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Glioma is the most commonly diagnosed primary brain malignancy in adults, with poor survival following standard treatment [1, 2]. Although surgery, concurrent radio-chemotherapy and target therapy have greatly advanced glioma patient survival, the tumor resistance and recurrence Xing-Na Bao, Shang-Wei Wang, Yongfeng Li 2 Arch Med Sci make glioma become an incurable disease [3]. Thus, it is highly necessary to understand underlying glioma pathogenesis for exploring novel effective therapeutic targets and finding precision diagnostic and prognostic biomarkers. Noncoding RNAs (ncRNAs) have been broadly explored due to their attributes of an unusual expression model and specific regulation mechanism in different diseases [4, 5]. NcRNAs are closely connected to diverse prospects of neoplasm cell performances [6]. LncRNAs more than 200 nt long are able to tune target gene transcription and translation via binding microRNAs [7, 8]. Hence, sponging microRNAs are dysregulated and then shape cancer advancement. So far, many lncRNAs have been investigated for their biological and clinical behavior in the pathophysiology of glioma through sponging miRNA [9, 10]. Li et al. reported that lncRNA linc00645 acted as an oncogene to promote glioma migration and invasion through modulating the miR205-3p-ZEB1 axis [11]. Dong et al. revealed that lncRNA EGFR-AS1 decreased miR-133b and upregulated RACK1 to promote glioma cell metastasis and growth [12]. Recently, the lncRNA TUBA4B has been widely studied in several malignant tumors including gastric cancer, NSCLC, ovarian cancer, and breast cancer [13–16]. However, the function of lncRNA TUBA4B in glioma still remains largely unclear. In this paper, we report for the first time that lncRNA TUBA4B was markedly decreased in glioma tissues and cells. We found that the patients with low expression of TUBA4B had shorter progression-free survival (PFS) and overall survival (OS) than those with high expression of TUBA4B. Functional analysis indicated that TUBA4B functioned as a  miR-183 sponge and could exert its tumor-suppressive impacts via the regulation of SMAD4. Collectively, our study is the first to demonstrate that TUBA4B could act as a  tumor suppressor by the TUBA4B/miR-183/SMAD4 axis to regulate glioma development and progression. Material and methods Tissue collection One hundred and sixteen paired clinical cancer tissues and adjoining non-tumor tissues (paired samples were from the same patients) were collected from glioma patients between January 2013 and January 2015 at Sishui Country People’s Hospital, Jining. The histological type of all patients included glioblastoma (n = 96) and astrocytoma (n = 26). All samples were instantly cryopreserved in liquid nitrogen for subsequent research. No systematic therapy, chemotherapy or radiotherapy was performed for any of the patients before surgical resection. Plasma samples from glioma patients (n = 102) and healthy controls (n = 102) were collected to evaluate the diagnostic value of TUBA4B. Informed consent was obtained from the patients or their family and this investigation was authorized by the Ethics Committee of Sishui Country People’s Hospital, Jining. Cell cultivation and transfection Six glioma cancer cell lines (U87, U251, LN229, U118, A172, T98), normal human astrocytes (NHAs) and HEK293 cells were obtained from the Cell Bank of Shanghai Institute for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) (Shanghai, China) and cultivated in RPMI 1640 (Gibco) medium or DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Life Technologies). HEK293 cells were cultured with MEM (Invitrogen) supplemented with glutamine, 10% heat inactivated horse serum, 1 mM Na pyruvate, and 0.1 mM non-essential amino acids. All cells were cultured in a humidified incubator at 37°C with 5% CO2. miR-183 mimics (miR-183, GI:28395069; accession number: AY194153.1), miR-183 inhibitor (anti-miR-183) and the negative control (NC) were purchased from Genepharma (Shanghai, China). The full-length sequences of TUBA4B (GI:1241766242; accession number: NM_001355221.1) were ligated into pcDNA3.1 (oe-TUBA4B) as an overexpression plasmid (Genepharma, China). The cloning sites of TUBA4B on vector pcDNA3.1 are BamH I and Xho I. Cell transfection was performed with Lipofectamine 2000 Reagent (Invitrogen) based on the manufacturer’s guide. RNA extraction and qRT-PCR TRIzol reagent (Invitrogen) was used to extract the total RNAs from tissues and cells. cDNA was synthesized with the PrimeScript RT Reagent Kit (Takara). RNA quantification was conducted with a  Nanodrop Spectrophotometer (Thermo Fisher Scientific). The relative expression of target gene and miRNA was detected by quantitative RT-PCR through SYBR Green PCR Master Mix (Takara) on the 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) according to the manufacturer’s instructions. Reaction conditions of qPRC were at 95°C for 10 min, followed by 40 cycles at 95°C for 10 s, 60°C for 30 s. The relative quantities of lncRNA/mRNA and miRNA were normalized to GAPDH and U6, respectively, and subsequently calculated by the 2–ΔΔCt method. The following primers were used in this study: TUBA4B, forward: 5’-CCCACAGGCTTTAAGGTTGA-3’, reverse 3’-AGGCCATAGTGATGGCTGTC-5’; miR-183: 5’-TATGGCACTGGTAGAATTCACT-3’; GAPDH, forward: Downregulated expression of lncRNA TUBA4B predicts unfavorable prognosis and suppresses glioma progression by sponging miR-183 to regulate SMAD4 expression Arch Med Sci 3 5’-TATGATGATATCAAGAGGGTAGT-3’, reverse, 5’-TGTATCCAAACTCATTGTCATAC-3’; U6, forward: 5’-CTCGCTTCGGCAGCACA-3’, reverse, 5’-AACGCTTCACGAATTTGCGT-3’. Cell MTS assay Cell proliferation was detected by MTS assay. Briefly, after 24 h transfection, the cells were plated in a 96-well plate at a density of 2000–3000 cells/well. After culturing for the indicated time, 20 μl of MTS was added into each well, and incubated for another 2 h at 37°C. An EnVision multimode plate reader (PerkinElmer) was used to evaluate absorbance at 490 nm. Soft-agar colony forming assay The anchorage-independent growth ability of cells was detected by CytoSelect 96-Well Cell Transformation Assay (Standard Soft Agar kit, Cell Biolabs) according to the manufacturer’s protocol. Briefly, 3 × 10 cells transfected with indicated vectors were plated to a 96-well plate coated with agar matrix and then incubated for another 14 days and the fresh medium was replaced every 3 days, and subsequently, the colonies were lysed and dyed with the Cyquant GR Dye, and analyzed using an EnVision multimode plate reader (PerkinElmer). Detection of cell apoptosis and cell cycle analysis The ApoAlert DNA Fragmentation Assay Kit was applied to detect apoptotic activity (Clontech) according to the manufacturer’s guide. Briefly, the cells were collected, incubated with Nucleotide Mix, stained with propidium iodide (PI) in 0.5 ml of PI/RNase/PBS (2.5  μg/ml), and detected with flow cytometry (FACSCalibur). For the cell cycle test, cells were digested, washed with PBS, fixed with cold ethanol, incubated with PI (100 ng/ml), and explored by flow cytometry (FACSCalibur). Migration and invasion assay A  24-well transwell chamber (8-μm pore size, Costar) with or without Matrigel was applied to assess the migration or invasion based on the manufacturer’s guide. The detailed process was consistent with a  previous study [17]. The migrated or invaded cells were fixed with 100% methanol, stained with 0.1% crystal violet, photographed with a microscope (Olympus) and counted with three fields selected at random. Dual-luciferase reporter assay MicroRNA.org, miRcode, TargetScan and Starbase V3.0 was used to predict the binding site of target. To verify this prediction, luciferase reporter assay was used. The presumptive miR-183 target binding segment in TUBA4B and the corresponding variant of the binding sequence were cloned and inserted into the pmirGLO Vector (Promega) to produce the TUBA4B-wild-type (WT-TUBA4B) vector and TUBA4B-mutated-type (Mut-TUBA4B). Likewise, the SMAD4-wild-type (WT-SMAD4) and SMAD4-mutated-type (Mut-SMAD4) reporter vectors were produced. Subsequently, mutated or wild type vector and miR-183 or anti-miR-183 were co-transfected into HEK293 cells in 96-well plates with Lipofectamine 2000 (Invitrogen) and then incubated for 24 h. Dual-Luciferase Reporter Assay (Promega) was applied to detect the relative luciferase activity luminescence based on the manufacturer’s guide. RNA pull down assay The RNA pull-down assay was used to analyze the binding situation between TUBA4B and miR-183, and SMAD4 and miR-183. Briefly, the commercially synthesized biotinylated TUBA4B or SMAD4 probe (Bio-TUBA4B-probe, Bio-SMAD4probe) or the negative control probes (Bio-NCprobe) were purchased from GenePharma. The cell lysates were incubated with Dynabeads M-280 Streptavidin (Invitrogen) and different biotin-labeled probes according to the guide. Finally, the binding miRNA was eluted and analyzed through qRT-PCR with SYBR Green PCR Master Mix (Takara) on the 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). RNA immunoprecipitation (RIP) Glioma cells were lysed, and then nurtured with RIP buffer supplemented with magnetic beads binding with human anti-Argonaute2 (Ago2) antibody (CST), and negative control normal mouse IgG (CST). Subsequently, the obtained immunoprecipitated RNA was extracted. A  NanoDrop (Thermo Scientific) was used to measure the RNA concentration and a bio-analyzer (Thermo Scientific) was applied to evaluate the RNA quality. Finally, qRT-PCR analysis was applied to detect RNA to validate the presence of the binding targets through SYBR Green PCR Master Mix (Takara) on the 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). Western blot analysis The detailed procedures were as previously described [18]. Briefly, the total protein was separated from cells with RIPA lysis buffer. Lysed protein was quantified with a Pie

archives of medical science/archives of medical science (online)

Downregulated expression of lncRNA TUBA4B predicts unfavorable prognosis and suppresses glioma progression by sponging miR-183 to regulate SMAD4 expression