ONCOLOGY LETTERS 18: 49-56, 2019 Homeobox A10 promotes the proliferation and invasion of bladder cancer cells via regulation of matrix metalloproteinase‑3 CHUNLEI LIU1*, MINGZHU GE2*, JUN MA1*, YANHUI ZHANG1, YANHUI ZHAO1 and TAO CUI1 Departments of 1Urology and 2Ultrasound, Qingdao Central Hospital, Qingdao, Shandong 266042, P.R. China Received February 9, 2018; Accepted January 31, 2019 DOI: 10.3892/ol.2019.10312 Abstract. Homeobox A10 (HOXA10) belongs to the family Smoking and obesity are risk factors for BC (2), and genetic of HOX genes, which are closely connected with embryonic mutations and abnormal protein expression serve important development and serve important roles in various tumors. roles in the genesis, development and progression of BC (4). However, the role of HOXA10 in bladder cancer (BC) remains Therefore, exploring new anomalous molecules involved in unclear. In the present study, the role of HOXA10 in BC and the development of BC may advance the understanding of the underlying mechanisms by which it promotes the disease the mechanisms behind this disease and contribute to the progression were investigated. Immunohistochemical analysis improvement of treatment strategies. demonstrated that the expression of the HOXA10 protein Homeobox A10 (HOXA10) belongs to the family of HOX was significantly higher in BC tissues as compared with that genes, which are classified into four subgroups, namely HOX in adjacent normal tissues. Subsequent statistical analysis A-D (5), and are closely connected with embryonic develop- revealed that upregulation of HOXA10 was significantly ment (6). HOXA10 encodes a DNA-binding transcription factor associated with the pathological grade and clinical stage of that serves vital roles in regulating gene expression, viability BC patients. In the BC cell lines T24 and 5637, silencing of and differentiation of cells in the embryo and morphogenesis. HOXA10 by small interfering RNA transfection suppressed An increasing number of studies have demonstrated that HOX the proliferation, migration and invasion of BC cells, and led genes, including HOXA10, serve important roles in oncology to decreased matrix metalloproteinase-3 expression. Taken and in the development of various tumors (7). Previous studies together, overexpression of HOXA10 may be associated with have identified abnormal HOXA10 expression and reported poor prognosis in BC, and may serve as a novel antitumor the cancer-promoting activities of HOXA10 in various malig- therapy target for the treatment of this disease. nant disorders, including endometrial, nasopharyngeal, gastric and breast cancer (7-11). However, the expression and role of Introduction HOXA10 in BC has not been previously reported. Based on the abnormal expression and cancer-promoting Bladder cancer (BC) is the most common malignancy of roles of HOXA10 in the development of various cancers, the urinary system and its incidence presents an increasing we hypothesized that HOXA10 may play vital roles in the trend (1). Urothelial carcinoma is the most common subtype development of BC. Initially, immunohistochemistry was of bladder cancer and is categorized as muscle invasive or used to determine the expression of HOXA10 in BC and non-muscle invasive (2). Despite the advances in surgical adjacent normal tissues. After revealing the high expression techniques and the use of chemotherapy as a treatment option, level of HOXA10 in BC, cell proliferation, migration and a great number of patients experience little clinical benefit due invasion assays were performed in T24 and 5637 cells with to the likelihood of recurrence and early tumor infiltration (3). HOXA10 knocked down by small interfering RNA (siRNA). Furthermore, western blotting analysis was carried out to explore the mechanism by which HOXA10 promotes BC progression and facilitates the malignant behavior of BC cells. Correspondence to: Dr Yanhui Zhao or Dr Tao Cui, Department of Materials and methods Urology, Qingdao Central Hospital, 127 Si Liu Nan Road, Qingdao, Shandong 266042, P.R. China Patients and specimens. A total of 78 paired fresh specimens E-mail: [email protected] of BC and adjacent normal tissues) were obtained from E-mail: [email protected] patients diagnosed with BC and underwent total cystectomy *Contributed equally in Qingdao Central Hospital (Qingdao, China) between March 2011 and August 2015. None of the patients underwent any Key words: homeobox A10, bladder cancer, cell invasion, matrix preoperative therapy, such as chemotherapy or radiation metalloproteinase-3 therapy. The exclusion criteria also included presence of tumors with histologically identified extensive necrosis. The diagnosis and pathological grades of the patients were verified 50 LIU et al: HOXA10 PROMOTES BLADDER CANCER VIA MMP-3 REGULATION using hematoxylin and eosin (H&E) staining, according to Ltd., (Suzhou, China). The siRNA sequences were as follows: the World Health Organization classification guidelines (12). si-HOXA10, 5'-CAC GGA CAG ACA AGT GAA ATT-3' (sense) Patients were assigned to three pathological grades (GI-III) and 5'-GTG CCT GTC TGT GTT CAC TTT TT-3' (antisense); according to the differential degree of tumor differen- si-Ctrl, 5'-UUC UCC GAA CGU GUC ACG UTT-3' (sense) tiation (12), and the patient population included 17, 25 and 36 and 5'-ACG UGA CAC GUU CGG AGA ATT-3' (antisense). cases with a pathological grade of I, II and III, respectively. T24 and 5637 cells were transfected with the siRNAs using The clinical stage of BC patients was determined according Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, to the American Joint Committee on Cancer guidelines (13), Inc.) according to the manufacturer's protocol. Western blot- including stages I, II, III and IV. The patient population in the ting was subsequently used to assess the silencing efficiency of present study consisted of 38 and 40 cases with clinical stages si‑HOXA10. Initially, the silencing efficiency of si‑HOXA10 of I+II and III+IV, respectively. All patients agreed to the use on HOXA10 expression in T24 and 5637 cells compared with of their samples in the present study and signed an informed si-Ctrl and untreated control groups was detected. As si-Ctrl consent prior to surgery. The current study was approved by the had little effect on HOXA10 and was similar to the untreated Ethical Reviewing Committee of Qingdao Central Hospital. group, cells were treated only using si-HOXA10 and si-Ctrl in Patient characteristics are listed in Table I. subsequent experiments. Immunohistochemical analysis. HOXA10 expression was Western blotting. Cells were lysed for 30 min on ice using detected in the 78 paired fresh specimens using immuno- radioimmunoprecipitation assay buffer (Beijing Solarbio histochemistry. Briefly, the fresh specimens were fixed in Science & Technology Co., Ltd., Beijing, China), and total 10% formalin for 24 h at room temperature and routinely protein was quantified using the BCA Protein Assay kit processed for paraffin embedding. Serial histologic sections (Beijing Solarbio Science & Technology Co., Ltd.) according were cut to a thickness of 4 µm, initially stained with hema- to the manufacturer's protocol. For western blot analysis, a toxylin for 5 min and eosin for 1 min at room temperature, total of 20 µg total protein was loaded per lane and separated and then analyzed by a senior pathologist to identify the in a 12% gel by SDS-PAGE. Subsequent to transferring the diagnosis and pathological grades of the patients. Subsequent proteins onto a nitrocellulose membrane (EMD Millipore, to dewaxing and rehydration, the sections were washed three Billerica, MA,USA), the membrane was blocked with 5% times with 1X phosphate-buffered saline (5 min/wash). fat-free milk for 1.5 h at room temperature and incubated with Next, the sections were blocked with normal goat serum for primary antibodies at 4˚C overnight. The primary antibodies 25 min at room temperature and subsequently incubated with used in the present study were as follows: Anti-HOXA10 HOXA10 monoclonal antibodies (1:50 dilution; Santa Cruz monoclonal antibody (cat. no. sc-271428; 1:500 dilution; Santa Biotechnology, Inc., Santa Cruz, CA, USA) in a moisture Cruz Biotechnology, Inc.); and anti-MMP-3 (cat. no. 14351), chamber overnight at 4˚C. Following incubation, immunode- anti-MMP-2 (cat. no. 40994), anti-MMP-7 (cat. no. 3801), tection was performed using the Envision ABC kit (Genetech anti-MMP-9 (cat. no. 15561) and anti-GAPDH (cat. no. 2118) Co., Ltd., Shanghai, China). Subsequently, the HOXA10 monoclonal antibodies (all 1:1,000 dilution), obtained from staining score of the specimens was assigned as previously Cell Signaling Technology, Inc., (Danvers, MA, USA). Next, described (9). Initially, the staining intensity score was the membrane was incubated with goat anti-rabbit IgG (H+L) assigned as follows: 0, negative staining; 1, weak staining; horseradish peroxidase-conjugated secondary antibody for 2, moderate staining; and 3, strong staining. In addition, the 2 h at room temperature (cat. no. GAR0072; 1:5,000 dilu- percentage of stained tumor cells was scored as follows: 0, tion; Multisciences Lianke Biotech Co., Ltd., Hangzhou, negative; 1, 1-10%; 2, 10-50%; 3, 50-80%; and 4, 80-100%. China). Subsequently, the proteins were detected by enhanced The HOXA10 staining score was then calculated as the sum chemiluminescence using a chemiluminescent and fluorescent of the scores for the staining intensity and percentage of imaging system (Sagecreation, Hangzhou, China). GAPDH stained tumor cells. Finally, BC and adjacent normal tissues was used as an internal control. were categorized into the high HOXA10 expression group when the total staining score was 5-7 and the low HOXA10 Cell proliferation assays. Subsequent to cell transfection with expression group when the score was 0-4. siRNAs for 24 h, a total of 1.5x103 cells in 200 µl RPMI-1640 medium were seeded into 96-well plates and maintained for Cell lines and cell culture. T24 and 5637 cell lines were used 5 days. Between days 1 and 5, cell proliferation was exam- in the current study.