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A Novel Function of NUCB2 in Promoting the Development and Invasion of Renal Cell Carcinoma

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A Novel Function of NUCB2 in Promoting the Development and Invasion of Renal Cell Carcinoma ONCOLOGY LETTERS 15: 2425-2430, 2018 A novel function of NUCB2 in promoting the development and invasion of renal cell carcinoma HUAN XU1*, WENZHI LI1,2*, KAI QI3, JUAN ZHOU1, MENG GU1 and ZHONG WANG1 1Department of Urology, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011; Departments of 2Urology and 3Surgery, Linyi People's Hospital, Shandong University, Linyi, Shandong 276003, P.R. China Received April 2, 2016; Accepted June 15, 2017 DOI: 10.3892/ol.2017.7563 Abstract. Previous studies have assessed nucleobindin cell carcinoma. NUCB2 may be an important prognostic 2 (NUCB2) expression in multiple urological cancer cell factor and target in the diagnosis and treatment of human lines and detected its effect on renal cancer cell apoptosis. renal cancer. Additionally, certain reports have indicated a novel function of NUCB2 in promoting invasion in renal cancer. The levels of Introduction NUCB2 expression in different tumor cell lines were detected by quantitative reverse transcription polymerase chain reac- Renal cell carcinoma (RCC) is the most common malignant tion (RT-qPCR). Human NUCB2 and β-actin (ACTB) cDNA neoplasms of the kidney (1). RCC represents 3-4% of adult plasmids were inserted into lentivirus plasmids, which were solid tumors globally, and metastasis is detected in ~30% of then transfected into 786-O cells. Western blotting and patients during initial diagnosis due to the lack of symptoms RT-qPCR were then performed to determine the gene expres- typical of the disease at the early stage (1). Additionally, the sion in NUCB2-knocked down cells. Apoptosis was also 5-year survival of the patients with metastatic RCC has been examined by flow cytometry subsequent to successful trans- identified to be <10% (2). The major therapeutic tool for RCC fection. Finally, a transwell invasion assay was performed to is surgery, though targeted therapy may be opted for in cases investigate the effects on invasive abilities in renal cancer where surgery does not prove effective. Therefore, attention cells. The RT-qPCR results demonstrated a high expression has been focused on the diagnosis and targeted treatment of of NUCB2 in 786-O, ACHN and LNCaP cells, and there RCC. As previously described, RCC development is induced was particularly high expression in renal cancer 786-O cells. by metabolic factors (3). Genes, including Von Hippel-Lindau Following successful transfection, downregulation of NUCB2 tumor suppressor, MET, folliculin, fumarate hydratase, SDH, facilitated renal carcinoma cell apoptosis, as demonstrated by tuberous sclerosis (TSC) 1 and TSC2, are typically mutated an increased apoptosis rate in the lenti-NUCB2-KD 786-O in RCC, all of which have fundamental roles in the regula- cells (13.72±0.84 vs. 3.32±0.10; lenti-NUCB2-KD group vs. tion of metabolic processes (3). This suggests that metabolic negative control). Notably, a significant decreased invasion rate disorders involved in energy and nutrient sensing are key was observed in the NUCB2 knocked-down cells compared factors in the development of RCC, offering novel targets for with negative control, suggesting an invasion-promoting tumor diagnosis and treatment. Therefore, metabolic factors, effect of NUCB2. These results suggested a novel function of which underlie RCC development, may provide novel targets NUCB2 in the process of development and invasion in renal for screening and developing more effective therapeutic strategies. Nucleobindin 2 (NUCB2), as a metabolic factor, is a neuro- peptide that serves important roles in regulating food intake and energy homeostasis (4). The protein, NUCB2/nesfatin-1 Correspondence to: Dr Zhong Wang or Dr Meng Gu, Department was identified by Oh et al (4) in 2006 as a satiety molecule of Urology, Shanghai Ninth People's Hospital, Shanghai expressed in the hypothalamus. The NUCB2 gene is located on Jiaotong University School of Medicine, 639 Zhizaoju Road, chromosome 11, which consists of 14 exons spanning 54,785 Shanghai 200011, P.R. China nucleotides and codes a precursor 396-amino-acid protein E-mail: [email protected] E-mail: [email protected] NUCB2 (5,6). NUCB2 was first reported to be expressed in the arcuate nucleus, lateral hypothalamus, paraventricular *Contributed equally nucleus and supraoptic nucleus of the hypothalamus (4,5). Following this, a number of studies have suggested that the Key words: nucleobindin 2, renal cell carcinoma, invasion, primary peripheral source of NUCB2 was originated from apoptosis, urological cancer stomach mucosa, and NUCB2 was also peripherally expressed in pancreas and fat tissues (7,8). In the hypothalamus, NUCB2 inhibits food intake and promotes body metabolism (4). As 2426 XU et al: NOVEL FUNCTION OF NUCB2 IN PROMOTING THE DEVELOPMENT AND INVASION OF RCC previously described, it has been report that the fasting plasma Table I. List of primer sequences for polymerase chain reaction. NUCB2 levels were significantly decreased in patients with type 2 diabetes compared with healthy or type 1 diabetes Size of subjects (9). Additionally, plasma NUCB2 appears to regu- Target Primer sequence (5'-3') product (bp) late glucose and fatty acid metabolism (10,11). NUCB2 also inhibits the apoptosis of pancreatic cells, which tends to accel- h ACTB-F2 GCACCCAGCACAATG 254 erate the development of metabolic disorders (12). In previous AAGA years, an increasing number of studies have focused on the h ACTB-R2 AATAAAGCCATGCCAA association between NUCB2 and carcinoma development. TCTCA Suzuki et al (13) suggested that NUCB2, as a tumor promoter, h NUCB2-F1 AAAAGGCAAGAAGTA 124 served an important role in the process of development and GGAAGGTT metastasis in breast carcinomas. Additionally, an association between NUCB2 expression and biochemical recurrence and h NUCB2-R1 CAGGATTCAGGTGGTT progression of prostatic cancer has been reported (6,14,15). TAGGTG In a recent study, clear cell RCC tissues were observed to ACTB, β-actin; NUCB2, nucleobindin 2; F, forward; R, reverse. express NUCB2 protein, which served a critical function in tumorigenesis and tumor development (16). However, there is a lack of studies investigating the effects of NUCB2 protein on the invasion and apoptosis rates of tumors, particularly in RCC. In the present study, NUCB2 Quantitative reverse transcription polymerase chain reac‑ expression was first analyzed in various urological carcinoma tion (RT‑qPCR). The total RNA was extracted using TRIzol® cell lines, and an in vitro study investigating the effect of reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, NUCB2 on RCC invasion and development was designed. MA, USA) according to the manufacturer's protocol. Following this, 20 µl RNase‑free water was added into the Materials and methods RNA, and ultraviolet analysis was used to measure the RNA content. RNA was then subjected to RT to obtain cDNA for Cell culture. All of the cell lines (293, 786-O, ACHN, PC-3 qPCR analysis. The sequences of primers are summarized and LNCaP) were purchased from the American Type in Table I, and the human NUCB2 and ACTB primers were Culture Collection (Manassas, VA, USA) and maintained at designed according to the sequences listed. Subsequent to the ‑80˚C with Cell Culture Freezing medium (Thermo Fisher synthesis of the probes, RT-qPCR was performed according Scientific, Inc., Waltham, MA, USA). All the cell lines were to the manufacturer's protocol of the PrimeScript™ RT-PCR cultured and maintained in RPMI-1640 medium (HyClone; kit (Takara Biotechnology Co., Ltd., Dalian, China). The GE Healthcare Life Sciences, Logan, UT, USA), 10% fetal qPCR was performed with the PCR mix containing 2X SYBR bovine serum (Thermo Fisher Scientific, Inc.) and 50 µg/ml Green mix (Takara Biotechnology Co., Ltd.). The Cq value streptomycin. All cells were cultured at 37˚C in an atmo- was automatically recorded using the PCR machine. Thermal sphere of 5% CO2 under saturated humidity. The 786-O cycler PCR conditions consisted of heating samples to 95˚C cells were divided into three groups: i) non-transfected for 2 min; 40 cycles of 95˚C for 15 sec, 60˚C for 20 sec, and control cells (NT group); ii) β-actin (ACTB)-transfected cells 72˚C for 20 sec; and 72˚C for 30 min. The relative expres- (lenti-NC cells) and iii) NUCB2-knocked down 786-O cells sion levels of NUCB2 mRNA were calculated using Quantity (lenti-NUCB2-KD cells). One software (version 4.6.2; Bio-Rad Laboratories, Inc., -ΔΔCq Hercules, CA, USA) using the 2 method: ΔΔCq = (Cqtarget Western blotting. The cells were lysed in ice-cold standard gene - Cqreference gene)test group ‑ (Cqtarget gene - Cqreference gene)blank control RIPA lysis buffer (Beyotime Institute of Biotechnology, group (17). Three replicates were performed and the HEK-293 Haimen, China), and the protein concentration was determined cell line was used as the control group. using a bicinchoninic protein assay kit. In total, 50 µg of each protein was loaded per lane and separated using SDS-PAGE Lentivirus infection. 5'-GAG GAC CAC TGC TAC AGT A-3', with a 10% gel and then transferred onto polyvinylidene 5'-TTT AGT AAC ACC ATG TGA G-3' and 5'-GCT CAG AAT fluoride membranes (EMD Millipore, Billerica, MA, USA). GGA ATA TCA T-3', (lenti-NUCB2-KD1, lenti-NUCB2-KD2 Following blocking with 5% bovine serum albumin (Solarbio, and lenti-NUCB2-KD3, respectively) were selected to be Beijing, China) for 1 h at 37˚C, primary antibodies against knocked down in the present study. A β-actin (ACTB) cDNA NUCB2 (1 µg/ml; cat. no. AF5949; R&D Systems, Inc., plasmid was used as a negative control, and the lenti-NC cells Minneapolis, MN, USA) and GAPDH (1:3,000; cat. no. 8884; were treated as the negative control group. Human NUCB2 Cell Signaling Technology, Inc., Danvers, MA, USA) were used. and ACTB cDNA plasmids were transfected with ViraPower The secondary antibodies were peroxidase‑conjugated immu- packaging mix using Lipofectamine 2000 reagent (Thermo noglobulin G (rabbit anti-sheep, ZDR-5308; sheep anti-rabbit, Fisher Scientific, Inc.) into 786‑O cells for 48 h to generate the ZDR‑5306; Beijing Zhongshan Jinqiao Biotechnology Co., lentivirus according to the manufacturer's protocol.
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