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Low Expression of miR-126 Is a Prognostic Marker for Metastatic Clear Cell Renal Cell Carcinoma

2015; Elsevier BV; Volume: 185; Issue: 3 Linguagem: Inglês

10.1016/j.ajpath.2014.11.017

ISSN

1525-2191

Autores

Heba Khella, Andreas Scorilas, Roy Mozes, Lorna Mirham, Evi Lianidou, Sergey N. Krylov, Jason Y. Lee, Michael Ordon, Robert Stewart, Michael A.S. Jewett, George M. Yousef,

Tópico(s)

MicroRNA in disease regulation

Resumo

Clear cell renal cell carcinoma (ccRCC) is an aggressive tumor with unpredictable behavior. Clinical parameters are not always accurate for predicting prognosis. miR-126 is differentially expressed in many cancers, including RCC, and is down-regulated in metastatic versus primary ccRCC. We assessed the prognostic significance of miR-126 in 264 primary ccRCCs. We also compared its expression in normal kidney, primary and metastatic ccRCC, and RCC subtypes. We validated our results on an independent set of 481 ccRCCs. miR-126 was down-regulated in metastatic versus primary tumors and in tumors of higher stage (P = 0.005) or higher grade (P = 0.002). miR-126 up-regulation was associated with significantly prolonged disease-free survival (P < 0.001) and overall survival (P = 0.015). For larger tumors (>4 cm), patients with higher miR-126 expression had significantly longer survival. Restoration of miR-126 expression decreased cellular migration and proliferation in RCC cell lines. The ccRCCs exhibited the highest miR-126 expression, and papillary RCCs exhibited the lowest expression. We identified a number of miR-126 targets and pathways that are involved in carcinogenesis, including the apoptosis signaling pathway. miR-126 is a promising prognostic marker in ccRCC that can distinguish between clear cell and papillary subtypes. In addition, miR-126 has potential therapeutic applications. Clear cell renal cell carcinoma (ccRCC) is an aggressive tumor with unpredictable behavior. Clinical parameters are not always accurate for predicting prognosis. miR-126 is differentially expressed in many cancers, including RCC, and is down-regulated in metastatic versus primary ccRCC. We assessed the prognostic significance of miR-126 in 264 primary ccRCCs. We also compared its expression in normal kidney, primary and metastatic ccRCC, and RCC subtypes. We validated our results on an independent set of 481 ccRCCs. miR-126 was down-regulated in metastatic versus primary tumors and in tumors of higher stage (P = 0.005) or higher grade (P = 0.002). miR-126 up-regulation was associated with significantly prolonged disease-free survival (P < 0.001) and overall survival (P = 0.015). For larger tumors (>4 cm), patients with higher miR-126 expression had significantly longer survival. Restoration of miR-126 expression decreased cellular migration and proliferation in RCC cell lines. The ccRCCs exhibited the highest miR-126 expression, and papillary RCCs exhibited the lowest expression. We identified a number of miR-126 targets and pathways that are involved in carcinogenesis, including the apoptosis signaling pathway. miR-126 is a promising prognostic marker in ccRCC that can distinguish between clear cell and papillary subtypes. In addition, miR-126 has potential therapeutic applications. Renal cell carcinoma (RCC) is the most common form of kidney cancer in adults. Recent reports show its increased incidence worldwide and marked increase of years of life lost due to kidney cancer in the past two decades.1US Burden of Disease CollaboratorsThe state of US health, 1990–2010: burden of diseases, injuries, and risk factors.JAMA. 2013; 310: 591-608Crossref PubMed Scopus (1815) Google Scholar, 2Chow W.H. Dong L.M. Devesa S.S. Epidemiology and risk factors for kidney cancer.Nat Rev Urol. 2010; 7: 245-257Crossref PubMed Scopus (983) Google Scholar From 20% to 30% of RCC patients have metastatic disease at the time of diagnosis, and 20% to 40% develop metastasis later, after nephrectomy.3Nerich V. Hugues M. Paillard M.J. Borowski L. Nai T. Stein U. Nguyen Tan H.T. Montcuquet P. Maurina T. Mouillet G. Kleinclauss F. Pivot X. Limat S. Thiery-Vuillemin A. 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The prognosis is unpredictable, because these small renal masses can be either progressive or nonprogressive.7Kurban G. Gedye C. Morales C. Yousef G.M. Almatar A. Jewett M.A. Diagnosis and treatment of small renal masses: the role for molecular biology.Arch Esp Urol. 2013; 66: 505-516PubMed Google Scholar Although larger tumors are generally associated with worse prognosis, tumor size alone is not an accurate prognostic predictor in RCC.8Pahernik S. Ziegler S. Roos F. Melchior S.W. Thuroff J.W. Small renal tumors: correlation of clinical and pathological features with tumor size.J Urol. 2007; 178: 414-417Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 9Turun S. Banghua L. Zheng S. Wei Q. Is tumor size a reliable predictor of histopathological characteristics of renal cell carcinoma?.Urol Ann. 2012; 4: 24-28Crossref PubMed Scopus (23) Google Scholar There is urgent need for molecular biomarkers that can help accurately predict disease outcome. Such biomarkers, used alone or in combination with other clinical parameters, could significantly improve patient management. miRNAs are short noncoding RNAs that regulate gene expression by binding to the 3′-UTR of their target genes. miRNAs are involved in a variety of biological functions, including cellular proliferation, differentiation, and apoptosis, which points to a role of miRNAs in tumor development and progression in different cancers.10Deng G. Sui G. Noncoding RNA in oncogenesis: a new era of identifying key players.Int J Mol Sci. 2013; 14: 18319-18349Crossref PubMed Scopus (92) Google Scholar, 11White N.M. Fatoohi E. Metias M. Jung K. Stephan C. Yousef G.M. Metastamirs: a stepping stone towards improved cancer management.Nat Rev Clin Oncol. 2011; 8: 75-84Crossref PubMed Scopus (161) Google Scholar miRNAs have also been shown to be potential prognostic markers in RCC.12Faragalla H. Youssef Y.M. Scorilas A. Khalil B. White N.M. Mejia-Guerrero S. Khella H. Jewett M.A. Evans A. Lichner Z. Bjarnason G. Sugar L. Attalah M.I. Yousef G.M. The clinical utility of miR-21 as a diagnostic and prognostic marker for renal cell carcinoma.J Mol Diagn. 2012; 14: 385-392Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 13Jung M. Mollenkopf H.J. Grimm C. Wagner I. Albrecht M. Waller T. Pilarsky C. Johannsen M. Stephan C. Lehrach H. Nietfeld W. Rudel T. Jung K. Kristiansen G. MicroRNA profiling of clear cell renal cell cancer identifies a robust signature to define renal malignancy.J Cell Mol Med. 2009; 13: 3918-3928Crossref PubMed Scopus (216) Google Scholar, 14Slaby O. Redova M. Poprach A. Nekvindova J. Iliev R. Radova L. Lakomy R. Svoboda M. Vyzula R. Identification of microRNAs associated with early relapse after nephrectomy in renal cell carcinoma patients.Genes Chromosomes Cancer. 2012; 51: 707-716Crossref PubMed Scopus (98) Google Scholar In previous work, we identified a miRNA signature associated with metastatic RCC and provided preliminary evidence that these small molecules are promising prognostic biomarkers in kidney cancer. miR-126 was one of the significantly down-regulated miRNAs in metastatic versus primary ccRCC.15Khella H.W. White N.M. Faragalla H. Gabril M. Boazak M. Dorian D. Khalil B. Antonios H. Bao T.T. Pasic M.D. Honey R.J. Stewart R. Pace K.T. Bjarnason G.A. Jewett M.A. Yousef G.M. Exploring the role of miRNAs in renal cell carcinoma progression and metastasis through bioinformatic and experimental analyses.Tumour Biol. 2012; 33: 131-140Crossref PubMed Scopus (56) Google Scholar, 16White N.M. Khella H.W. Grigull J. Adzovic S. Youssef Y.M. Honey R.J. Stewart R. Pace K.T. Bjarnason G.A. Jewett M.A. Evans A.J. 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Comprehensive microRNA profiling of prostate cancer.J Cancer. 2013; 4: 350-357Crossref PubMed Scopus (177) Google Scholar miR-126 has also been shown to have a tumor suppressor effect in melanoma.23Felli N. Felicetti F. Lustri A.M. Errico M.C. Bottero L. Cannistraci A. De F.A. Petrini M. Pedini F. Biffoni M. Alvino E. Negrini M. Ferracin M. Mattia G. Care A. miR-126&126∗ restored expressions play a tumor suppressor role by directly regulating ADAM9 and MMP7 in melanoma.PLoS One. 2013; 8: e56824Crossref PubMed Scopus (78) Google Scholar miR-126 is expressed in endothelial cells and controls angiogenesis in cancer.24Agudo J. Ruzo A. Tung N. Salmon H. Leboeuf M. Hashimoto D. Becker C. Garrett-Sinha L.A. Baccarini A. Merad M. Brown B.D. The miR-126-VEGFR2 axis controls the innate response to pathogen-associated nucleic acids.Nat Immunol. 2014; 15: 54-62Crossref PubMed Scopus (107) Google Scholar, 25Huang T.H. Chu T.Y. 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The miR-126-VEGFR2 axis controls the innate response to pathogen-associated nucleic acids.Nat Immunol. 2014; 15: 54-62Crossref PubMed Scopus (107) Google Scholar miR-126 was also shown to decrease cellular migration and invasion in colorectal cancer by targeting CXCR4,27Li Z. Li N. Wu M. Li X. Luo Z. Wang X. Expression of miR-126 suppresses migration and invasion of colon cancer cells by targeting CXCR4.Mol Cell Biochem. 2013; 381: 233-242Crossref PubMed Scopus (68) Google Scholar RhoA/ROCK,28Li N. Tang A. Huang S. Li Z. Li X. Shen S. Ma J. Wang X. miR-126 suppresses colon cancer cell proliferation and invasion via inhibiting RhoA/ROCK signaling pathway.Mol Cell Biochem. 2013; 380: 107-119Crossref PubMed Scopus (51) Google Scholar VCAM-1, and the PI3K/Akt signaling pathway.29Banerjee N. Kim H. Talcott S. Mertens-Talcott S. Pomegranate polyphenolics suppressed azoxymethane-induced colorectal aberrant crypt foci and inflammation: possible role of miR-126/VCAM-1 and miR-126/PI3K/AKT/mTOR.Carcinogenesis. 2013; 34: 2814-2822Crossref PubMed Scopus (93) Google Scholar miR-126 decreases cellular proliferation in osteosarcoma.30Xu J.Q. Liu P. Si M.J. Ding X.Y. MicroRNA-126 inhibits osteosarcoma cells proliferation by targeting Sirt1.Tumour Biol. 2013; 34: 3871-3877Crossref PubMed Scopus (31) Google Scholar In the present study, we examined the clinical utility of miR-126 as a prognostic marker in RCC. We correlated the expression of miR-126 with various clinical parameters and survival data. We validated our results on another independent set of patients from The Cancer Genome Atlas (TCGA). We also tested the ability of miR-126 to predict disease progression in subgroups of patients with stage I RCC (tumor size ≤4 cm versus >4 cm) and tested the effect of miR-126 overexpression on cellular migration and proliferation in RCC cell-line models. We identified miR-126–predicted targets and pathways and tested the effect of restoration of miR-126 expression level on apoptosis signaling pathway. Finally, we compared miR-126 among subtypes of RCC. We examined expression of miR-126 in 264 primary ccRCC and 20 metastatic ccRCC formalin-fixed, paraffin-embedded tissues. Specimens were collected from St. Michael's Hospital and University Health Network, Toronto, ON, Canada. Areas of pure tumor tissue with no hemorrhage or necrosis were selected by a pathologist (L.M. or G.M.Y.). Multiple sections were mixed from the same tumor, to compensate for tumor heterogeneity. Pure tumor areas were excised using laser-capture microdissection. Tumor classification and staging were according to the 2002 TNM system and the 2004 World Health Organization Classification of Tumours.31Tumours of the kidney. Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs.in: Eble J.N. Sauter G. Epstein J.I. Sesterhenn I. World Health Organization Classification of Tumours. IARCPress, Lyon2004: 12-87Google Scholar Distribution of the numerical variables of the study is shown in Supplemental Table S1. All procedures were approved by the Research Ethics Board at St. Michael's Hospital and University Health Network. RNA was extracted from 40 pairs of normal and cancer fresh tissues from the same patient, for comparison of miR-126 expression in the paired tissues. We also compared expression of miR-126 in normal kidney, oncocytoma, and RCC subtypes using fresh tissues obtained from 20 samples for each group. Fresh specimens were collected immediately after resection, snap-frozen in liquid nitrogen, and stored at −80°C until total RNA extraction. Total RNA was isolated using an miRNeasy kit (Qiagen, Mississauga, ON, Canada; Valencia, CA) according to the manufacturer's protocol and as described previously.15Khella H.W. White N.M. Faragalla H. Gabril M. Boazak M. Dorian D. Khalil B. Antonios H. Bao T.T. Pasic M.D. Honey R.J. Stewart R. Pace K.T. Bjarnason G.A. Jewett M.A. Yousef G.M. Exploring the role of miRNAs in renal cell carcinoma progression and metastasis through bioinformatic and experimental analyses.Tumour Biol. 2012; 33: 131-140Crossref PubMed Scopus (56) Google Scholar RNA quality and concentration were determined spectrophotometrically (NanoDrop 1000 spectrophotometer; Thermo Fisher Scientific, Waltham, MA). Samples optimal for analysis were stored at −80°C. For real-time quantitative RT-PCR (RT-qPCR), miR-126–specific reverse transcription was performed with 5 ng total RNA using a TaqMan microRNA reverse transcription kit (Life Technologies, Carlsbad, CA) as described by the manufacturer for miR-126. RT-qPCR was performed using a TaqMan microRNA assay kit on a Step One Plus real-time PCR system (Life Technologies). Thermal cycling conditions were as specified by the manufacturer's fast protocol, and all reactions were performed in triplicate. Relative expression was determined using the ΔΔCT method, and expression values were normalized to small nucleolar RNAs RNU48 and RNU44 (Life Technologies). Gene expression analysis was performed using the comparative CT method. Expression levels were normalized to the geometrical mean of two reference genes, SNORD44 (RNU44) and SNORD48 (RNU48). Because miR-126 expression levels do not exhibit Gaussian distribution, we performed U testing to examine differences in miR-126 expression status between primary and metastatic tumors. Correlations between continuous variables of the study (ie, miR-126 expression levels and tumor size) were assessed by Spearman's correlation coefficient. The relation of miR-126 expression levels as a continuous variable with many clinicopathological parameters, nominal and ordinal, was examined by U test and Jonckheere–Terpstra test, respectively. Converting a continuous variable to a dichotomous one is often helpful (eg, for classifying a patient cohort into high and low categories). Using the X-tile algorithm, which produces an optimal cutpoint and corrects for use of minimum P-value statistics, we established a cutoff point of 2.15 relative quantification units (equivalent to the 20th percentile) and we classified the patients into two groups, miR-126+ and miR-126−. Using either Fisher's exact test or Pearson's χ2 test, we evaluated associations between miR-126 status and various clinicopathological variables. Survival analysis and appraisal of the prognostic value of miR-126 was performed not only by developing univariate and multivariate Cox proportional hazard regression models, but also by constructing Kaplan–Meier disease-free survival (DFS) and overall survival (OS) curves. DFS was defined as the time between the initial resection of the kidney tumor and the event of recurrence or metastasis. OS was defined as the time between the initial resection of the kidney tumor and the date of death or date of last contact. The multivariate model was adjusted for patient age, histological stage, and tumor grade, and P values were calculated by the test for trend approach. P < 0.05 was considered indicative of statistical significance. The RCC cell lines 786-O and ACHN were obtained from ATCC (Manassas, VA) and were grown according to the manufacturer's protocol. Pre-miR miRNA precursor for miR-126 was purchased from Life Technologies. Cells were transfected using Ambion siPORT NeoFX transfection agent (Life Technologies) as recommended by the manufacturer and as described previously.32White N.M. Chow T.F. Mejia-Guerrero S. Diamandis M. Rofael Y. Faragalla H. Mankaruous M. Gabril M. Girgis A. Yousef G.M. Three dysregulated miRNAs control kallikrein 10 expression and cell proliferation in ovarian cancer.Br J Cancer. 2010; 102: 1244-1253Crossref PubMed Scopus (83) Google Scholar, 33White N.M. Bui A. Mejia-Guerrero S. Chao J. Soosaipillai A. Youssef Y. Mankaruos M. Honey R.J. Stewart R. Pace K.T. Sugar L. Diamandis E.P. Doré J. Yousef G.M. Dysregulation of kallikrein-related peptidases in renal cell carcinoma: potential targets of miRNAs.Biol Chem. 2010; 391: 411-423Crossref PubMed Google Scholar The transfection agent was diluted in Opti-MEM reduced serum medium (Life Technologies) and incubated for 10 minutes at room temperature. miR-126 precursor was diluted in the same medium to a final concentration of 30 nmol/L, combined with the transfection agent, and incubated for 10 minutes at room temperature. Transfection mixtures were added to the cell-culture plate and overlaid with cell suspensions. Cells were incubated at 37°C and 5% CO2. Three separate transfections were performed, and each was analyzed in triplicate. Transfection efficiency was confirmed using BLOCK-iT Fluorescent Oligo oligomer (Life Technologies). 786-O cells were seeded in a 12-well plate, and transfected with siPORT NeoFX transfection agent, negative control, or miR-126. At 24 hours after transfection, the cell monolayer was wounded using a 200-μL pipette tip. Hydroxyurea (100 mmol/L) was added to the cell culture to inhibit cell proliferation. Photomicrographs were taken every 30 minutes starting at the time of wounding (0 hour) and ending at 9 hours. ImageJ software version 1.47v (NIH, Bethesda, MD) was used for cell migration analysis. Percent cell-free area was calculated as (cell-free area9hours/cell-free area0hours) × 100, and cell migration rate was expressed as a percentage of the cell-covered area (ie, as 100 − percent cell-free area). Each experiment was performed in triplicate. Cellular proliferation was measured by using a cell proliferation reagent WST-1 (Roche Applied Science, Indianapolis, IN) colorimetric assay. Cells were plated at 6.0 × 103 cells per well in a 96-well plate and transfected with siPORT NeoFX transfection agent, negative control, or miR-126. The cell proliferation reagent WST-1 was added to each well, and cells were incubated for 2 hours at 37°C. The absorbance of each well was measured at a wavelength of 440 nm. Each test was repeated in six replicates. We compiled miR-126 read counts and clinical variables associated with ccRCC patients from TCGA (https://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp, last accessed November 7, 2014). Clinical variables that were analyzed in relation to miR-126 read counts included OS time, pathological stage, and tumor size in 481 patients. Read counts of miR-126 were compared in 68 matched pairs of ccRCC and normal kidney tissues. Data were obtained from TCGA. We also obtained the expression levels of a number of miR-126–predicted targets (including PIK3CD, PIK3R2, and VEGFA) in 481 ccRCCs, and we correlated the expression levels of these targets with OS. Cutoff points were determined, and Kaplan–Meier curves were constructed using Cutoff Finder software (http://molpath.charite.de/cutoff/index.jsp, last accessed November 7, 2014). Target prediction was performed using TargetScanHuman software release 6.2 (http://www.targetscan.org, last accessed November 7, 2014). We also used miRecords34Xiao F. Zuo Z. Cai G. Kang S. Gao X. Li T. miRecords: an integrated resource for microRNA-target interactions.Nucleic Acids Res. 2009; 37: D105-D110Crossref PubMed Scopus (1149) Google Scholar software that combines the results of 11 prediction programs; only predictions made by at least three programs were included. We filtered the predicted gene targets list through extensive literature search and pathway analysis using DIANA-miRPath software version 2.0 (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=mirpath/index, last accessed November 7, 2014) and the Gene Functional Classification tool from the DAVID Bioinformatics Database version 6.7 (http://david.abcc.ncifcrf.gov/gene2gene.jsp, last accessed November 7, 2014). The University of California Santa Cruz Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway, last accessed November 24, 2013) was used for sequence comparison of miR-126. Conservation among species of these miRNAs was examined with sequence alignment in the genomes of 46 vertebrate species. We tested the effect of miR-126 overexpression on apoptosis by TaqMan array human gene expression assays using a StepOnePlus real-time PCR system (Life Technologies). 786-O cells transfected with miR-126 were compared with untransfected cells. Total RNA was isolated using an RNeasy mini kit (Qiagen) according to the manufacturer's protocol. Reverse transcription was performed using a high-capacity cDNA reverse transcription kit (Life Technologies). This was followed by PCR amplification using TaqMan Array human gene expression assays specific for human apoptosis. Fold change was calculated by the 2−ΔΔCt method. First, ΔCT values were calculated for each target by normalizing its threshold cycle to the average of four endogenous controls including 18S, GAPDH, HPRT1, and GUSB. Then, ΔΔCT was calculated as 786-OmiR-126 transfected cells − 786-Ountransfected cells × ΔCT. We used RT-qPCR analysis to compare miR-126 expression in 40 pairs of normal kidney and ccRCC tissues from the same patient. miR-126 was up-regulated in ccRCC, compared with normal tissue counterparts (Figure 1). We validated our results on 68 pairs of normal kidney and ccRCC tissues using data from TCGA, which confirmed the up-regulation of miR-126 in cancer (P < 0.0001) (Supplemental Figure S1, A and B). We assessed miR-126 expression in 264 primary and 20 metastatic ccRCC tumors by RT-qPCR. miR-126 expression was decreased in metastatic versus primary ccRCC (10.45 ± 3.10 versus 12.47 ± 1.17, mean ± SEM), although the difference was not statistically significant (P = 0.827) (Table 1).Table 1miR-126 Expression As a Continuous Variable Stratified According to Classical Clinicopathological ParametersVariableNo. of patientsmiR-126 expression (RQ units)P valueMeans ± SEMMedianAge (years) ≤6113011.10 ± 1.266.560.839 >6113013.82 ± 1.986.25Sex Male17112.66 ± 1.346.710.621 Female9111.94 ± 2.255.94Tumor status Primary26412.41 ± 1.176.510.827 Metastatic2010.45 ± 3.106.95Tumor size (cm) ≤4.08815.46 ± 3.166.960.069 >4.017112.52 ± 1.755.47Laterality Left14214.32 ± 1.876.840.185 Right11810.11 ± 1.186.16Tumor stage I/II16713.22 ± 1.486.780.012 III/IV9011.18 ± 1.995.23Tumor grade I/II12112.12 ± 1.297.230.016 III/IV13412.93 ± 1.975.82RQ, relative quantification.P values were calculated by Mann-Whitney U test. Open table in a new tab RQ, relative quantification. P values were calculated by Mann-Whitney U test. We tested the association between miR-126 expression and the different clinicopathological characteristics in 264 primary ccRCC cases. As a continuous variable, miR-126 was significantly down-regulated in tumor stages III/IV compared with stages I/II (P = 0.012). Also, miR-126 expression was significantly lower in tumor grade III/IV, compared with grade I/II (P = 0.016). There was no significant association between miR-126 expression level and age, sex, tumor size, or laterality (Table 1). As a binary variable, miR-126 overexpression negatively correlated with tumor size; miR-126 positivity was observed in 88% of cases with smaller tumors (≤4 cm), compared with 77% of cases with larger tumors (>4 cm) (P = 0.048) (Table 2). Furthermore, there was a stepwise decrease of miR-126 positivity with increasing tumor stage (P < 0.001). The percentage of cases with tumor grade I and miR-126 positivity was similar to that for grade II, and this was significantly higher than for grade III and IV (P = 0.002) There was no significant association between miR-126 expression and patient age, sex, or tumor laterality (Table 2).Table 2Associations between miR-126 Status As a Binary Variable and Clinicopathological ParametersVariableNo. of patients (%)P valueTotalmiR-126−miR-126+Age (years) ≤6113025 (19.2)105 (80.8)1.000∗Fisher's exact test. >6113025 (19.2)105 (80.8)Sex Male17132 (18.7)139 (81.3)0.5∗Fisher's exact test. Female9120 (22.0)71 (78.0)Laterality Left14225 (17.6)117 (82.4)0.528∗Fisher's exact test. Right11825 (21.2)93 (78.8)Tumor size (cm) ≤4.08811 (12.5)77 (87.5)0.048∗Fisher's exact test. >4.017139 (22.8)132 (77.2)Tumor stage I12616 (12.7)110 (87.3)<0.001†Pearson's χ2 test. II417 (17.1)34 (82.9) III8120 (24.7)61 (75.3) IV97 (77.8)2 (22.2)Tumor grade I152 (13.3)13 (86.7)0.002†Pearson's χ2 test. II10613 (12.3)93 (87.7) III10018 (18.0)82 (82.0) IV3414 (41.2)20 (58.8)Cutoff point: 2.15 relative quantification units (equivalent to the 20th percentile). Relative quantification to the geometrical mean of two reference genes RNU44 and RNU48.∗ Fisher's exact test.† Pearson's χ2 test. Open table in a new tab Cutoff point: 2.15 relative quantification units (equivalent to the 20th percentile). Relative quantification to the geometrical mean of two reference genes RNU44 and RNU48. In the univariate analysis, patients with higher miR-126 expression exhibited a significant increase in both DFS (hazard ratio HR = 0.30, 95% CI = 0.18 to 0.50, P < 0.001) and OS (HR = 0.40, 95% CI = 0.19 to 0.86, P = 0.019). The same trend toward better prognosis was observed in the multivariate analysis for both DFS (HR = 0.58, 95% CI = 0.32 to 1.04) and OS (HR = 0.78, 95% CI = 0.32 to 1.87), although the trend did no

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