UDC 616-006.487.48-039.36-037-053.2: 575.24 SIGNIFICANCE OF miR-885-5p IN NEUROBLASTOMA OUTCOME

Neuroblastoma is the most common extracranial malignant solid tumor in children. This disease displays a remarkable heterogeneity in clinical behavior, ranging from spontaneous regression to rapid progression and resistance to therapy. Recent evidence has shown that microRNAs are often involved in regulation of tumor development and progression. MiR-885-5p has a tumor suppressive role in neuroblastoma, interfering with cell cycle progression and cell survival. MiR-885-5p leads to the accumulation of p53 protein and activates p53-mediated pathway of cell cycle arrest, resulting in upregulation of its targets. We have analyzed association of miR-885-5p expression in 58 neuroblastoma tumors with different clinical characteristics and disease outcome. In tumor samples of patients with unfavorable clinical characteristics lower miR-885-5p expression levels were observed. Event-free survival analysis showed that low miR885-5p expression was tightly associated with a significantly poorer outcome than in those with high expression of miR-885-5p. In this study, evidence is presented on miR885-5p dysregulation in neuroblastoma. As follows, along with other clinical features, it can be used as an independent prognostic and possibly therapeutic approach for optimization of neuroblastoma treatment.


INTRODUCTION
Neuroblastoma (NB) is a malignant tumor of the sympathetic nervous system reaching 11.7 % of all malignancies in children and taking fourth place in structure of cancer mortality after acute leukemia, central nervous system tumors and malignant lymphomas [5]. Age distribution of the disease is heterogeneous and frequency of tumor detection decreases with age. A characteristic feature of NB is its clinical heterogeneityfrom localized tumors to widespread forms and early hematogenous metastasing. However, ~40 % of NB rapidly progress despite multimodal treatment regimens. This high clinical heterogeneity reflects a complexity of genomic abnormalities that are characterized for NB tumors [7].
Multiple segmental aberrations of chromosomes and individual gene amplification, particular MYCN oncogene, are inherent for aggressive NB. Amplification of MYCN gene is observed in ~25 % of primary tumors and is associated with rapid tumor progression and poor prognosis of the disease in patients of any age and stage. Although, status of MYCN gene is a central stratification biological marker for risk group determination, it is important to emphasize that MYCN gene amplification is not detected in most of metastatic NB [4].
Despite extensive knowledge of somatically acquired genomic rearrangements in NB and their correlation with clinical tumor phenotype, very few data are known about the factors leading to these genetic events. Inactivation of p53 tumor suppressor pathway allows cell survival during stress and occurs in many human cancers; however, normal embryonic stem cells and some cancers such as NB maintain wild-type human TP53 [9,13]. Recent studies demonstrate that p53 is generally functional, it accumulates in nucleus in response to DNA damage, and is efficiently degraded by MDM2 in NB. There are reports that p53 function may be compromised as a consequence of aberrant MDM2 expression levels in NB [8]. In the last years, new mechanisms of TP53 gene activity regulation have been identified, among which epigenetic factors, namely microRNA -a new class of RNA, were discovered in 1993 [11].
The occurrence of many cancers is a result of accumulation of genetic and epigenetic changes. While genetic alterations are almost impossible to reverse, epigenetic changes can dynamically respond to signals from physical, biological and social environments [3]. This characteristic confers the importance of epigenetic research in various cellular processes, particularly in gene expression regulation. New functional studies suggest that microRNAs regulate important genes involved in NB pathogenesis [10]. MicroRNA is a class of non-coding RNAs consisting of approximately 22 nucleotides, which play an important role in regulation of mRNA translation and degradation, resulting in genes suppressing. MicroRNAs are known to regulate oncogenes, tumor suppressor genes, genes involved in cell cycle regulation, cell migration, apoptosis and angiogenesis [12].
Several commonly lost genomic regions have been characterized in NB, without identification of the genes responsible for tumour suppression. 3p25.3 region is deleted in ~14 % of NB, and houses miR-885-5p [1]. Afanasyeva E.A. et al. [2] has provided evidence that miR-885-5p is a candidate for NB tumour suppressor and involved in directing cell phenotype of NB. Experimentally enforced expression of miR-885-5p in NB cell lines resulted in growth inhibition, senescence and apoptosis. MicroRNA miR-885-5p inhibits proliferation and survival, and positively regulates p53 pathway.
The aim of our study was to analyze level of miR-885-5p expression in NB tumours and to investigate its association with clinical characteristics and disease outcome to determine a possibility of its application as a prognostic marker of NB.

MATERIALS AND METHODS
Patients. 58 patients with verified NB diagnosis have been enrolled into this study. The age of patients ranged from 1 month to 17 years. Mean age was 37.2±5.9 months. Tumor samples were collected before any cytoreductive treatment by biopsy or tumor resection. Fresh tumor samples for qPCR analysis were stored in "RNA-later" (Ambion, USA) to stabilize RNA and DNA; for fluorescent in situ hybridization (FISH), formalin-fixed, paraffin embedded tissue (FFPE) samples were used. Written informed consent was obtained from patients' parents for tissue sampling according to the requirements of the protocol N 35 from 1.03.2015 approved by Ethical Commission of the National Cancer Institute.

Real-time quantitative PCR (Real-time qPCR).
Nucleic acids were extracted from tumor samples using silica gel-based membrane purification method (NucleoSpin MiRNA, Macherey-Nagel) according to the manufacturer's instructions. Briefly, 20 ng of total RNA were reverse transcribed to cDNA with stem-loop primers and the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, USA). TaqMan MicroRNA Assays (Applied Biosystems, USA) were used to quantify endogenous miR-885-5p. Data were normalized to U6 microRNA expression. Real-time RT-qPCR was performed with 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and TaqMan Universal PCR MasterMix. Calculations were performed using the ΔΔCt relative quantification method. The thresholds and baselines were set manually in SDS and Ct values were extracted.
Genomic MYCN status was assessed in NB fresh tumors by real-time qPCR analysis and FISH for FFPE samples. MDM2 expression level was detected by real-time RT-qPCR using TaqMan primers and probes (primers and probes sequences are given in [8]). MYCN status and MDM2 expression in NB tumor samples were analyzed, as previously described [8]. Results were normalized to relevant controls.
Statistical analysis. All experiments were repeated independently at least three times. Gaussian distribution of the group was checked with Kolmogorov-Smirnov tests. Analysis of the association between the studied markers and clinical characteristics of patients was performed using U-Mann-Whitney test. Prognostic significance of markers was verified with the ROC-curve (Receiver Operating Characteristic curve). Event-free survival was evaluated by Kaplan-Meier estimator, statistical significance of parameters differences was determined using F-Cox criterion. The data were processed with the software package Statistica 6.1 using parametric and nonparametric methods of statistical analysis and MedCalc 12.1.4.0. The difference was considered statistically significant at p<0.05.

RESULTS AND DISCUSSION
It was found that the value of miR-885-5p expression in tumor cells of patients with NB varied in a wide range. The lowest miR-885-5p expression level was established in the recurrent and metastatic tumor samples compared with primary tumors, p = 0.04 (Fig. 1, A). This suggests that miR-885-5p is involved in progression of the disease.
Patients were subdivided by age into patients younger than 12 months, patients from 12 to 24, and patients 24 months of age and older (Fig. 1, B). Stage was dichotomized for the entire NB cohort with respect to metastatic progression (i.e., stage 1, 2, and 3 vs stage 4) (Fig. 1, C). We also defined groups with different MYCN status causing clinically aggressive NB course (Fig. 1, D). Virtually all NB have wild-type TP53 before chemotherapy treatment, suggesting that p53 may be attenuated by another mechanism in these tumors e.g. by its direct antagonist -MDM2 [14]. Patients were divided into 2 groups according to level of expression of MDM2 [8] (high and low) (Fig. 1, E).
The occurrence age of patient is an independent prognostic marker of NB and is used in different stratification systems. Patients older than 1 year constitute a high risk group [6]. In this study, we observed not statistically significant decrease of miR-885-5p expression depending on patients' age, p>0.05 (Fig. 1, E). Despite that no significant difference of miR-885-5p expression was found in tumors with different MYCN status, however statistically insignificant decrease in MYCN-amplified samples was observed, p = 0.26 (Fig. 1, D). It was also found that in patients with a diffused late-stage disease miR-885-5p expression level was lower than in patients with early-stages NB, p = 0.04 (Fig. 1, C). In addition, tumors with high MDM2 expression level had significantly lower expression of miR-885-5p, p = 0.02 (Fig. 1, E). This testifies to theory of the important role of miR-885-5p in functioning of p53-mediated pathway of cell cycle arrest.
To assess the prognostic significance of miR-885-5p expression in NB progression, ROC-analysis was conducted. As the criterion of clinical efficacy, event-free survival rate was applied for patient distribution on groups according to miR-885-5p expression level. According to ROC-analysis, low miR-885-5p expression was shown as a marker of unfavorable NB and risk of disease recurrence. This marker had sufficient sensitivity and specificity (AUC: 0.69, p = 0.01, Se: 89.7 %, Sp: 42.9 %, optimal criterion: ≤0.0713 a.u.), and may serve as an independent marker of prognosis and stratification of NB patients on risk groups.
Patients were divided into 2 groups according to miR-885-5p expression level (high >0.0713 a.u. and low ≤0.0713 a.u. expression level) and the event-free survival of this groups was analyzed (Fig. 2). As the result, miR-885-5p was under-expressed in 74 % of patients. We found that reduced miR-885-5p level was associated with a significant decrease in event-free survival of NB patients, p = 0.02. Accordingly, the rate of 5-year event-free survival of patients with a reduced miR-885-5p expression was 5.6 times lower than in high expression group (9.3 and 63.6 %, respectively). Therefore, a decreased miR-885-5p expression is associated with risk of the disease recurrence. Such a significant difference suggests that this marker has a substantial role in the pathogenesis and progression of NB. Thus, miR-885-5p is NB tumor suppressor. Along with other clinical features, it can be used as a prognostic marker and potential therapeutic approach for optimization of NB treatment.

CONCLUSIONS
The obtained results are promising for further miR-885-5p evaluation as a prognostic marker of NB progression. Modulation of miR-885-5p expression may provide novel therapeutic opportunities for improvement of NB treatment. After conducting similar studies of larger patients' cohort, this marker can be recommended for inclusion as the molecular genetic component of NB risk group stratification.