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Study of the Expression and Function of ACY1 in Patients with Colorectal Cancer

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Study of the Expression and Function of ACY1 in Patients with Colorectal Cancer ONCOLOGY LETTERS 13: 2459-2464, 2017 Study of the expression and function of ACY1 in patients with colorectal cancer BING YU1,2, XUEZHONG LIU3, XIUZHEN CAO2, MINGYUE ZHANG2 and HONG CHANG1 1Department of Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021; 2Department of Colorectal Surgery, Tai'an Central Hospital, Tai'an, Shandong 271000; 3Department of Gastrointestinal Surgery, Liaocheng People's Hospital, Liaocheng, Shandong 252000, P.R. China Received February 22, 2016; Accepted October 19, 2016 DOI: 10.3892/ol.2017.5702 Abstract. Aminoacylase 1 (ACY1) is important for regulating area of intense study; however, it is a complex issue. Previous the proliferation of numerous types of cancer. However, the studies indicate that tumor cells of different clinical stages expression and mechanisms underlying the function of ACY1 may exhibit different expression levels of certain biomarkers, in colorectal cancer remain unclear. In order to investigate which could be used as a target for the diagnosis or treatment the expression and function of ACY1 in colorectal cancer, of the tumor (3-5). In addition, with ongoing and advancing tumor tissue and blood samples were collected for analysis research into tumor biology, new targets are being identified from 132 patients diagnosed with colorectal cancer. Reverse and studied, including aminoacylase 1 (ACY1) (6). transcription-quantitative polymerase chain reaction analysis ACY1 is a cytosolic enzyme that deacylates the α-acylated and western blotting identified significantly increased expres- amino acid from the N-terminal peptide of intracellular sion of ACY1 mRNA in colorectal tumor tissue (P<0.05 vs. proteins (6). Following this, terminal acetylated proteins are adjacent normal tissue) and notably increased ACY1 protein more stable (7-9). In addition to its function in scavenging levels. This ACY1 mRNA expression was found to be posi- N-acylated amino acids, ACY1 has been studied in a number of tively correlated with tumor stage. In addition, plasma ACY1 types of human cancer. In small cell lung cancer (SCLC), renal concentration was increased in patients with colorectal cancer cell carcinoma and liver cancer ACY1 expression has been compared with healthy controls. Furthermore, in vitro knock- demonstrated to be significantly reduced (10-13), suggesting down of ACY1 in human colorectal cancer HT-29 cells was that ACY1 serves a role in inhibiting tumorigenesis. Limited shown to inhibit proliferation and increase apoptosis. This studies have investigated the detailed function and mechanism effect was found to be associated with the activation of ERK1 of ACY1 in tumor proliferation, and certain previous studies and TGF-β1 signaling. In conclusion, the results of the present have reported that activation of ERK 1/2 and expression of study suggest that ACY1 promotes tumor progression, and TGF-β may be implicated in this process (13-16). However, thus may be a potential target for the diagnosis and treatment the role of ACY1 in gastrointestinal cancer remains unclear. of colorectal cancer. In the present study, the function and regulation of ACY1 in colorectal cancer was investigated through analyzing clinical Introduction samples. Colorectal cancer, the third most common cancer worldwide, is Materials and methods a major public health issue in China and globally (1). Although advances have been made in surgical and medical treatments, Patients and clinical specimens. In total, 160 patients who ~40% of patients with colorectal cancer succumb to the underwent colectomy in the Department of Gastrointestinal cancer (2). The early diagnosis and assessment of tumors is an Surgery at Liaocheng People's Hospital (Liaocheng, China) between January 2010 and January 2015 were included in the present study and evaluated for the presence of tumor cells. This led to 28 cases being excluded for the absence of tumor cells. Tumor cells were found in the remaining 132 cases. Correspondence to: Dr Hong Chang, Department of Hepatobiliary Colorectal adenocarcinoma and adjacent normal colorectal Surgery, Shandong Provincial Hospital Affiliated to Shandong tissue specimens were collected from these patients, along University, 324 Jingwuweiqi Street, Jinan, Shandong 250021, P. R. Ch i na with serum samples, between January 2010 and January 2015. E-mail: [email protected] The tissues were immediately put into ice-cold RPMI-1640 medium (Hyclone; GE Healthcare Life Sciences, Logan, Key words: aminoacylase 1, colorectal cancer, tumor proliferation, UT, USA) and transported to the lab for further protein clinical diagnosis and RNA extraction. The serum and tissue specimens were collected and stored at ‑80˚C for ELISA, western blotting and reverse transcription-quantitative polymerase chain reaction 2460 YU et al: ACY1 IN COLORECTAL CANCER (RT-qPCR) analysis. In addition, 120 cases of serum samples Table I. Clinicopathological characteristics of the patients. from healthy volunteers were collected (between January 2010 and January 2015) as a control for the serum of the patients. Patients with Healthy Clinicopathological characteristics of the patients and control Characteristic colorectal cancer control group group are listed in Table I. This protocol was approved by the Institutional Review Board for Human Research of the Age (mean ± SD; 52.3±12.1; 32-69 50.1±13.8; 30-68 School of Medicine, Shandong University (Jinan, China) and range, years) informed consent was obtained from all patients. Gender (no. of patients) ACY1 RT‑qPCR. Total RNA was isolated from the tissues Male 81 72 using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, Female 51 48 MA, USA) and then reverse transcribed using the PrimeScript TNM stage (no. RT reagent kit (Clontech Laboratories, Inc., Mountain View, of patients) CA, United States), according to the manufacturer's protocol. Stage I 44 N/A Subsequently, the mRNA expression of ACY1 and β-actin Stage II 62 N/A (the internal control) was quantified through qPCR analysis (Applied Biosystems). The following primers, described Stage III 18 N/A previously (13,14), were used: ACY1 forward, 5'-GGC TGC Stage IV 8 N/A ATG AGG CTG TGTT-3' and reverse, 5'-CTT GGC ACT GGT TNM, tumor-node-metastasis; N/A, not applicable. TGG GAT G-3'; and β-actin forward, 5'-TGG CAC CCA GCA CAA TGAA-3' and reverse, 5'-CTAA GTC ATA GTC CGC CTA GAA GCA-3'. The thermocycling conditions were as follows, for a total of 40 cycles: Denaturing at 95˚C for 30 sec; annealing at 60˚C for 5 sec; and extension at 72˚C for 45 sec. and regulatory proteins, in the clinical samples and in cells The 2-ΔΔCq method was used for the quantification of the PCR in vitro. A total of 1 mg tissue was solubilized in 100 µl lysis results (13,17,18). buffer (30 mM Tris, 2 M Thiourea, 4% CHAPS, 7 M urea; pH 8.5) on ice for 30 min. The lysis buffer was subsequently Cell culture and small interfering RNA (siRNA) silencing. centrifuged at 1,000 x g for 15 min and the supernatant was Human colorectal adenocarcinoma HT-29 cells were used for collected for further testing. Subsequently, western blotting in vitro analysis of the function and regulation of ACY1. The was performed as previously described (18), and primary anti- cells were cultured in the McCoy's 5A Medium (cat. no. 16600; bodies were used for incubation at 4˚C overnight and secondary Gibco; Thermo Fisher Scientific, Inc.) supplemented with antibodies at room temperature for 1 h. Primary antibodies 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life targeting the following proteins were used: ACY1 (1:1,000; Sciences), 100 U/ml penicillin and 100 g/ml streptomycin, mouse; monoclonal; cat. no. ab54960; Abcam, Cambridge, as previously described (19). Cells were incubated at 37˚C in UK); Transforming growth factor-β1 (TGF-β1; 1:1,000; cat. 5% CO2. For the siRNA silencing, cells were treated with the no. 3709s; rabbit; monoclonal; Cell Signaling Technology, following previously described siRNA sequences (13) targeting Inc., Danvers, MA, USA); extracellular signal-related kinase the human ACY1 mRNA sequence: Sense, 5'-UCA ACA CGG 1 (ERK1; 1:1,000; cat. no. 4094; rabbit; monoclonal; Cell UCA CCA CAU AGC CAGG-3' and antisense, 5'-CCU GGC Signaling Technology, Inc.); phosphorylated-ERK1 (p-ERK1; UAU GUG GUG ACC GUG UUGA-3'. The siRNA-NC was 1:1,000; cat. no. 4370; rabbit; monoclonal; Cell Signaling used as the negative control [scrambled control siRNA (cat. Technology, Inc.); and β-actin (1:1,000; cat. no. 8H10D10; no. sc-37007) was obtained from Santa Cruz Biotechnology, mouse; monoclonal; Cell Signaling Technology, Inc.). The Inc., Dallas, TX, USA]. Related recombined adenoviral following secondary antibodies were used: anti-rabbit expression vector were constructed by Invitrogen; Thermo IgG-biotin (cat. no. BA1020); and anti-mouse IgG-biotin (cat. Fisher Scientific, Inc. Lipofectamine RNAiMAX Transfection no. BM2001) (both Boster Biological Technology, Wuhan, Reagent was used for the transfection of the siRNA (Invitrogen; China). Bands were detected using the DAB Chromogenic Thermo Fisher Scientific, Inc.), according to the manufac- Reagent kit (cat. no. AR1021; Boster Biological Technology). turer's protocol. Briefly, 0.25x106 cells per well (6-well plate) were seeded prior to transfection. The transfection reagent was Serum ACY1 concentration ELISA. Serum levels of ACY1 diluted by the medium according to the kit protocol and then protein were measured by ELISA. Prior to the ELISA, serum the siRNA was added to the diluted transfection reagent at a samples were centrifuged at 800 x g at 4˚C for 10 min and 1:1 ratio. The cocktail was subsequently incubated for 5 min the supernatant was collected. The ELISA was then conducted at room temperature and added to the cells. The transfection using the ACY1 ELISA kit (cat. no. ABIN417990; Cloud-Clone system was kept at 37˚C in 5% CO2 for 48 h (20-22). After Corporation, Houston, TX, USA), according to the manufac- 48 h, downregulation of ACY1 was measured by qPCR.
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