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UBA2 Promotes Proliferation of Colorectal Cancer

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UBA2 Promotes Proliferation of Colorectal Cancer 5552 MOLECULAR MEDICINE REPORTS 18: 5552-5562, 2018 UBA2 promotes proliferation of colorectal cancer PING HE1, XUN SUN2, HONG-JING CHENG1, YA‑BIN ZOU2, QUAN WANG3, CHANG‑LI ZHOU1, WAN-QI LIU1, YUE-MING HAO1 and XIANG-WEI MENG1 Departments of 1Gastroenterology, 2Pathology and 3Gastrointestinal Surgery, Bethune First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China Received December 18, 2017; Accepted August 13, 2018 DOI: 10.3892/mmr.2018.9613 Abstract. Small ubiquitin‑like modifier proteins are involved Introduction in tumorigenesis; however, the potential effects and func- tions of the family member ubiquitin‑like modifier‑activating Colorectal cancer is the third most common cancer world- enzyme 2 (UBA2) on colorectal cancer are not clear. The wide (1); the lifetime risk of developing colorectal cancer is present study aimed to examine the effects of UBA2 on the 4.7% for men and 4.4% for women. Although the mortality proliferation of colorectal cancer cells in vitro and in vivo. The rate from colorectal cancer has been declining for several mRNA and protein expression levels of UBA2 in patients with decades owing to the early diagnosis and improved treatment, colorectal cancer were measured by reverse transcription‑quan- >1 million novel cases are diagnosed each year. Therefore, it titative polymerase chain reaction and immunohistochemistry, is crucial to identify novel biomarkers and therapeutic targets respectively. UBA2 expression levels in colorectal cancer for colorectal cancer to improve the prognosis of the disease. tissues were significantly increased compared with the para- Sumoylation is a transient post‑translational modifica- cancerous normal tissues. The expression of UBA2 was also tion process that is highly regulated by the balance between associated with higher stage colorectal cancer and poor prog- enzyme‑mediated conjugating and deconjugating activities. nosis. MTT and colony formation assays were used to examine Small ubiquitin‑like modifier (SUMO) conjugation involves proliferation in colorectal cancer cell lines. Flow cytometry an associated enzymatic cascade that includes an E1 ubiq- was performed to examine the effects of UBA2 on the cell uitin‑activating enzyme, an E2 ubiquitin‑conjugating enzyme cycle and apoptosis of colorectal cancer cell lines and protein and an E3 ubiquitin ligase (2). It has been demonstrated that expression levels were examined by western blotting. Athymic sumoylation is associated with a range of cellular processes, nude mice were used to examine the ability of transfected including cell cycle progression, apoptosis regulation, colorectal cancer cells to form tumors in vivo. Downregulation maintenance of genome integrity, DNA repair, cell survival, of UBA2 inhibited the proliferation of colorectal cancer cell modulation of subcellular transport and transcription (3,4). lines in vitro and in vivo through the regulation of cell cycle A previous study reported that sumoylation regulates the associated protein expression and apoptosis. Furthermore, cell cycle in glioblastoma by stabilizing cyclin‑dependent downregulation of UBA2 decreased the expression levels kinase (CDK) 6 (5). Other studies have demonstrated the of cyclin B1, B‑cell lymphoma‑2, phosphorylated protein interaction between CDKs and sumoylation during cell cycle kinase B and E3 ubiquitin‑protein ligase MDM2 in colorectal regulation (6,7). The imbalance between sumoylation and cancer cells, whereas the expression levels of p21 and p27 were desumoylation is associated with a variety of diseases, such increased. UBA2 was demonstrated to serve an essential role as cancer (8-10). For example, sumo‑conjugating enzyme in the proliferation of colorectal cancer and may be used as a UBC9 is upregulated in a number of types of cancer, including potential biomarker to predict prognosis and as a therapeutic prostate cancer, breast cancer, advanced melanomas and colon target in colorectal cancer. cancer (11,12). UBA2/SAE2 was reported to promote the growth of colon tumors in mice (13), and tumor aggressiveness was associated with increased MDM2 expression induced by the upregulation of SUMO1 in patients with oral squamous cell carcinoma (14). Ubiquitin‑like modifier‑activating enzyme 2 (UBA2), Correspondence to: Dr Xiang‑Wei Meng, Department of Gastroenterology, Bethune First Hospital of Jilin University, also known as sumo‑activating enzyme subunit 2 (SAE2), is 71 Xinmin Street, Changchun, Jilin 130021, P.R. China a subcomponent of the sumoylated E1 enzyme. The human E‑mail: [email protected] UBA2 gene is located on chromosome19q12, which is one of the most important enzymes that directly affect the levels Key words: ubiquitin‑like modifier activating enzyme 2, colorectal of sumoylation in the body (3,15,16). A number of previous cancer, cell proliferation, apoptosis studies have implicated UBA2 in clinical diseases; for example, haploinsuffiency of the UBA2 gene is associated with cutis aplasia (17,18). In addition, UBA2 was reported to form a fusion protein with the DNA dC>dU‑editing enzyme HE et al: ROLE OF UBA2 IN COLORECTAL CANCER 5553 APOBEC3G to prevent the degradation of the APOBEC3G The concentrations and purity of the extracted RNA were protein by human immunodeficiency virus (HIV)‑viral infec- measured on a spectrophotometer at 260 and 280 nm wave- tivity factor (19). Therefore, UBA2 may serve an inhibitory lengths. role in HIV infectivity, which suggested that UBA2 may exert its biological function by stabilizing certain proteins. Recent Reverse transcription‑quantitative polymerase chain reaction studies have demonstrated that UBA2 was highly expressed in (RT‑qPCR) assay. Total RNA from frozen colorectal cancer certain malignant diseases, including liver cancer (20), small tissues or cell cultures was reverse transcribed into cDNA cell lung cancer (21) and gastric cancer (22). Another study using a Reverse Transcriptase kit (Qiagen, Inc., Valencia, CA, reported that the expression levels of UBA2 were different USA) according to the manufacturer's protocol. UBA2 mRNA in metastatic colorectal cancer compared with the primary expression levels were measured by qPCR using 12.5 µl tumor (23). However, the function and clinical significance of SYBR‑Green (cat. no. DRR041B; Takara Biotechnology UBA2 in colorectal cancer are not clear. Co., Ltd., Dalian, China) 1 µl each primer, 2 µl cDNA and In the present study, the expression level of UBA2 was 8.5 µl diethyl pyrocarbonate‑treated water. The thermocy- examined in patients with colorectal cancer, and UBA2 cling conditions were as follows: Initial denaturation at 95˚C expression was associated cancer stage. Furthermore, the for 15 sec, followed by 40 cycles of 95˚C (5 sec) and 60˚C function of UBA2 on cell proliferation in vitro and in vivo (30 sec). The primers used were as follows: GAPDH, forward, was examined. The results of the present study indicated that 5 ' ‑ T G A CTT CAA CAG CGA CAC CCA‑3' and reverse, 5'‑CAC UBA2 may serve important roles in tumorigenesis and may CCT GTT GCT GTA GCC AAA‑3'; UBA2, forward, 5'‑CAC act as a potential therapeutic target in colorectal cancer. AGG TTG CCA AGG AA‑3' and reverse, 5'‑GAC ACT CAT AAC ACT CGG TCA‑3'; GAPDH was used as an internal control. Materials and methods UBA2 mRNA expression levels were quantified using the 2-ΔΔCq method and normalized to the expression levels of Patients. Colorectal cancer tissues were collected from GAPDH (26). All experiments were performed three times in 237 patients with colorectal cancer in Bethune First Hospital triplicate. of Jilin University (Changchun, China) between January 2009 and December 2015; patient clinicopathological characteristics IHC analysis. IHC was performed to examine the protein are presented in Table I. Patients who received preoperative expression of UBA2 in colorectal cancer specimens. The radiotherapy or chemotherapy and patients with multiple specimen sections (5 µm) were deparaffinized using xylene for tumors were excluded from the present study. The pathological 5 min at room temperature three times, which was followed by stage of cancer was determined according to the American rehydration using 100% ethanol for 10 min, 95% ethanol for Joint Committee on Cancer stage. All tumors were confirmed 10 min and water for 10 min. Antigen retrieval via steam treat- by a pathologist and have been collected intraoperatively. The ment in citrate buffer was then performed at 120˚C for 10 min. fresh specimens for molecular analysis were immediately Endogenous peroxidase activity was blocked by 3% hydrogen frozen in liquid nitrogen and stored in ‑80˚C. Specimens for peroxide at room temperature for 10 min. Non‑specific protein immunohistochemistry (IHC) were fixed in formalin for 24 h binding was blocked by 10% normal sheep serum for 30 min at room temperature and embedded in paraffin for further at room temperature (Fuzhou Maixin Biotech Co., Ltd., experiments. The present study was approved by the Ethics Fuzhou, China). Sections were incubated with anti‑UBA2 Review Committee at Jilin University. All patients were primary antibody (1:100; Abcam, Cambridge, MA, USA; cat. informed their participation rights and signed the written no. ab185955) at 4˚C overnight. Subsequently, the sections informed consent. were incubated with a secondary antibody conjugated with HRP [cat. no. KIT‑9706; UltraSensitive™ SP (Rabbit) IHC kit; Bioinformatic analysis. Analysis of UBA2 mRNA expression Fuzhou Maixin Biotech Co., Ltd.] for 40 min at room tempera- levels was performed
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