*P 0

*P 0.05 and **P 0.01 vs. western blotting assays were performed to detect the effects of NEAT1 on cell biological behavior. TPEN Knockdown of NEAT1 in glioma cell lines was associated with cell cycle arrest at the G0/G1 phase, decreased proliferation and elevated apoptosis and by functioning as a ceRNA for miR-324-5p, which, in turn, TPEN regulated the expression of potassium channel tetramerization protein domain name made up of 20 (KCTD20). The inhibition of NEAT1 and KCTD20 inhibited the proliferation of glioma cells, which was consistent with the results of miR-324-5p overexpression. Therefore, the results revealed a novel NEAT1/miR-324-5p/KCTD20 regulatory axis in glioma, and identified a potential target for the development of diagnostic, prognostic and/or therapeutic tools for this disease. Materials and methods Human tissue samples All 43 human glioma tissues (age range, 26C71 years; mean age, 49 years), as well as their paired adjacent noncancerous tissues, were obtained from patients SHCC who underwent surgical resection at The First Affiliated Hospital of Nanjing Medical University (Nanjing, China) between January 2013 and December 2016. The distance between the tumor and the matched normal adjacent tissue was ~2 cm. The inclusion criteria were as follows: i) Diagnosed with glioma; and ii) had not received pre-operative radiotherapy, chemotherapy or other adjuvant treatments before surgery. The exclusion criteria were as follows: i) Diagnosed with other diseases; and ii) failed to cooperate with researchers. Histological grade was classified by pathologists using the World Health Organization criteria (28). All tissue samples were collected during surgery, frozen immediately in liquid nitrogen, and stored at ?80C for total RNA or protein extraction. The clinicopathological characteristics of the patients are summarized in Table I. The present study was approved by the Institutional Review Board and Ethics Committee of Nanjing Medical University (Nanjing, China) and Fourth Military Medical University (Xi’an, China), and written informed consent was obtained from all patients. Table I. Association between the expression levels of NEAT1 and clinicopathological features of 43 patients with glioma. luciferase activity. All experiments were performed in triplicate. Orthotopic xenograft studies A total of 24 male immunodeficient nude mice (n=6 for each group; age, 6 weeks; weight, ~20 g) were purchased from Shanghai Laboratory Animal Research Center. The mice were maintained under specific pathogen-free conditions in a temperature-controlled room (~20C; humidity, 20%), with a 12-h light/dark cycle, and with access to commercially available mouse food and sterilized water. To examine tumor growth in the orthotopic xenograft model, a total of 100 l of PBS made up of U251 cells (2.5105) were stably transfected with shNEAT1 or shKCTD20 and the corresponding negative control (shNC) was injected intracranially into the striatum of NOD/SCID mice by a stereotactic device (coordinates, 2 mm behind the anterior fontanel, 2 mm lateral to the sagittal suture, at a 3-mm depth from the TPEN dura). A bioluminescence imaging system (IVIS Spectrum; PerkinElmer, Inc.) was used to confirm tumor formation and measure tumor growth weekly. At 7, 14, 21 and 28 days after cell implantation, the mice were injected intraperitoneally with D-luciferin at 50 mg/ml and then subjected to bioluminescence imaging to visualize tumor growth for 10C120 sec. The whole experiment lasted ~2 months. During this process, according to development of neurological indicators (hunching, weight loss, rough coat), the moribund mice were anesthetized by an intraperitoneal injection of 10% chloral hydrate (400 mg/kg body weight). The mice showed no indicators of peritonitis, pain or other discomfort, and were then euthanized by means of cervical dislocation. Their brains were harvested, cut into sections, paraffin-embedded and stained with H&E to confirm the presence of tumors, and subjected to immunohistochemistry (IHC). For H&E staining, brain tissues were fixed with 10% formaldehyde at room heat for 24 h. Subsequently, paraffin-embedded specimens were cut into 5-m sections and dewaxed with xylene and cleared with a series of changing alcohol concentrations. The samples were washed three times in PBS (5 min each time) at room temperature, and then stained with hematoxylin (Sigma-Aldrich; Merck KGaA) for 5 min at room temperature. Sections were stained with eosin (Sigma-Aldrich; Merck KGaA) for 2 min at room temperature to observe the clarity of the nucleus and cytoplasm under a light microscope (Leica Microsystems GmbH). The images were assessed using Image-Pro Plus 6.0 (Media Cybernetics, Inc.). For IHC, TPEN tumor tissues were fixed with 10% formaldehyde for 24 h at room heat and sectioned into 5-m-thick slides, followed by incubation with citrate buffer (Thermo Fisher Scientific, Inc.) under high pressure to repair the antigen for 3 min. The slides were incubated with 0.3% H2O2 for 20 min at room temperature to quench the endogenous peroxidase. Next, blocking.