miR-182 as a Prognostic Marker for Glioma Progression and Patient Survival
2010; Elsevier BV; Volume: 177; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2010.090812
ISSN1525-2191
AutoresLili Jiang, Pu Mao, Libing Song, Jueheng Wu, Jieting Huang, Chuyong Lin, Jie Yuan, Liang‐Hu Qu, Shi‐Yuan Cheng, Jun Li,
Tópico(s)Cancer-related molecular mechanisms research
ResumoAccumulating evidence indicates that miRNA expression can be used as a diagnostic and prognostic marker for human cancers. We report that the expression level of miR-182 was markedly up-regulated in glioma cell lines and in human primary glioma specimens. Quantitative PCR analysis showed that miR-182 was significantly increased by up to 32-fold in glioma tumors compared with the adjacent nontumor brain tissues obtained from the same patient. Elevated expression of miR-182 was further identified by in situ hybridization in 248 of 253 (98%) archived human glioma biopsies tested. Statistical analysis revealed a significant correlation between miR-182 expression and World Health Organization glioma grading (P < 0.001). The cumulative 5-year survival rate of glioma patients was 51.54% (95% confidence interval, 0.435 to 0.596) in the low miR-182-expression group, whereas it was only 7.23% (95% confidence interval, 0.027 to 0.118) in the high miR-182-expression group (P = 0.001), and multivariate Cox regression analysis indicated that miR-182 expression was an independent prognostic indicator for the survival of glioma patients. Moreover, the correlations of miR-182 level with the clinical features of glioma suggested in the in situ hybridization analysis were further verified by the real-time RT-PCR analysis. Taken together, our results suggest that miR-182 could be a valuable marker of glioma progression and that high miR-182 expression is associated with poor overall survival in patients with malignant glioma. Accumulating evidence indicates that miRNA expression can be used as a diagnostic and prognostic marker for human cancers. We report that the expression level of miR-182 was markedly up-regulated in glioma cell lines and in human primary glioma specimens. Quantitative PCR analysis showed that miR-182 was significantly increased by up to 32-fold in glioma tumors compared with the adjacent nontumor brain tissues obtained from the same patient. Elevated expression of miR-182 was further identified by in situ hybridization in 248 of 253 (98%) archived human glioma biopsies tested. Statistical analysis revealed a significant correlation between miR-182 expression and World Health Organization glioma grading (P < 0.001). The cumulative 5-year survival rate of glioma patients was 51.54% (95% confidence interval, 0.435 to 0.596) in the low miR-182-expression group, whereas it was only 7.23% (95% confidence interval, 0.027 to 0.118) in the high miR-182-expression group (P = 0.001), and multivariate Cox regression analysis indicated that miR-182 expression was an independent prognostic indicator for the survival of glioma patients. Moreover, the correlations of miR-182 level with the clinical features of glioma suggested in the in situ hybridization analysis were further verified by the real-time RT-PCR analysis. Taken together, our results suggest that miR-182 could be a valuable marker of glioma progression and that high miR-182 expression is associated with poor overall survival in patients with malignant glioma. Gliomas, which represent approximately 70% of all brain tumor cases, are the most common and malignant tumors in the brain of humans, and the overall prognosis for patients inflicted with malignant gliomas is poor.1Deorah S Lynch CF Sibenaller ZA Ryken TC Trends in brain cancer incidence and survival in the United States: surveillance, epidemiology, and end results program, 1973 to 2001.Neurosurg Focus. 2006; 20: E1Crossref PubMed Scopus (263) Google Scholar The cumulative 1-year survival rate of glioma patients is less than 30%. The median survival time for glioblastoma multiforme (GBM), the grade IV gliomas, is only 15 months.2Stupp R Mason WP van den Bent MJ Weller M Fisher B Taphoorn MJ Belanger K Brandes AA Marosi C Bogdahn U Curschmann J Janzer RC Ludwin SK Gorlia T Allgeier A Lacombe D Cairncross JG Eisenhauer E Mirimanoff RO European Organization for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.N Engl J Med. 2005; 352: 987-996Crossref PubMed Scopus (14644) Google Scholar Although significant improvements have been made in neurosurgical techniques, development of new chemotherapeutic agents, and exploitation of accurate radiotherapy, extremely poor prognosis of malignant gliomas remains unchanged over the last three decades.3Furnari FB Fenton T Bachoo RM Mukasa A Stommel JM Stegh A Hahn WC Ligon KL Louis DN Brennan C Chin L DePinho RA Cavenee WK Malignant astrocytic glioma: genetics, biology, and paths to treatment.Genes Dev. 2007; 21: 2683-2710Crossref PubMed Scopus (1810) Google Scholar, 4Wen PY Kesari S Malignant gliomas in adults.N Engl J Med. 2008; 359: 492-507Crossref PubMed Scopus (3243) Google Scholar Therefore, it is an urgent clinical challenge to identify sensitive and specific early biomarkers for the diagnosis and prognosis of this malignancy, as well as to develop new therapeutic strategies for this deadly disease. MicroRNAs (miRNAs) are single stranded and noncoding RNAs that play important roles in many biological processes.5Bartel DP MicroRNAs: genomics, biogenesis, mechanism and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29453) Google Scholar During the initiation and progression of human cancers, miRNAs have been shown to modulate cell proliferation, survival, and tumor angiogenesis, invasion, and metastasis.6Gregory RI Shiekhattar R MicroRNA biogenesis and cancer.Cancer Res. 2005; 65: 3509-3512Crossref PubMed Scopus (570) Google Scholar, 7Calin GA Croce CM MicroRNA signatures in human cancers.Nat Rev Cancer. 2006; : 857-866Crossref PubMed Scopus (6605) Google Scholar, 8Esquela-Kerscher A Slack FJ Oncomirs: microRNAs with a role in cancer.Nat Rev Cancer. 2006; 6: 259-269Crossref PubMed Scopus (6186) Google Scholar, 9Nicoloso MS Calin GA MicroRNA involvement in brain tumors: from bench to bedside.Brain Pathol. 2008; 18: 122-129Crossref PubMed Scopus (83) Google Scholar Dysregulation of miRNA expression has been found in various types of human cancers including cancers occurring in breast, colon, lung, liver, pancreas, chronic lymphocytic leukemia, and malignant gliomas.10Iorio MV Ferracin M Liu CG Veronese A Spizzo R Sabbioni S Magri E Pedriali M Fabbri M Campiglio M Ménard S Palazzo JP Rosenberg A Musiani P Volinia S Nenci I Calin GA Querzoli P Negrini M Croce CM MicroRNA gene expression deregulation in human breast cancer.Cancer Res. 2005; 65: 7065-7070Crossref PubMed Scopus (3474) Google Scholar, 11Schetter AJ Leung SY Sohn JJ Zanetti KA Bowman ED Yanaihara N Yuen ST Chan TL Kwong DL Au GK Liu CG Calin GA Croce CM Harris CC MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma.JAMA. 2008; 299: 425-436Crossref PubMed Scopus (1431) Google Scholar, 12Yu SL Chen HY Chang GC Chen CY Chen HW Singh S Cheng CL Yu CJ Lee YC Chen HS Su TJ Chiang CC Li HN Hong QS Su HY Chen CC Chen WJ Liu CC Chan WK Chen WJ Li KC Chen JJ Yang PC MicroRNA signature predicts survival and relapse in lung cancer.Cancer Cell. 2008; 13: 48-57Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 13Jiang J Gusev Y Aderca I Mettler TA Nagorney DM Brackett DJ Roberts LR Schmittgen TD Association of microRNA expression in hepatocellular carcinomas with hepatitis infection, cirrhosis, and patient survival.Clin Cancer Res. 2008; 14: 419-427Crossref PubMed Scopus (475) Google Scholar, 14Bloomston M Frankel WL Petrocca F Volinia S Alder H Hagan JP Liu CG Bhatt D Taccioli C Croce CM MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis.JAMA. 2007; 297: 1901-1919Crossref PubMed Scopus (1052) Google Scholar, 15Ciafrè SA Galardi S Mangiola A Ferracin M Liu CG Sabatino G Negrini M Maira G Croce CM Farace MG Extensive modulation of a set of microRNAs in primary glioblastoma.Biochem Biophys Res Commun. 2005; 334: 1351-1358Crossref PubMed Scopus (948) Google Scholar, 16Fulci V Chiaretti S Goldoni M Azzalin G Carucci N Tavolaro S Castellano L Magrelli A Citarella F Messina M Maggio R Peragine N Santangelo S Mauro FR Landgraf P Tuschl T Weir DB Chien M Russo JJ Ju J Sheridan R Sander C Zavolan M Guarini A Foà R Macino G Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukemia.Blood. 2007; 109: 4944-4951Crossref PubMed Scopus (420) Google Scholar While the molecular mechanisms of miRNA-mediated gene regulation are still under investigation, emerging studies suggest that miRNA expression signatures can be diagnostically and/or prognostically indicative for human cancers.17Lu J Getz G Miska EA Alvarez-Saavedra E Lamb J Peck D Sweet-Cordero A Ebert BL Mak RH Ferrando AA Downing JR Jacks T Horvitz HR Golub TR MicroRNA expression profiles classify human cancers.Nature. 2005; 435: 834-838Crossref PubMed Scopus (8201) Google Scholar For example, miRNA profiles of acute lymphocytic leukemia could categorize patients with different molecular pathologies into different subgroups.18Garzon R Volinia S Liu CG Fernandez-Cymering C Palumbo T Pichiorri F Fabbri M Coombes K Alder H Nakamura T Flomenberg N Marcucci G Calin GA Kornblau SM Kantarjian H Bloomfield CD Andreeff M Croce CM MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia.Blood. 2008; 111: 3183-3189Crossref PubMed Scopus (529) Google Scholar A unique miRNA signature was found to be associated with prognostic factors and disease progression in chronic lymphocytic leukemia.19Calin GA Ferracin M Cimmino A Di Leva G Shimizu M Wojcik SE Iorio MV Visone R Sever NI Fabbri M Iuliano R Palumbo T Pichiorri F Roldo C Garzon R Sevignani C Rassenti L Alder H Volinia S Liu CG Kipps TJ Negrini M Croce CM A microRNA signature associated with prognosis and progression in chronic lymphocytic leukemia.N Engl J Med. 2005; 353: 1793-1801Crossref PubMed Scopus (2083) Google Scholar A statistical miRNA profile that differed with tumor histology distinguished lung adenocarcinomas from noncancerous lung tissues and correlated with the prognosis of patients with lung cancers.20Yanaihara N Caplen N Bowman E Seike M Kumamoto K Yi M Stephens RM Okamoto A Yokota J Tanaka T Calin GA Liu CG Croce CM Harris CC Unique microRNA molecular profiles in lung cancer diagnosis and prognosis.Cancer Cell. 2006; 9: 189-198Abstract Full Text Full Text PDF PubMed Scopus (2690) Google Scholar A five-miRNA signature was identified as a potential independent predictor of cancer relapse and survival of non-small-cell lung cancer patients.12Yu SL Chen HY Chang GC Chen CY Chen HW Singh S Cheng CL Yu CJ Lee YC Chen HS Su TJ Chiang CC Li HN Hong QS Su HY Chen CC Chen WJ Liu CC Chan WK Chen WJ Li KC Chen JJ Yang PC MicroRNA signature predicts survival and relapse in lung cancer.Cancer Cell. 2008; 13: 48-57Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar An onco-miRNA, miR-21, was drastically up-regulated in various types of human cancers, including gliomas, and associated with prognosis and therapeutic outcome in colon and breast cancers.15Ciafrè SA Galardi S Mangiola A Ferracin M Liu CG Sabatino G Negrini M Maira G Croce CM Farace MG Extensive modulation of a set of microRNAs in primary glioblastoma.Biochem Biophys Res Commun. 2005; 334: 1351-1358Crossref PubMed Scopus (948) Google Scholar, 20Yanaihara N Caplen N Bowman E Seike M Kumamoto K Yi M Stephens RM Okamoto A Yokota J Tanaka T Calin GA Liu CG Croce CM Harris CC Unique microRNA molecular profiles in lung cancer diagnosis and prognosis.Cancer Cell. 2006; 9: 189-198Abstract Full Text Full Text PDF PubMed Scopus (2690) Google Scholar, 21Chan JA Krichevsky AM Kosik KS MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells.Cancer Res. 2005; 65: 6029-6033Crossref PubMed Scopus (2228) Google Scholar, 22Yan LX Huang XF Shao Q Huang MY Deng L Wu QL Zeng YX Shao JY MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis.RNA. 2008; 14: 2348-2360Crossref PubMed Scopus (972) Google Scholar Acute myeloid leukemia patients with high expression of miR-191 and miR-199a had significantly lower overall and event-free survival rates than AML patients with low expression of the miRNAs.18Garzon R Volinia S Liu CG Fernandez-Cymering C Palumbo T Pichiorri F Fabbri M Coombes K Alder H Nakamura T Flomenberg N Marcucci G Calin GA Kornblau SM Kantarjian H Bloomfield CD Andreeff M Croce CM MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia.Blood. 2008; 111: 3183-3189Crossref PubMed Scopus (529) Google Scholar Reduced expression of let-7 miRNA in human lung cancer and ovarian cancer was associated with shortened postoperative survival.23Takamizawa J Konishi H Yanagisawa K Tomida S Osada H Endoh H Harano T Yatabe Y Nagino M Nimura Y Mitsudomi T Takahashi T Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival.Cancer Res. 2004; 64: 3753-3756Crossref PubMed Scopus (2151) Google Scholar, 24Lu L Katsaros D de la Longrais IA Sochirca O Yu H Hypermethylation of let-7a-3 in epithelial ovarian cancer is associated with low insulin-like growth factor-II expression and favorable prognosis.Cancer Res. 2007; 67: 10117-10122Crossref PubMed Scopus (267) Google Scholar Taken together, a body of evidence has suggested that miRNA expression might be a clinically useful marker for analyzing molecular pathogenesis of human cancers and developing targeted therapies or selecting high-risk cancer patients for adjuvant chemotherapies. Recently, we examined the levels of 450 miRNAs in human primary glioma tissues of varying World Health Organization grades by microarray analyses and found that miR-182 was significantly up-regulated in glioma tissues as compared with normal brain tissues. In the present study, we characterized the miR-182 expression in a cohort of 253 gliomas specimens including World Health Organization grade I to IV tumors. Our results suggested that expression of miR-182 was associated with the progression of human gliomas with significant prognostic value and thus might represent a useful prognostic marker and a potential target for gliomas therapies. Primary normal human astrocytes (NHA) were purchased from the Sciencell Research Laboratories (Carlsbad, CA) and cultured as recommended by the manufacturer. Glioma cell lines LN18, U87, U118, T98 (American Type Culture Collection, Manassas, VA), SNB19 (from Y.-H. Zhou, University of California, Irvine, CA), A172, U138, U251, and U373 (our own collections25Jarzynka MJ Hu B Hui KM Bar-Joseph I Gu W Hirose T Haney LB Ravichandran KS Nishikawa R Cheng SY ELMO1 and Dock180, a bipartite Rac1 guanine nucleotide exchange factor, promote human glioma cell invasion.Cancer Res. 2007; 67: 7203-7211Crossref PubMed Scopus (118) Google Scholar) were grown in the Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and 100 units penicillin-streptomycin at 37°C with 5% CO2 atmosphere in a humidified incubator. A total of 253 paraffin-embedded glioma specimens (only astrocytomas were included and no oligodendrogliomas were used), which had been clinically and histopathologically diagnosed at the First Affiliated Hospital of Sun Yat-Sen University from 2000 to 2005, included World Health Organization grade I to IV tumors. The grade I gliomas used in the study were pilocytic astrocytomas. Among the 253 cases collected, survival information for 119 cases was available. For the use of these clinical materials for research purposes, patient’s consents and approval from the Institutional Research Ethics Committee were obtained. Clinical information of these samples is described in detail in Table 1. The purity in sections adjacent to the regions used for in situ hybridization (ISH) analysis and RNA extraction was validated through routine histopathological analysis. Three normal brain tissue samples were obtained by collecting donations with consents from individuals who died in traffic accidents and were confirmed to be free of any prior pathological lesions.Table 1Clinicopathological Characteristics of Studied Patients and Expression of miR-182 in GliomasCharacteristicsNo. of cases%Sex Male17267.98 Female8132.02Age, years ≤4515460.87 >459939.13Gliomas histopathology (World Health Organization grading) Grade I2911.46 Grade II7931.23 Grade III9939.13 Grade IV4618.18Patient survival (n = 119) Alive4134.45 Deceased7865.55Survival time for low miR-182 expression group, monthsMedian42.00Survival time for high miR-182 expression group, monthsMedian23.00miR-182 expression (in situ hybridization) Negative51.98 Positive24898.02 Low expression (SI < 6)12348.62 High expression (SI ≥ 6)13051.38 Open table in a new tab Total miRNA from cultured cells, fresh surgical glioma tissues, and paraffin-embedded, archived clinical glioma specimens was extracted by using the mirVana miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer’s instructions. CDNA was synthesized from 5 ng of total RNA by using the Taqman miRNA reverse transcription kit (Applied Biosystems, Foster City, CA), and the expression levels of miR-182 were quantified by using miRNA-specific TaqMan MiRNA Assay Kit (Applied Biosystems). Real-time RT-PCR was performed by using the Applied Biosystems 7500 Sequence Detection system. The expression of miRNA was defined based on the threshold cycle (Ct), and relative expression levels were calculated as 2−[(Ct of miR-182) − (Ct of U6)] after normalization with reference to expression of U6 small nuclear RNA. In situ hybridization analysis was performed as previously described.11Schetter AJ Leung SY Sohn JJ Zanetti KA Bowman ED Yanaihara N Yuen ST Chan TL Kwong DL Au GK Liu CG Calin GA Croce CM Harris CC MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma.JAMA. 2008; 299: 425-436Crossref PubMed Scopus (1431) Google Scholar Briefly, thin sections in 4-μm thickness of paraffin-embedded specimens were deparaffinized with xylene and rehydrated with an ethanol dilution series from 100% to 25%. Sections were treated with 40 μg/ml proteinase K in 0.2% glycine at 37°C for 5 minutes and 30 seconds and re-fixed in 4% paraformaldehyde. Slides were prehybridized in a hybridization solution (50% formamide, 5× standard saline citrate, 0.1% Tween, 9.2 mmol/L citric acid for adjustment to pH 6.0, 50 μg/ml heparin, 500 μg/ml yeast RNA) at 49.5°C for 2 hours. Subsequently, 20 nmol/L of a Locked Nucleic Acid-modified, 5′digoxigenin (DIG) labeled oligonucleotide probe complementary to miR-182 or a scrambled control probe was added to 100 μl of the hybridization solution and hybridized at a temperature of 49.5°C overnight. Sections were rinsed twice in 5× standard saline citrate, followed by three washes of 20 minutes at 50°C in 50% formamide/2× standard saline citrate and subsequently five times washes in PBS/0.1% Tween-20, and blocked in blocking solution (2% goat serum, 2 mg/ml bovine serum albumin in Phosphate Buffered Saline with Tween-20) at room temperature for 1 hour. An anti-DIG antibody (1:1000; Roche, Mannheim, Germany) was applied, and the sections were incubated at 4°C overnight. After washing in a staining solution (10 mmol/L Tris-HCl pH 9.0, 50 mmol/L MgCl2, 100 mmol/L NaCl, 0.1% Tween-20), the sections were incubated with the Nitro-Blue Tetrazolium Chloride/5-Bromo-4-Chloro-3′-Indolyphosphate p-Toluidine (NBT/BCIP) developing solution (50 ml staining solution, 240 μl of 50 mg/ml NBT, 175 μl of 50 mg/ml BCIP) and rinsed in PBST followed by double distilled water. The sections were then dehydrated and mounted with Entellan. The expression of miR-182 in a total of 253 paraffin-embedded glioma specimens, randomly divided into two cohorts, were examined by the same group of four investigators, using in situ hybridization analyses. Furthermore, the signals of in situ hybridization of tissue sections were examined and scored separately by two independent investigators blinded to the histopathological features and patient data of the samples. The scores were determined by combining the proportion of positively stained tumor cells and the intensity of staining. The proportion of positively stained tumor cells was graded as follows: 0, no positive tumor cells; 1, 50% positive tumor cells. The cells at each intensity of staining were recorded on a scale of 0 (no staining), 1 (weak staining, light blue), 2 (moderate staining, blue), and 3 (strong staining, dark blue). For tumors that showed heterogeneous staining, the predominant pattern was taken into account for scoring. The staining index (SI) was calculated as follows: staining index = staining intensity × proportion of positively stained tumor cells. Using this method, the expression of miR-182 in gliomas was evaluated by SI, scored as 0, 1, 2, 3, 4, 6, or 9. Cutoff values to define the high- and low-expression of miR-182 were chosen on the basis of a measurement of heterogeneity with the log-rank test statistic with respect to overall survival. Because the optimal cutoff SIs were identified from the current study as 6, an SI score ≥6 was taken to define tumors as high expression, and SI <6 to define tumors as low expression of miR-182. In situ hybridization signals for miR-182 expression in tumor and normal tissues were quantified by using the AxioVision Rel.4.6 computerized image analysis system assisted with the automatic measurement program (Carl Zeiss, Oberkochen, Germany). The method of Mean Optical Density (MOD) was preformed as previously reported.26Bao S Ouyang G Bai X Huang Z Ma C Liu M Shao R Anderson RM Rich JN Wang XF Periostin potently promotes metastatic growth of colon cancer by augmenting cell survival via the Akt/PKB pathway.Cancer Cell. 2004; 5: 329-339Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar Briefly, the stained slides were evaluated at 200× magnification by using the SAMBA 4000 computerized image analysis system assisted with Immuno 4.0 quantitative program (Image Products International, Chantilly, VA), and 10 randomly picked fields in each specimen were analyzed to determine the MOD, which represents the strength of staining signals as measured per positive pixels, based on which the mean MOD of a study group of samples was further generated for subsequent intergroup comparative analysis. This program takes into consideration of the areas and numbers of positively stained tumor cells to obviate the impact of cell density on MOD value. A negative control with each batch of staining was used for background subtraction in the quantitative analysis. The representative staining fields of each specimen were analyzed and scored independently by two observers who were blinded to each other and to the diagnosis of the specimens. The MOD data were statistically analyzed by using t-test to compare the MOD differences between groups, and P < 0.05 was considered to be statistically significant. To generate an mir-182 expression vector, approximately 200-bp genomic fragment up and downstream of the pre-mir-182 form was amplified by PCR and cloned into Plasmid of Murine Stem Cell Virus (pMSCV). Retroviral production and infection were performed as described previously.27Hahn WC Dessain SK Brooks MW King JE Elenbaas B Sabatini DM DeCaprio JA Weinberg RA Enumeration of the simian virus 40 early region elements necessary for human cell transformation.Mol Cell Biol. 2002; 22: 2111-2123Crossref PubMed Scopus (519) Google Scholar After 48 hours infection in NHA, the expression of miR-182 was examined either by real-time RT-PCR analysis or in situ hybridization analysis. All statistical analyses and the patient age cutoff values were performed by using the SPSS 10.0 statistical software package (SPSS Inc, Chicago, IL). The χ2 test was used to analyze the relationship between miR-182 expression and the clinicopathological characteristics. Bivariate correlations between study variables were calculated via Spearman’s rank correlation coefficients. Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test. Survival data were evaluated by using univariate and multivariate Cox regression analyses. In all cases, P < 0.05 was considered statistically significant. We performed initial screening on the expression profile of a total of 450 miRNAs by using normal brain tissues and primary tumor specimens of four different grades of human gliomas (one specimen per grade). When comparing expression levels of the miRNAs in gliomas samples with those in the normal brain tissues, we found that nine miRNAs displayed more than 1.61-fold increase and levels of eight miRNAs were decreased by 1.28-fold or lower in their expression, among which miR-182 showed the most robust change in expression (3.93-fold increase; Table 2). To further characterize the expression of miR-182 in gliomas, we performed real-time RT-PCR analyses and found that miR-182 was markedly up-regulated to various levels in all nine glioma cell lines examined compared with NHA (Figure 1A). To determine whether miR-182 up-regulation correlated with clinical glioma progression, we assessed the levels of miR-182 expression by real-time RT-PCR analyses in three normal brain tissues and 12 fresh-frozen glioma tissues of varying World Health Organization grades (grades I to IV, with three samples per grade). miR-182 was found to be up-regulated in all 12 human primary glioma tissues compared with the normal brain tissue samples (Figure 1B). Importantly, the trend of miR-182 expression in these human primary glioma tissues correlated with the malignancy of these gliomas.Table 2The Most Significantly Altered MiRNAs in Varying World Health Organization Grades Gliomas Compared with Normal Brain TissuesNo.MiRNALog2*Positive and negative log2 scores indicate up-regulation and down-regulation of miR-182, respectively, in gliomas.P1miR-1823.930.0232miR-10b2.910.0353miR-6302.570.0434miR-4862.500.0245miR-4512.080.0326miR-6711.980.0167miR-130a1.800.0228miR-1881.730.0439miR-251.610.04110miR-124a−3.500.02311miR-129−3.010.02612miR-139−2.960.03413miR-33−2.510.03614miR-7−2.470.03815miR-136−1.360.04216miR-219−1.350.04417miR-218−1.280.029* Positive and negative log2 scores indicate up-regulation and down-regulation of miR-182, respectively, in gliomas. Open table in a new tab To validate our finding of miR-182 up-regulation, we performed a comparative analysis of miR-182 expression by real-time RT-PCR by using paired glioma tissues and their corresponding adjacent nontumor tissues (ANT) from the four individual glioma patients. As shown in Figure 2A, miR-182 was overexpressed in all four human primary glioma samples compared with the counterparts of paired adjacent nontumorous brain tissues. Importantly, all four tumors displayed greater than fivefold increases of miR-182 compared with adjacent nontumor tissues. In a GBM specimen, a 32-fold increase of the tumor/adjacent nontumorous tissue (T/ANT) ratio of miR-182 expression was found (Figure 2A). To further evaluate whether miR-182 up-regulation was linked to the clinical progression of gliomas, we sought to examine the expression of miR-182 in a large cohort of clinical glioma samples by using ISH. The specificity of the miR-182 probe was first examined by ectopically overexpressing miR-182 by retroviral-mediated transduction in NHA that had been shown to express miR-182 at very low level (Figure 1A), followed by in situ hybridization analysis. As shown in Figure 2B (right panel), miR-182 staining was clearly shown in the miR-182-infected NHA, which was further confirmed by real-time RT-PCR analysis (Figure 2B, left panel), but was undetectable in the vector infected-NHA or the uninfected NHA. Consistent with the abovementioned result obtained from real-time RT-PCR, in situ hybridization analysis also showed miR-182 overexpression in four human primary glioma samples compared with the paired nontumor adjacent tissues, whereas a scramble control probe did not yield any signal in sister sections of these tumors (Figure 2C and data not shown), validating the specificity of the miR-182 probe used in the study (Figure 2C). Two hundred fifty-three paraffin-embedded, archived clinical glioma specimens including 29 World Health Organization grade I, 79 grade II, 99 grade III, and 46 grade IV tumors were examined for miR-182 expression by using in situ hybridization analyses (Table 2). The study population of gliomas consisted of 253 patients (172 male patients; 81 female patients) with a median age at diagnosis of 45 years (range, 21 to 72 years). Figure 3A showed representative data of the in situ hybridization results. miR-182 was found to be up-regulated in these glioma samples of all grades compared with normal brain tissues. As summarized in Table 2, miR-182 expression was detected in 248 of 253 (98%) tumors. In contrast, expression of miR-182 was marginally or not detectable in normal brain tissues (Figure 3A). Quantitative analyses showed that MODs of miR-182 staining in grades I to IV tumors were significantly higher than those in normal brain tissues, and that MODs of miR-182 staining were also significantly different among various clinical grades (P < 0.05, Figure 3B). Levels of miR-182 expression by in situ hybridization were further analyzed to determine its relationship with the clinical features of gliomas. The median expression of miR-182, using real-time PCR analysis, of 119 patient samples was used as the threshold, and patien
Referência(s)