagent Logan, UT, USA) containing 10% fetal bovine serum (FBS) (Invitrogen) in accordance with the manufacturer's instructions, was used to culture the cells. All cells were maintained at 37̊C and then cDNA was synthesized from the total RNA. Reverse in a humidified atmosphere with 5% CO2. transcription was performed using M-MLV reverse transcrip- Twenty-five osteosarcoma and 8 cartilage tumor tissues tase (Promega, Madison, WI, USA). The expression level of samples were obtained from patients who underwent surgery CPNE1 was detected by qPCR using an SYBR-Green Master at the Affiliated Yixing Hospital of Jiangsu University between Mixture (Takara Biotechnology Co., Ltd.). qPCR was performed January 2005 and December 2015. All cases had been clini- on a Bio-Rad Connect Real-Time PCR system. GAPDH was cally and pathologically confirmed. Immunohistochemistry used as an internal control. Relative gene expression levels were was performed by using 25 samples of osteosarcoma (13 males calculated using 2-ΔΔCt analysis. Two sets of primers were used and 12 females; mean age, 37 years) and 8 samples of carti- for PCR: GAPDH forward, 5'-TGACTTCAACAGCGAC lage tumor (4 males and 4 females; mean age, 28 years). The ACCCA-3' and reverse, 5'-CACCCTGTTGCTGTAGCC experimental protocols for the present study were approved by AAA-3'; CPNE1 forward, 5'-ACCCACTCTGCGTCCTT-3' and the Hospital's Protection of Human Subjects Committee. reverse, 5'-TGGCGTCTTGTTGTCTATG-3'. Immunohistochemistry. Paraffin-embedded histological Cellomics ArrayScan assay. Briefly, Saos-2 cells infected specimens were cut into 4-µm thick sections. Then, sections with the lentiviral-mediated CPNE1-siRNA or scr-siRNA were routinely dewaxed and rehydrated in xylol, and graded were seeded into 96-well plates at a density of 2x103 cells/ alcohol. Endogenous peroxidase activity was blocked with 3% well and cultured at 37̊C in a humidified atmosphere with 5% hydrogen peroxide in phosphate-buffered solution (PBS) for CO2. The infected Saos-2 cells with green fluorescence were 15 min
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