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• W2896488950 abstract "miR-135a-5p has been reported as a tumor suppressor in several extracranial tumors. However, its exact roles in gliomagenesis and relevance to the patients' prognoses are largely unknown. Herein, we detected the miR-135a-5p and tumor necrosis factor receptor–associated factor 5 (TRAF5) levels in 120 human glioma specimens and 20 nontumoral brain tissues; we found the miR-135a-5p level decreased, whereas the TRAF5 level increased, with the elevation of glioma grade. Their labeling indexes were inversely correlated with each other and showed strong negative (miR-135a-5p) and positive (TRAF5) correlation with the Ki-67 index. Cox regression demonstrated that both of their expression levels were independent survival predictors, whereas Kaplan-Meier analysis showed that subgrouping the glioma patients according to their levels could perfectly reflect the patients' prognoses regardless of the similarities in pathologic, molecular, and clinical features. In the following in vitro and in vivo studies, it was demonstrated that miR-135a-5p induced G1 arrest and inhibited the proliferation of glioma cells by targeting TRAF5 and subsequently blocking AKT phosphorylation as well as c-Myc and cyclin D1 expression. These effects could be reversed by TRAF5 overexpression and simulated by specific TRAF5 silencing. This study highlights the importance of miR-135a-5p and TRAF5 in gliomagenesis and progression and implies their potential prognostic and therapeutic values in malignant glioma. miR-135a-5p has been reported as a tumor suppressor in several extracranial tumors. However, its exact roles in gliomagenesis and relevance to the patients' prognoses are largely unknown. Herein, we detected the miR-135a-5p and tumor necrosis factor receptor–associated factor 5 (TRAF5) levels in 120 human glioma specimens and 20 nontumoral brain tissues; we found the miR-135a-5p level decreased, whereas the TRAF5 level increased, with the elevation of glioma grade. Their labeling indexes were inversely correlated with each other and showed strong negative (miR-135a-5p) and positive (TRAF5) correlation with the Ki-67 index. Cox regression demonstrated that both of their expression levels were independent survival predictors, whereas Kaplan-Meier analysis showed that subgrouping the glioma patients according to their levels could perfectly reflect the patients' prognoses regardless of the similarities in pathologic, molecular, and clinical features. In the following in vitro and in vivo studies, it was demonstrated that miR-135a-5p induced G1 arrest and inhibited the proliferation of glioma cells by targeting TRAF5 and subsequently blocking AKT phosphorylation as well as c-Myc and cyclin D1 expression. These effects could be reversed by TRAF5 overexpression and simulated by specific TRAF5 silencing. This study highlights the importance of miR-135a-5p and TRAF5 in gliomagenesis and progression and implies their potential prognostic and therapeutic values in malignant glioma. Gliomas are the most common primary brain tumors.1Ricard D. Idbaih A. Ducray F. Lahutte M. Hoang-Xuan K. Delattre J.Y. Primary brain tumours in adults.Lancet. 2012; 379: 1984-1996Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 2Louis D.N. Perry A. Reifenberger G. von Deimling A. Figarella-Branger D. Cavenee W.K. Ohgaki H. Wiestler O.D. Kleihues P. Ellison D.W. The 2016 World Health Organization classification of tumors of the central nervous system: a summary.Acta Neuropathol. 2016; 131: 803-820Crossref PubMed Scopus (9405) Google Scholar High-grade gliomas, especially glioblastoma (GBM), are highly recurrent and lethal because of their rampant growth and invasion, which makes radical resection almost clinically unfeasible.3Wen P.Y. Kesari S. Malignant gliomas in adults.N Engl J Med. 2008; 359: 492-507Crossref PubMed Scopus (3245) Google Scholar Despite the benefits of alkylating agents, such as temozolomide, and the advances in neurosurgery, the patients' prognoses remain poor, with a median survival of no more than 20 months.4Marosi C. Preusser M. Milestones of the last 10 years: CNS cancer.Memo. 2017; 10: 18-21Crossref PubMed Scopus (9) Google Scholar In recent years, the great efforts in biomarker screening and therapeutic target selection have been constantly improving the diagnosis, prognosis, and therapy of gliomas.2Louis D.N. Perry A. Reifenberger G. von Deimling A. Figarella-Branger D. Cavenee W.K. Ohgaki H. Wiestler O.D. Kleihues P. Ellison D.W. The 2016 World Health Organization classification of tumors of the central nervous system: a summary.Acta Neuropathol. 2016; 131: 803-820Crossref PubMed Scopus (9405) Google Scholar, 5Brennan C.W. Verhaak R.G. McKenna A. Campos B. Noushmehr H. Salama S.R. et al.The somatic genomic landscape of glioblastoma.Cell. 2013; 155: 462-477Abstract Full Text Full Text PDF PubMed Scopus (3052) Google Scholar, 6Li H. Yu L. Liu J. Bian X. Shi C. Sun C. Zhou X. Wen Y. Hua D. Zhao S. Ren L. An T. Luo W. Wang Q. Yu S. miR-320a functions as a suppressor for gliomas by targeting SND1 and beta-catenin, and predicts the prognosis of patients.Oncotarget. 2017; 8: 19723-19737PubMed Google Scholar, 7Shi C. Ren L. Sun C. Yu L. Bian X. Zhou X. Wen Y. Hua D. Zhao S. Luo W. Wang R. Rao C. Wang Q. Yu S. miR-29a/b/c function as invasion suppressors for gliomas by targeting CDC42 and predict the prognosis of patients.Br J Cancer. 2017; 117: 1036-1047Crossref PubMed Scopus (43) Google Scholar, 8Szopa W. Burley T.A. Kramer-Marek G. Kaspera W. Diagnostic and therapeutic biomarkers in glioblastoma: current status and future perspectives.Biomed Res Int. 2017; 2017: 8013575Crossref PubMed Scopus (184) Google Scholar The biological functions and prognostic values of some miRNAs have been demonstrated in various kinds of tumors.6Li H. Yu L. Liu J. Bian X. Shi C. Sun C. Zhou X. Wen Y. Hua D. Zhao S. Ren L. An T. Luo W. Wang Q. Yu S. miR-320a functions as a suppressor for gliomas by targeting SND1 and beta-catenin, and predicts the prognosis of patients.Oncotarget. 2017; 8: 19723-19737PubMed Google Scholar, 7Shi C. Ren L. Sun C. Yu L. Bian X. Zhou X. Wen Y. Hua D. Zhao S. Luo W. Wang R. Rao C. Wang Q. Yu S. miR-29a/b/c function as invasion suppressors for gliomas by targeting CDC42 and predict the prognosis of patients.Br J Cancer. 2017; 117: 1036-1047Crossref PubMed Scopus (43) Google Scholar, 9Lu J. Getz G. Miska E.A. Alvarez-Saavedra E. Lamb J. Peck D. Sweet-Cordero A. Ebert B.L. Mak R.H. Ferrando A.A. Downing J.R. Jacks T. Horvitz H.R. Golub T.R. MicroRNA expression profiles classify human cancers.Nature. 2005; 435: 834-838Crossref PubMed Scopus (8202) Google Scholar, 10Calin G.A. Croce C.M. MicroRNA-cancer connection: the beginning of a new tale.Cancer Res. 2006; 66: 7390-7394Crossref PubMed Scopus (956) Google Scholar, 11Croce C.M. miRNAs in the spotlight: understanding cancer gene dependency.Nat Med. 2011; 17: 935-936Crossref PubMed Scopus (28) Google Scholar, 12Barciszewska A.M. MicroRNAs as efficient biomarkers in high-grade gliomas.Folia Neuropathol. 2016; 54: 369-374Crossref PubMed Scopus (31) Google Scholar However, the effects of miR-135a-5p, a miR-135 family member, is somewhat complicated, because it may exert opposite roles in different tumors. miR-135a-5p has been confirmed as a tumor suppressor in non–small-cell lung cancer,13Shi H. Ji Y. Zhang D. Liu Y. Fang P. miR-135a inhibits migration and invasion and regulates EMT-related marker genes by targeting KLF8 in lung cancer cells.Biochem Biophys Res Commun. 2015; 465: 125-130Crossref PubMed Scopus (72) Google Scholar gastric cancer,14Cheng Z. Liu F. Zhang H. Li X. Li Y. Li J. Cao Y. Cao L. Li F. miR-135a inhibits tumor metastasis and angiogenesis by targeting FAK pathway.Oncotarget. 2017; 8: 31153-31168Crossref PubMed Scopus (36) Google Scholar pancreatic carcinoma,15Dang Z. Xu W.H. Lu P. Wu N. Liu J. Ruan B. Zhou L. Song W.J. Dou K.F. MicroRNA-135a inhibits cell proliferation by targeting Bmi1 in pancreatic ductal adenocarcinoma.Int J Biol Sci. 2014; 10: 733-745Crossref PubMed Scopus (41) Google Scholar and metastatic prostate cancer.16Xu B. Tao T. Wang Y. Fang F. Huang Y. Chen S. Zhu W. Chen M. hsa-miR-135a-1 inhibits prostate cancer cell growth and migration by targeting EGFR.Tumour Biol. 2016; 37: 14141-14151Crossref PubMed Scopus (30) Google Scholar However, in colorectal cancer, higher miR-135a-5p is often associated with more malignant phenotypes.17Nagel R. le Sage C. Diosdado B. van der Waal M. Oude Vrielink J.A. Bolijn A. Meijer G.A. Agami R. Regulation of the adenomatous polyposis coli gene by the miR-135 family in colorectal cancer.Cancer Res. 2008; 68: 5795-5802Crossref PubMed Scopus (418) Google Scholar These findings prompted us to investigate this miRNA specifically in gliomas. As a potent activator of the mitogen-activated protein kinase and NF-κB pathway, tumor necrosis factor receptor–associated factor 5 (TRAF5) participates in the onset and progression of several tumors.18Li M. Long C. Yang G. Luo Y. Du H. miR-26b inhibits melanoma cell proliferation and enhances apoptosis by suppressing TRAF5-mediated MAPK activation.Biochem Biophys Res Commun. 2016; 471: 361-367Crossref PubMed Scopus (30) Google Scholar, 19Tao T. Cheng C. Ji Y. Xu G. Zhang J. Zhang L. Shen A. Numbl inhibits glioma cell migration and invasion by suppressing TRAF5-mediated NF-kappaB activation.Mol Biol Cell. 2012; 23: 2635-2644Crossref PubMed Scopus (69) Google Scholar, 20Wang B. Zhao H. Zhao L. Zhang Y. Wan Q. Shen Y. Bu X. Wan M. Shen C. Up-regulation of OLR1 expression by TBC1D3 through activation of TNFalpha/NF-kappaB pathway promotes the migration of human breast cancer cells.Cancer Lett. 2017; 408: 60-70Crossref PubMed Scopus (39) Google Scholar This conclusion is further supported by the findings that miR-26b inhibits melanoma cell proliferation and numb-like protein inhibits glioma cell migration/invasion by directly targeting TRAF5.18Li M. Long C. Yang G. Luo Y. Du H. miR-26b inhibits melanoma cell proliferation and enhances apoptosis by suppressing TRAF5-mediated MAPK activation.Biochem Biophys Res Commun. 2016; 471: 361-367Crossref PubMed Scopus (30) Google Scholar, 19Tao T. Cheng C. Ji Y. Xu G. Zhang J. Zhang L. Shen A. Numbl inhibits glioma cell migration and invasion by suppressing TRAF5-mediated NF-kappaB activation.Mol Biol Cell. 2012; 23: 2635-2644Crossref PubMed Scopus (69) Google Scholar Bioinformatics predicts that TRAF5 is a candidate target of miR-135a-5p. However, to the best of our knowledge, whether this miRNA suppresses glioma cell proliferation by silencing TRAF5 has not been reported. In this study, we reported, for the first time, that the low expression of miR-135a-5p is responsible for the abnormal increase of TRAF5 in glioma cells. Introducing exogenous miR-135a-5p effectively suppressed TRAF5 expression and thereby obstructed the proliferation of glioma cells. Our findings also indicated the prognostic values of these molecules as independent predictors and the usefulness of them in glioma therapy. The surgical specimens of human gliomas and 20 nontumoral control brain tissues were collected from Tianjin Medical University General Hospital (Tianjin, China) with written consents. For the glioma specimens, IDH1/2 gene mutations were detected by Sanger sequencing, and the statuses of chromosome arms 1p and 19q were detected by either fluorescence in situ hybridization or comparative genomic hybridization. Then, the diagnoses were updated according to the 2016 World Health Organization classification of central nervous system tumors,2Louis D.N. Perry A. Reifenberger G. von Deimling A. Figarella-Branger D. Cavenee W.K. Ohgaki H. Wiestler O.D. Kleihues P. Ellison D.W. The 2016 World Health Organization classification of tumors of the central nervous system: a summary.Acta Neuropathol. 2016; 131: 803-820Crossref PubMed Scopus (9405) Google Scholar and only 120 patients with astrocytic gliomas without 1p/19q codeletion were enrolled in the present study. All the glioma patients with complete information were followed up since the date of operation to December 31, 2013, with a follow-up time of 4.5 to 89 months. The clinical and pathologic features are summarized in Supplemental Table S1. This study was performed in accordance with the principles of the Declaration of Helsinki and approved by the Ethics Committee of Tianjin Medical University General Hospital. The miRNA microarray expression data from the Chinese Glioma Genome Atlas (CGGA; http://www.cgga.org.cn, last accessed June 20, 2018) were used to further verify the expression change of miR-135a-5p and its interrelationship with patients' survival. Meanwhile, the data from the CGGA, The Cancer Genome Atlas (https://cancergenome.nih.gov, last accessed June 20, 2018), and Oncomine (https://www.oncomine.org, last accessed June 20, 2018) were used for TRAF5 mRNA expression analysis and prognostic value assessment. In situ hybridization (ISH) and immunohistochemistry (IHC) were performed as described previously.6Li H. Yu L. Liu J. Bian X. Shi C. Sun C. Zhou X. Wen Y. Hua D. Zhao S. Ren L. An T. Luo W. Wang Q. Yu S. miR-320a functions as a suppressor for gliomas by targeting SND1 and beta-catenin, and predicts the prognosis of patients.Oncotarget. 2017; 8: 19723-19737PubMed Google Scholar The locked nucleic acid–modified and digoxin-labeled oligonucleotide probe for miR-135a-5p and the corresponding scrambled control sequence were purchased from Exiqon (Vedbaek, Denmark) (Table 1). Mouse anti-human TRAF5 antibody and rabbit anti-human Ki-67 antibody were purchased from Santa Cruz Biotechnology (Dallas, TX) and Millipore (Billerica, MA), respectively. The ISH and IHC images were captured under a DM6000B microscope (Leica, Wetzlar, Germany), and the ratios [labeling index (LI) (%)] of the positive cell number/the total cell number in five randomly selected ×400 fields were calculated with Image Pro Plus 5.0 (Media Cybernetics, Rockville, MD).Table 1The Probes of miR-135a-5p and Scr Used for ISHOligonucleotide probeSequenceDIG-labeled LNA miR-135a-5p5′-TCACATAGGAATAAAAAGCCATA-3′DIG-labeled LNA Scr5′-CGTATAGGCCCAAGAATTAGG-3′DIG, digoxin; ISH, in situ hybridization; LNA, locked nucleic acid; Scr, scrambled control sequence. Open table in a new tab DIG, digoxin; ISH, in situ hybridization; LNA, locked nucleic acid; Scr, scrambled control sequence. Human glioblastoma cell lines U251 and U87MG were purchased from the China Academic Sinica Cell Repository (Shanghai, China) and ATCC (Manassas, VA), respectively. For patient-derived culture (primary GBM), fresh glioblastoma sample was minced in phosphate-buffered saline, digested by trypsin on a 37°C shaker for 20 minutes, filtered through nylon mesh filters, and washed with phosphate-buffered saline three times. Both the cell lines and primary GBM were maintained in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) with 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel). For neurosphere culture, U87MG cells were cultivated in Dulbecco's modified Eagle's medium/F12 medium (Corning, Corning, NY) supplemented with 20 ng/mL epidermal growth factor (PeproTech, Rocky Hill, NJ), 20 ng/mL basic fibroblast growth factor (PeproTech), and B27 (Gibco). All the cells were incubated at 37°C in 5% CO2 in a humidified incubator. The miR-135a-5p–expressing lentivirus and the control (Con) lentivirus were purchased from Genechem (Shanghai, China). The TRAF5-expressing lentivirus was constructed and packaged by Applied Biological Materials (Vancouver, BC, Canada). The stable sub–cell lines of U251 (U251-Con, -miR-135a-5p, and -miR-135a-5p+TRAF5), U87MG (U87MG-Con, -miR-135a-5p, and -miR-135a-5p+TRAF5), and primary GBM (primary GBM-Con, -miR-135a-5p, and -miR-135a-5p+TRAF5) were established by corresponding lentivirus infection. The expression efficiencies of the exogenous genes were confirmed by real-time quantitative RT-PCR (RT-qPCR) and Western blot analysis. The specific siRNAs of TRAF5 (siTRAF5#1 and siTRAF5#2) and the scrambled control (Ribobio, Guangzhou, China) (Table 2) were used to transfect U251 and U87MG cells with X-tremeGENE siRNA Transfection Reagent (Roche, Indianapolis, IN).Table 2The siRNAs and Scrambled Control SequencedsRNA oligonucleotideSequenceTRAF5 siRNA (siTRAF5#1)F: 5′-GGAUGUAAUGCCAAGGUUATT-3′R: 5′-UAACCUUGGCAUUACAUCCTT-3′TRAF5 siRNA (siTRAF5#2)F: 5′-GGAGCACAUGCGUUUGGUUTT-3′R: 5′-AACCAAACGCAUGUGCUCCTT-3′Scrambled control sequenceF: 5′-UUCUCCGAACGUGUCACGUTT-3′R: 5′-ACGUGACACGUUCGGAGAATT-3′dsRNA, double-stranded RNA; F, forward; R, reverse. Open table in a new tab dsRNA, double-stranded RNA; F, forward; R, reverse. RT-qPCR was performed as previously described.21Liu J. Xu J. Li H. Sun C. Yu L. Li Y. Shi C. Zhou X. Bian X. Ping Y. Wen Y. Zhao S. Xu H. Ren L. An T. Wang Q. Yu S. miR-146b-5p functions as a tumor suppressor by targeting TRAF6 and predicts the prognosis of human gliomas.Oncotarget. 2015; 6: 29129-29142Crossref PubMed Scopus (71) Google Scholar miR-135a-5p was quantified by the Stem-Loop Detection Kit (GenePharma, Shanghai, China) using U6 as the internal control, and TRAF5 mRNA was quantified by the GoTaq qPCR Master Mix Kit (Promega, Fitchburg, WI) using GAPDH as the internal control. The primers (Table 3) were synthesized by Invitrogen (Shanghai, China). The coarse data were then converted by 2−ΔΔCt transformation to represent the fold changes of the RNAs.Table 3Primers Used for miR-135a-5p and TRAF5 mRNA RT-qPCR DetectionsPrimerSequencemiR-135a-5pF: 5′-CCGGCGTATGGCTTTTTATTCC-3′R: 5′-CAGTGCAGGGTCCGAGGT-3′U6F: 5′-CGCAAGGATGACACGCAAATTCG-3′R: 5′-CAGTGCAGGGTCCGAGGT-3′TRAF5F: 5′-CCTACGGAAAGACCTGAAAGAGC-3′R: 5′-GGGTATTCAGGACACAAGTTTTCC-3′GAPDHF: 5′-TGCACCACCAACTGCTTAGC-3′R: 5′-GGCATGGACTGTGGTCATGAG-3′F, forward; R, reverse; RT-qPCR, real-time quantitative RT-PCR. Open table in a new tab F, forward; R, reverse; RT-qPCR, real-time quantitative RT-PCR. For 5-ethynyl-2′-deoxyuridine assays, cells (5 × 103 per well) were seeded into 96-well plates or poly-l-lysine/laminin–coated 96-well plates (neurosphere culture). The stable sub–cell lines and the siRNA-transfected cells were stained 24 hours after seeding and 48 hours after transfection, respectively, with the Cell-Light EdU Apollo567 in Vitro Imaging kit (Ribobio), according to the manufacturer's instruction. For 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) proliferation assays, cells (1 × 103 per well) were cultivated for 24, 48, 72, 96, and 120 hours. At the indicated intervals, 20 μL of Cell Titer 96AQeuous One Solution Reagent (Promega) was added to each well and incubated for 2 hours at 37°C. The absorbance at 490 nm was measured with a Synergy microplate reader (BioTek Instruments, Winooski, VT). The candidate targets of miR-135a-5p were predicted using TargetScan version 7.0 (http://www.targetscan.org/vert_70, last accessed June 20, 2018). As a member of the TRAF family, TRAF5 was focused on the basis of the site type, combination of the context score, context score percentile, and probability of conserved targeting. The cDNA fragment corresponding to the 3′-untranslated region (UTR) of TRAF5 (TRAF5–3′-UTR–wild type) and its mutant (TRAF5–3′-UTR–mutant) lacking the miR-135a-5p target sequence were synthesized by GenScript (Nanjing, China) and inserted into the multiple cloning site of the pEZX-MT01 vector (GeneCopoeia, Rockville, MD) downstream of the firefly luciferase reporter gene to construct the recombinant reporter plasmids wild type and mutant type. These plasmids were then used to transfect the Con and the miR-135a-5p overexpression sub–cell lines with X-tremeGENE HP DNA Transfection Reagent (Roche). Firefly and renilla luciferase activities were detected with the Dual-Luciferase Reporter Assay System (Promega). The results were presented as the firefly luciferase activities normalized against those of renilla. Western blot analysis was performed as previously described.22Li Y. Wang Y. Yu L. Sun C. Cheng D. Yu S. Wang Q. Yan Y. Kang C. Jin S. An T. Shi C. Xu J. Wei C. Liu J. Sun J. Wen Y. Zhao S. Kong Y. miR-146b-5p inhibits glioma migration and invasion by targeting MMP16.Cancer Lett. 2013; 339: 260-269Crossref PubMed Scopus (99) Google Scholar The mouse anti-human TRAF5 and cyclin D1 antibodies (Santa Cruz Biotechnology); rabbit anti-human phosphorylated AKT and AKT antibodies (CST, Boston, MA); rabbit anti-human c-Myc antibody (Bioworld, Nanjing, China); and mouse anti-human glyceraldehyde-3-phosphate dehydrogenase antibody (Boster, Wuhan, China) were used as the primary antibodies. The stable sub–cell lines and siRNA-transfected cells were harvested and fixed in 70% ethanol at 4°C overnight. The cells were incubated with 40 μg/mL propidium iodide (Sigma, St. Louis, MO) at room temperature for 30 minutes and analyzed with an Accuri C6 flow cytometer (BD, Franklin Lakes, NJ). The data were processed with ModFit LT software version 3.1 (Verity Software House, Topsham, ME). The animal experiments were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University General Hospital. Six-week–old BALB/C athymic nude mice (National Laboratory Animal Center, Beijing, China) were anesthetized and intracranially injected with 5 × 105 U87MG sub–cell lines (U87MG-Con, -miR-135a-5p, or -miR-135a-5p+TRAF5) and primary GBM (primary GBM-Con, -miR-135a-5p, and -miR-135a-5p+TRAF5). The growth of the xenografts was monitored with the IVIS Lumina Imaging System (PerkinElmer, Waltham, MA) 7, 14, 21, and 28 days after implantation. The mice were sacrificed at day 35, and the brains were collected for hematoxylin and eosin and IHC staining. Statistical analyses were performed using SPSS software version 21.0 (IBM, Chicago, IL). Data were presented as means ± SD. One-way analysis of variance, t-test, Pearson's correlation, Kaplan-Meier analysis, log-rank test, and Cox proportional hazards regression were used to analyze corresponding data. The medians of the miR-135a-5p and TRAF5 labeling indexes were used as cutoffs in survival analyses. Statistical significance was assigned at P < 0.05. All experiments in vitro and in vivo were performed at least three times with triplicate samples. ISH results showed that the expression level of miR-135a-5p was significantly lower in the gliomas than in the control brains (P < 0.001). Among the glioma groups, the LI of miR-135a-5p declined gradually with the elevation of the grade (P < 0.001) (Figure 1, A and B ) and showed an inverse correlation with the Ki-67 proliferation index (r = −0.782, P < 0.0001) (Figure 1C and Supplemental Figure S1, A and B). Kaplan-Meier analysis revealed that the disease-free survival (DFS) and overall survival (OS) of the patients with a high miR-135a-5p level were significantly longer than those of the patients with a low miR-135a-5p level (DFS, P < 0.0001; OS, P < 0.0001) (Figure 1D). These differences remained obvious even within the patients with the same tumor grade, same IDH status, similar ages, and similar Karnofsky performance scores (DFS, P < 0.01 to approximately P < 0.0001; OS, P < 0.01 to approximately P < 0.0001) (Figure 1, E–I, and Supplemental Figure S2). The above findings were further confirmed by the data from the CGGA (Supplemental Figure S3A). Furthermore, Cox regression demonstrated that miR-135a-5p was an independent predictor for DFS and OS of glioma patients (Table 4 and Supplemental Table S2). These results indicate the practical value of miR-135a-5p in prognosis assessment and imply its potential antiproliferation effect.Table 4Multivariate Analysis for DFS and OS in Patients with GliomasFactorDFSOSHR (95% CI)P valueHR (95% CI)P valueSex0.971 (0.653–1.443)0.8831.003 (0.677–1.485)0.989Age1.001 (0.987–1.016)0.8591.001 (0.987–1.016)0.852Predominant side1.099 (0.786–1.536)0.5801.033 (0.741–1.441)0.848Predominant location1.076 (0.815–1.421)0.6041.041 (0.787–1.376)0.780KPS1.001 (0.976–1.028)0.9141.003 (0.978–1.029)0.811IDH status0.186 (0.095–0.363)<0.00010.153 (0.079–0.296)<0.0001miR-135a-5p LI0.960 (0.932–0.989)0.0070.969 (0.940–0.998)0.038TRAF5 LI1.061 (1.039–1.084)<0.00011.072 (1.048–1.096)<0.0001DFS, disease-free survival; HR, hazard ratio; KPS, Karnofsky performance score; LI, labeling index; OS, overall survival. Open table in a new tab DFS, disease-free survival; HR, hazard ratio; KPS, Karnofsky performance score; LI, labeling index; OS, overall survival. Prompted by the inverse correlation between miR-135a-5p LI and the Ki-67 index, miR-135a-5p–overexpressing U251 and U87MG sub–cell lines and the corresponding controls were established by recombinant lentivirus infection. miR-135a-5p expression was monitored by RT-qPCR (Supplemental Figure S4A). 5-Ethynyl-2′-deoxyuridine and MTS assays showed that compared with the controls, the proliferation activities of the miR-135a-5p–overexpressing sub–cell lines were much lower (P < 0.01 to approximately P < 0.001) (Figure 2, A–C ). Combining with the above ISH and IHC results, the data confirm the adverse effect of miR-135a-5p on glioma cell proliferation and suggest its existence in both cell culture models and human glioma tumors. By TargetScan version 7.0, it was found that among the target regions of miR-135a-5p in the 3′-UTR of the TRAF family members that were primarily concerned (TRAF1, TRAF4, TRAF5, and TRAF6), the score and ranking of TRAF5 were the highest (Supplemental Table S3). The silencing effect of the complementary pairing between miR-135a-5p and the 3′-UTR of TRAF5 mRNA was verified by dual-luciferase assays in the miR-135a-5p–overexpressing U251 and U87MG sub–cell lines (Figure 2, D and E). Moreover, the results of RT-qPCR and Western blot analysis showed that both the levels of TRAF5 mRNA and its protein were significantly lower in miR-135a-5p–overexpressing cell lines than in the controls (P < 0.05 to approximately P < 0.01) (Figure 2, F and G). These results demonstrate that TRAF5 is a natural target of miR-135a-5p in glioma cells. TRAF5 immunohistochemistry was performed in the above glioma and brain specimens, and its level was found to be higher in gliomas than in brain tissues and was increased with the elevatory tumor grade (P < 0.001) (Figure 3, A and B ). Furthermore, the TRAF5 LI correlated negatively with miR-135a-5p LI (r = −0.831, P < 0.0001) (Figure 3C) but positively with the Ki-67 index (r = 0.946, P < 0.0001) (Supplemental Figure S1C). Kaplan-Meier analyses found that stratifying the glioma patients with the TRAF5 levels perfectly reflected their differences in DFS (P < 0.0001) and OS (P < 0.0001) (Figure 3D). Even within the patients with the same tumor grade and IDH status as well as similar age and Karnofsky performance score, higher TRAF5 LI was always associated with shorter survival (DFS, P < 0.01 to approximately P < 0.0001; OS, P < 0.01 to approximately P < 0.0001) (Figure 3, E–I, and Supplemental Figure S5). Meanwhile, the data from the CGGA, The Cancer Genome Atlas, and Oncomine (three data sets, including Liang et al,23Liang Y. Diehn M. Watson N. Bollen A.W. Aldape K.D. Nicholas M.K. Lamborn K.R. Berger M.S. Botstein D. Brown P.O. Israel M.A. Gene expression profiling reveals molecularly and clinically distinct subtypes of glioblastoma multiforme.Proc Natl Acad Sci U S A. 2005; 102: 5814-5819Crossref PubMed Scopus (402) Google Scholar Bredel et al,24Bredel M. Bredel C. Juric D. Harsh G.R. Vogel H. Recht L.D. Sikic B.I. Functional network analysis reveals extended gliomagenesis pathway maps and three novel MYC-interacting genes in human gliomas.Cancer Res. 2005; 65: 8679-8689Crossref PubMed Scopus (258) Google Scholar and The Cancer Genome Atlas) all showed that the TRAF5 mRNA level was significantly higher in glioblastoma and predicted poorer prognosis (Supplemental Figure S3, B–D). Cox regression verified that TRAF5 LI was another independent survival predictor in parallel with miR-135a-5p LI and IDH status (Table 4 and Supplemental Table S2). These results also manifested that miR-135a-5p shortage was an important cause for the excessive expression of TRAF5 in glioma cells. To further interpret the inhibitory effect of miR-135a-5p on glioma cell proliferation, the cell cycle distribution of the miR-135a-5p–overexpressing sub–cell lines and their controls was compared, and the percentages of the G0/G1 cells were found to be significantly higher in the former (P < 0.01) (Figure 4, A and B ). The miR-135a-5p–overexpressing sub–cell lines were then infected with TRAF5 lentivirus, which had been proved to highly efficiently express TRAF5 in glioblastoma cell lines (Supplemental Figure S4, B–D). Flow cytometry assay results showed TRAF5 lentivirus infection completely abrogated the blocking effects of miR-135a-5p on G1/S-phase transition (P < 0.01) (Figure 4, A and B). Consistently, the miR-135a-5p–overexpressing sub–cell lines showed lower proliferation ability than their controls, whereas the proliferation activities of the TRAF5 lentivirus–infected sub–cell lines were restored almost to the control levels (P < 0.01 to approximately P < 0.001) (Figure 4, C–E). In addition, knocking down the endogenous TRAF5 with the specific siRNAs (siTRAF5#1 and siTRAF5#2) perfectly imitated the above effects of miR-135a-5p (P < 0.05 to approximately P < 0.001) (Figure 5, and Supplemental Figure S6). The above results of proliferation experiments were completely repeated in the U87MG sub–cell lines maintained in nonserum neurosphere medium (P < 0.01 to approximately P < 0.001) (Supplemental Figure S7). In nude mice experiments, the xenograft tumors overexpressing miR-135a-5p formed by U87MG cells (Figure 6) and primary GBM cells" @default.
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• W2896488950 date "2019-01-01" @default.
• W2896488950 modified "2023-10-14" @default.
• W2896488950 title "miR-135a-5p Functions as a Glioma Proliferation Suppressor by Targeting Tumor Necrosis Factor Receptor–Associated Factor 5 and Predicts Patients' Prognosis" @default.
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• W2896488950 cites W1814975936 @default.
• W2896488950 cites W1825871834 @default.
• W2896488950 cites W1967541628 @default.
• W2896488950 cites W1999876234 @default.
• W2896488950 cites W2018915744 @default.
• W2896488950 cites W2029990894 @default.
• W2896488950 cites W2036044314 @default.
• W2896488950 cites W2039872271 @default.
• W2896488950 cites W2098341282 @default.
• W2896488950 cites W2107318151 @default.
• W2896488950 cites W2109069210 @default.
• W2896488950 cites W2109597203 @default.
• W2896488950 cites W2109816625 @default.
• W2896488950 cites W2118769864 @default.
• W2896488950 cites W2124486266 @default.
• W2896488950 cites W2130979840 @default.
• W2896488950 cites W2142900503 @default.
• W2896488950 cites W2144035284 @default.
• W2896488950 cites W2151335185 @default.
• W2896488950 cites W2152091242 @default.
• W2896488950 cites W2171239378 @default.
• W2896488950 cites W2265951579 @default.
• W2896488950 cites W2366536035 @default.
• W2896488950 cites W2464778401 @default.
• W2896488950 cites W2517869136 @default.
• W2896488950 cites W2582203003 @default.
• W2896488950 cites W2585372137 @default.
• W2896488950 cites W2588386377 @default.
• W2896488950 cites W2590936958 @default.
• W2896488950 cites W2594731695 @default.
• W2896488950 cites W2742175163 @default.
• W2896488950 cites W2749694630 @default.
• W2896488950 cites W984156358 @default.
• W2896488950 doi "https://doi.org/10.1016/j.ajpath.2018.08.019" @default.
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