1068 MOLECULAR MEDICINE REPORTS 9: 1068-1074, 2014 Transmembrane protein with unknown function 16A overexpression promotes glioma formation through the nuclear factor‑κB signaling pathway JUN LIU1, YU LIU2, YINGANG REN1, LI KANG1 and LIHUA ZHANG1 Departments of 1Geriatrics and 2Neurology, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710038, P.R. China Received July 18, 2013; Accepted January 2, 2014 DOI: 10.3892/mmr.2014.1888 Abstract. Ion channels have been suggested to be important in Introduction the development and progression of tumors, however, chloride channels have rarely been analyzed in tumorigenesis. More In previous years, the association between ion channels and recently, transmembrane protein with unknown function 16A tumors has drawn particular attention. Increasing evidence has (TMEM16A), hypothesized to be a candidate calcium-acti- demonstrated that ion channels are involved in the regulation vated Cl- channel, has been found to be overexpressed in a of tumor progression, including potassium (1-3), calcium (4) number of tumor types. Although several studies have impli- and sodium channels (5,6). Therefore, understanding the cated the overexpression of TMEM16A in certain tumor types, underlying molecular mechanisms of ion channels in tumori- the exact role of TMEM16A in gliomas and the underlying genesis, and tumor progression and migration provides novel mechanisms in tumorigenesis, remain poorly understood. In insights into tumor pathogenesis, and also identifies potential the present study, the role of TMEM16A in gliomas and the targets for tumor prevention and treatment. potential underlying mechanisms were analyzed. TMEM16A Chloride channels are expressed ubiquitously and are was highly abundant in various grades of gliomas and important in various cellular processes, including the cell cycle cultured glioma cells. Knockdown of TMEM16A suppressed and proliferation, cell volume modulation, epithelial secretion cell proliferation, migration and invasion. Furthermore, and membrane excitability (7). To date, chloride channel nuclear factor-κB (NF-κB) was activated by overexpression of family members, including CLC proteins, ionotropic receptors TMEM16A. In addition, TMEM16A regulated the expression for γ‑aminobutyric acid and glycine, and the cystic fibrosis of NF-κB-mediated genes, including cyclin D1, cyclin E and transmembrane conductance regulator (CFTR), have been c-myc, involved in cell proliferation, and matrix metallopro- described (8). Calcium-activated chloride channels (CaCCs) teinases (MMPs)-2 and MMP-9, which are associated with the are reported to be major modulators of cell volume and epithe- migration and invasion of glioma cells. Collectively, results lial secretion (9). However, the composition of CaCCs remains of the present study provide evidence for the involvement of unknown. More recently, transmembrane protein with unknown TMEM16A in gliomas and the potential mechanism through function 16 (TMEM16) A, also known as anoctamin 1, has which TMEM16A promotes glioma formation. been hypothesized to be a candidate CaCC (10-12). TMEM16A belongs to the TMEM16 family, which consists of nine other members, including TMEM16B-K, characterized by eight transmembrane segments and a highly conserved domain of unknown function. TMEM16A has been demonstrated to be Correspondence to: Dr Lihua Zhang, Department of Geriatrics, involved in the regulation of gastrointestinal tract motility, Tangdu Hospital, Fourth Military Medical University, 1 Xinsi Road, epithelial fluid transport and saliva production (13-16). CaCCs Baqiao, Xi'an, Shanxi 710038, P.R. China have been found to be proapoptotic, acting as tumor suppressor E-mail: [email protected] genes in mammary epithelium (17). However, bestrophin, Abbreviations: CaCCs, calcium-activated chloride channels; a putative CaCC that promotes cell proliferation, has been TMEM16A, transmembrane protein with unknown function 16A; shown to be overexpressed in colon cancer (18). Notably, MMP, matrix metalloproteinase; NF-κB, nuclear factor-κB; CFTR, TMEM16A is localized on chromosome 11q13, a region that cystic fibrosis transmembrane conductance regulator; BrdU, is frequently amplified in human cancers, including those 5-bromo-2-deoxyuridine of the head and neck, esophagus, bladder and breast (19-21). Therefore, TMEM16A dysregulation may be associated with Key words: calcium-activated chloride channels, nuclear factor-κB, tumor progression. As hypothesized, several studies have glioma, transmembrane protein with unknown function 16A revealed that TMEM16A is overexpressed in various tumor types, including gastrointestinal stromal tumors, esophageal cancers, and head and neck cancers (22-24). However, the LIU et al: ROLE OF TMEM16A IN GLIOMAS 1069 molecular mechanisms of TMEM16A in the regulation of reverse, 5'-GTCCATGTTCTGCTGGGCCTG-3'; cyclin E tumors remains poorly understood. forward, 5'-GTCCTGGCTGAATGATACATC-3' and Several studies have hypothesized that chloride channels reverse, 5'-CCCTATTTTGTTCAGACAACATGGC-3'; are associated with the nuclear factor-κB (NF-κB) signaling c-myc forward 5'-ACACATCAGCACAACTACGC-3' and pathway (25-29). NF-κB, which is tightly controlled, regulates reverse, 5'-CCTCTTGACATTCTCCTCGGT-3'; GAPDH a wide range of cellular processes and is extensively involved forward, 5'-CGGAGTCAACGGATTTGGTCGTAT-3' and in cancer progression (30). Normally, the binding of NF-κB reverse 5'-AGCCTTCTCCATGGTGGTGAA-3'. The qPCR to inhibitory inhibitor of κB (IκB) proteins inactivates NF-κB mixture system contained 5 µl SsoFast™ EvaGreen Supermix in the cytoplasm (31). Degradation of IκB proteins releases (Bio-Rad, Hercules, CA, USA), 1 µl cDNA (diluted at 1:50) NF-κB and enables it to translocate to the nucleus and activate and 2 µl each of the forward and reverse primers (1 µM), to a target genes. Dysregulation of this process is involved in a final volume of 10 µl. The PCR procedure was as follows: 94˚C number of diseases (32,33). NF-κB has also been demonstrated for 4 min; 94˚C for 20 sec, 55˚C for 30 sec and 72˚C for 20 sec; to be a critical regulator of tumorigenesis involved in cell 2 sec for plate reading for 35 cycles; melting curve, 65‑95˚C. survival, metastasis and angiogenesis (31). However, although GAPDH was used as a control for normalizing the gene TMEM16A has been hypothesized to be a transmembrane expression. Three independent experiments were performed. protein regulating cellular ion exchanges, its role in the regula- The data obtained were analyzed by the 2-ΔΔCt method and tion of the NF-κB signal pathway remains unclear. statistically analyzed as described previously (35), followed by To date, no studies have determined the exact role of Student's unpaired sample t-test. TMEM16A in gliomas. The aim of the present study was to determine whether TMEM16A is involved in gliomas and the Nuclear protein extraction. Nuclear proteins were extracted potential underlying mechanisms. using a Nuclear Extraction kit (Sangon Biotech, Shanghai, China) according to the manufacturer's instructions. Briefly, Materials and methods cells were lysed in cytoplasmic buffer containing protease inhibitors, mixed and incubated for 15 min at 4˚C. Next, cells Cell lines and cell culture. One normal human astrocyte line were centrifuged at 12,000 rpm for 20 min at 4˚C. Supernatants (SVGp12) and four glioma cell lines (U87MG, U118, U251 containing cytoplasmic extraction products were collected. Cell and SHG44) were obtained from the American Type Culture sediments were collected and resuspended in nucleus buffer for Collection (Manassas, VA, USA). All cells were maintained 10 min at 4˚C. Next, the sample was centrifuged at 12,000 rpm according to standard protocols. Briefly, cells were cultured in for 10 min at 4˚C and the supernatant was collected for analysis. Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum to which 100 U/ml penicillin, Western blotting. A total of 20-30 µg protein was fractionated by 100 µg/ml streptomycin and 2 mM L-glutamate were added. 12% SDS-PAGE electrophoresis and transferred to nitrocellulose All cells were cultured at 37˚C with 5% CO2 in an incubator membranes (Amersham, Little Chalfont, UK). The membrane (Invitrogen Life Technologies, Carlsbad, CA, USA). was treated under agitation and blocking at room temperature, with 2% non-fat dry milk in Tris-buffered saline (TBS) for Tumor tissue preparation. All tumor tissues were obtained 1 h, followed by incubation with primary rabbit anti-human from Tangdu Hospital (Xi'an, China) according to institutional TMEM16A, IκB, p65, MMP-2, MMP-9, GAPDH and Histone guidelines for consent with the approval of patients and the polyclonal antibodies. (Santa Cruz Biotechnology, CA, USA). hospital. Brain tissue was obtained from 20 patients with Antibodies were diluted in blocking buffer (1:2,000) and incu- gliomas (eight females and 12 males; median age, 43.6 years; bated with the membrane at 4˚C overnight. Next, membranes range, 16‑78 years; five each of grade I, II, III and IV). Normal were washed three times with TBS and Tween 20 [TBST; brain tissue samples were obtained from five patients with 10 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.05% Tween-20] brain trauma (median age, 42 years; range, 21-68 years). for 10 min at room temperature. Subsequently, the membrane All samples were obtained from the initial surgery, prior to was incubated in horseradish peroxidase (HRP)-conjugated chemotherapy and radiotherapy. The malignant grades of the secondary