Detection and quantification of extracellular microRNAs in medulloblastoma

Introduction

Medulloblastoma (MB) is the most common malignant brain tumor in children. Metastatic MB carries a poor prognosis. Mechanisms that predict dissemination are poorly understood. Recently, several studies have revealed a critical role for microRNAs (miRNAs) during tumorigenesis and metastasis of several cancers, including MB.
Besides intracellular miRNAs with the traditional function of translation regulation, there is accumulating evidence that miRNAs exist extracellularly in body fluids, including cerebrospinal fluids (CSF).  Several reports have described that deregulated extracellular-miRNAs (ex-miRNAs) are closely associated with the clinical course of malignant tumors. Interestingly, such deregulation returns to a normal level after tumor resection. Hence, expression analysis of ex-miRNAs is of increasing interest for diagnostic and prognostic purposes.

Every cancer investigated has a distinct miRNA signature and deregulated levels of miRNAs have been detected in body fluids of patients, including those with lymphoma, leukemia,colon, breast, prostate,   ovarian,  pancreatic, gastric,  and lung cancer. In the context of brain tumors, recent studies have demonstrated a significant presence of certain miRNAs in CSF samples from patients with central nervous system lymphoma, glioma, and metastatic brain cancers.  Recent miRNA profiling of CSF has enabled early detection of glioblastoma and reflected disease activity.Therefore, ex-miRNAs may represent important minimally invasive candidate biomarkers in brain tumors. The presence and biological role of ex-miRNAs in MBs, however, remain unknown. This study was conducted to gain insight into the identity and quantity of MB-related ex-miRNAs and to speculate on their possible biological function in the context of MB metastasis.

Methods

Patient characteristics and CSF
CSF samples from patients with MB (n = 2), control patients with leukemia with no intracranial mass lesions and/or neurologic disorders (n = 3), CSF samples from patients with ependymoma (n = 3) and glioblastoma (n = 1) that were collected from patients treated at the University Children's Hospital of Zürich, Switzerland. Written informed consent was obtained from each patient. CSF samples from patients with MB were collected 3 weeks after surgery and before start of radiotherapy or chemotherapy. CSF samples were centrifuged (500 g, 10 min, room temperature) within 60 min after collection to remove cells and debris and were stored at -80 °C until further processing.
Human MB cell lines
Human MB cell lines (DAOY and D283) were purchased from American Type Culture Collection (Manassas, VA, USA). D341 human MB cells were the kind gift of Dr. Henry Friedman (Duke University, Durham, UK). MB cell lines were cultured as previously published and maintained at 37 °C in a humidified atmosphere with 5% CO ₂ . To isolate RNA from cultured medium, 10.000-20.000/mL DAOY cells or 20.000-40.000/mL D341, D283, and T293 cells were plated and left to grow in their conditioned media for 72 h in 24 wells plates. Conditioned medium (2 mL) of each cell lines were centrifuged at 1,200 rpm to remove cells. The supernatant was then centrifuged at 10,000 rpm to remove debris.
RNA extraction for microarray
Total RNA from cell cultures or CSF were extracted using a mix of Qiazol, Qiagen (Qiagen, Basel, Switzerland) and chloroform directly on cells. For small RNA in conditioned medium or CSF, the addition of miRNAs extraction reagent (Toray) was performed. In both situations, a centrifugation step was required to collect aqueous phase containing RNA that was finally transferred to miRNeasy Mini spin column from miRNeasy purification kit Qiagen (Qiagen, Basel, Switzerland). After subsequent washing steps, RNAs were eluted using 30 μL of nuclease-free water and concentrated up to 3 μL with vacuum concentrator. Quality was checked on Bioanalyzer using RNA 6000 Pico Chip (Agilent Chemical Analysis, Life Sciences, and Diagnostics. Basel, Switzerland) gel and quantified using Nanodrop Photometer [Figure 1]a and b.

Labeling and hybridization

Total RNA (250 ng) extracted from cells and 3 μL of concentrated small RNA extracted from medium were used with Toray 3D-Gene miRNA labeling kit (Toray, Japan) in presence of spikes used as positive controls. Briefly, 5'-phosphates were removed from miRNA end using alkaline phosphatase and a fluorescent label was enzymatically attached to the 3'-end of the miRNA. After an enzyme inactivation step and addition of a hybridization buffer, labeled miRNA was injected on 3D-Gene Human miRNA Oligo Chips (Toray, Japan) targeting 2019 miRNA based on miRBase release 19. Finally, arrays were placed in a hybridization chamber and set into a 32 °C oven for 16 h with a shaker adjusted to 250 rpm.

Washing and scanning

Arrays were washed using 3 solutions with different stringencies to remove non-specifically bound miRNAs. Then, arrays were scanned with the 3D-Gene Scanner 3000 instrument (Toray, Japan) to measure fluorescence. Scanning was carried out using 3 different photomultiplier sensitivities (PMT gain) to allow optimizing of signal detection and checking for consistency.

Microarray analysis

Images were analyzed with the 3D-Gene Extraction software (Toray, Japan). After completion of the auto-analysis work followed on image files, raw signals, and detection calls was produced in tabular files. GeneSpring GX12 (Agilent) was then used to apply quantile normalization and differential expression analysis using modified t-test implemented in the software. Experimental variability was assessed with principal component analysis (PCA) [Figure 2] and [Figure 3]b and Pearson correlation matrix [Figure 3]a generated using the same software.

MicroRNA isolation for reverse transcription polymerase chain reaction analysis

For precipitation of nucleic acids, the monovalent cation concentration of the solution was adjusted to 0.5 mol/L sodium acetate. Glycogen (AM9510, Ambion, Life Technology, NY, USA) was added to a final concentration of 100 μg/mL. The solution was then mixed with 1 volume of isopropanol. The mixture was chilled for 20 min at -20 °C, then centrifuged for 20 min at 13,000 rpm. The supernatant fluid was removed, and the nucleic acid resuspended in lysis buffer. Final purification of RNA enriched for small RNAs from 600 μL of conditioned media and CSF samples was obtained using the mirVanaTM miRNA Isolation Kit (Ambion, Life Technology) according to manufacturer's instructions for "Enrichment Procedure for Small RNAs." Using this approach consisting of two sequential filtrations with different ethanol concentrations, an RNA fraction highly enriched in RNA species ≤ 200 nt was obtained. First strand synthesis of mature miRNAs was followed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using miRNA-specific TaqMan MGB probes (Applied Biosystems, Life Technology). For the qRT-PCR reaction, the Gene Expression Master Mix was used and the protocol was optimized for the ABI7900HT reader (Applied Biosystems). Probe-primer solutions specific for the following miRNAs were used: miR-1290 (002863), miR-125a-3p (002199), miR-1298 (002861), miR-125b-1* (002378), miR-486-3p (002093), miR-572 (001614), miR-4476 (464702_mat), miR-615-5p (002353), and miR-3918 (464506_mat) (Applied Biosystems, Life Technology). The relative gene expression was calculated for each gene of interest using the ΔΔCT method, where cycle threshold values were normalized to the level of cel-miR-39-3p (4464066, Ambion, Life Technology), which was used as spike-in by adding it during the lysis step of miRNAs extraction.

  Results

Given that some human cancer cells secrete miRNAs into their extracellular environment and body fluids, it was hypothesized that MB cell lines may secrete miRNAs into their spent culture medium. To test this hypothesis, 3 cell lines representing MB subtypes D341 and D283 (metastasis-related group 3 and group 4 MB subtypes)and DAOY (sonic hedgehog-related) were cultured individually for 72 h in vitro and miRNAs expression was analyzed in the lysates of each MB cell line and in their corresponding culture media. We identified 1,662, 1,615, and 1,199 secreted miRNAs in the culture-medium (CM) of MB cell lines D283, D341 and DAOY, respectively, among them 1,083 miRNAs that were common in the CM of the 3 cell lines. In cell lysates of D283, D341 and DAOY, on the other hand, we detected 1,787, 1,394 and 1,761 miRNA respectively, with 1,347 miRNAs found common to all 3 cell lines [Figure 4]a. Interestingly, 950 miRNAs were commonly identified in CM of both groups and in lysates of the 3 cell lines tested, indicating that the level of ex-miRNAs may well reflect the expression level of tumor miRNAs. Using a fold-change > 2, we identified a group of 156 miRNAs that are commonly enriched in CM derived from the 3 cell lines compared to their respective cell lysates [Figure 4]b and [Supplementary Table 1- [Additional file 1]] and 57 miRNAs that were specific to the CM of D341 and D283, which represented the 2 metastasis-related group 3 and group 4 MB subtypes, respectively  compared to DAOY-derived CM [Figure 4]b and [Supplementary Table 2-[Additional file 2]]. We found 2 additional groups of miRNAs to be differentially enriched in CM of D341 and D283, represented by 60 miRNAs overrepresented and 52 underrepresented compared to DAOY-derived CM [Supplementary Tables 3- [Additional file 3] and 4 - [Additional file 4]. Overall, the results of this experiment demonstrate that MB cell lines secrete miRNAs into the CM and that certain ex-miRNAs retain different enrichment levels in the CM-derived from the 2 cell lines representing the metastasis-related group 3 and group 4 MB subtypes

Detection of ex-miRNAs in CSF of MB patients by microarray analysis

We next asked whether ex-miRNAs could be detected in CSF of MB patients, to test whether it would be technically possible to use the CSF as a source for diagnostic miRNA testing. Using microarray analysis, we screened cell-free CSF from a patient with MB and compared the results to controls (CSF from two different leukemia patients with no cerebral manifestation or neurological disease). PCA [Figure 2] showed clear separation between the miRNA spectrum in CSF of MB patient and controls. Microarray analysis identified 1,254 miRNAs in the MB-CSF sample [Table 1], of which 86 miRNAs were differentially expressed in CSF of the MB patient compared to the 2 CSF controls [Figure 4]c and [Supplementary Table 5 - [Additional file 5]]. Further analysis identified 268 miRNAs over-represented (with fold-change > 2) and 6 miRNAs under-represented in MB-CSF compared with the 2 different controls tested [Supplementary Table 6 -[Additional file 6] and Supplementary Table 7 -[Additional file 7]], indicating a trend toward miRNA enrichment in the MB-CSF sample.

Comparison between miRNA expressions in CM vs. CSF samples

An overlap of the spectra of ex-miRNA candidates detected in the CSF of the MB patient and those excreted by MB cell lines into the CM would support our hypothesis of miRNA secretion by MB cells. Indeed, Pearson correlations analysis showed that ex-miRNAs profiles in MB-CSF displayed a good homogeneity with the profile of miRNAs secreted in CM of MB cell lines [Figure 3]a. This conclusion was confirmed by PCA showing clear separation of miRNAs derived from lysates of MB cell lines from those derived of MB-CSF samples or of CM derived from MB cells [Figure 3]b, confirming the conclusions from Pearson correlations. Compiling expression tables allowed identification of 5 miRNAs (miR-486-3p, miR-572, miR-3918, miR-4476, and miR-615) that were significantly up-regulated in the CM of the 3 cell lines (D283, D341 and DAOY) and enriched in MB-CSF compared to control CSF [Figure 5]a. Moreover, 3 other miRNAs (miR-1290, miR-125a, miR-125b), known to be associated with metastasis, and miR-1298, were over-represented in the CM of metastasis-related cell lines (D283 and D341), but not in DAOY and were significantly over-represented in MB-CSF compared to control CSF [Figure 5]b.

To further validate the finding of selective enrichment of miR-1298 in MB-CSF, we tested it against an additional 5 different CSF controls from one leukemia patient, 3 ependymoma patients, and one glioblastoma patient (specifically chosen to control for brain surgery as a possible factor influencing miRNA secretion). Consistently, TaqMan analysis confirmed significant enrichment of miR-1298 in MB-CSF compared to the 5 controls [Figure 8]a. Together, using TaqMan analysis, we confirmed the microarray data result and demonstrated the feasibility of quantitative detection of miRNAs in culture medium and CSF using qRT-PCR (popular gene expression assay and efficient method for high-throughput used in most diagnostic labs). Importantly, we could detect ex-miRNAs by qRT-PCR in CM of as few as 100-500 MB cells [Figure 8]b, recommending qRT-PCR for the development of non-invasive detection of metastasis-predicting markers for MB.

Discussion

Aberrant expression of ex-miRNA circulating in CSF of certain brain tumor patients has recently been reported to be cancer biomarkers and potential regulators of the disease. However, the existence and role of ex-miRNAs in MB extracellular environment are unknown. Therefore, better understanding of ex-miRNA secretion and function in MB seems crucial for the development of novel insights for its diagnosis and prognosis. This study aimed to identify key miRNAs in culture medium of 3 cell lines, representing different MB subtypes. Our results identified a significant number (1,347) of hitherto unrecognized new miRNAs commonly expressed in CM of the 3 cell lines. A significant concordance of ex-miRNA spectra in CM and those expressed intracellularly was observed. Since deregulated miRNA expression is an early event in tumorigenesis, measuring miRNA levels in CSF may also be useful for early detection, which can contribute greatly to the success of treatment. Therefore, in order to use ex-miRNAs as biomarkers for MB, it is important to establish a signature capable of differentiating disease from healthy states. Our pilot microarray screening identified 86 miRNAs exclusively detected in CSF of MB patients but not in control CSF from patients with no brain tumor. We also identified 268 miRNAs that are over-represented and interestingly, only 6 miRNAs under-represented in MB-CSF compared with control CSF. These findings could be of great significance, providing the correlation between expression levels of these miRNAs in CSF of MB patients and their disease states can be established in future studies.
Tumor cell-derived ex-miRNAs are reported to be pro-tumorigenic.  Ex-miRNAs can transfer their oncogenic activity to recipient target cells to influence cancer stimulatory activities, thus contributing to the formation of a pre-metastatic niche and promotion of metastasis.  This exchange of miRNAs between primary tumors and target cells is an interesting and novel dimension to the regulation of a cell phenotype  and may be particularly important in cancers that have a propensity for dissemination, such as MB. MB includes various subtypes with group 3 and 4 subtypes being clinically distinct with regard to metastasis and prognosis, which may also manifest in a difference in their miRNA spectra. Hence, it was not surprising to find a group of miRNAs that were uniquely over-(60 miRNAs) or under-represented (52 miRNAs) in the CM of the 2 metastasis-related cell

lines D283 and D341. More importantly, we identified 4 miRNAs (miR-1290, miR-125a, miR-125b, miR-1298) that were over-represented in MB CSF and significantly enriched in the CM media of the 2 metastasis-related cell lines (D283 and D341). Remarkably, apart from miR-1298, where no functional information is publically available, the 3 other miRNAs (miR-1290, miR-125a, miR-125b) were detected in body fluids of various cancer patients, whereby their increased expression and/or secretion is associated with metastasis of multiple malignancies.^39] Consistently, detection of metastasis-related ex-miRNAs in extracellular environment of certain human malignancies, including breast and prostate cancers, were observed in other studies. Our observations provide indirect evidence supporting the hypothesis that ex-miRNA are possible facilitators of metastasis by modifying local or distal microenvironments.  However, further studies are needed using counter-regulation of key ex-miRNA expression to determine their effect on regulation of motility, migration, and invasion of MB cells.
To the best of our knowledge, this is the first study revealing the spectra of ex-miRNAs in cell CM conditioned by MB cell lines and in CSF of an MB patient. Although the number of samples studied here is very small, our identification of key secreted miRNAs that are specifically enriched in MB-CSF provides a rationale for future investigations. Such investigations, using larger sets of MB samples could lead in the near future to the discovery of CSF-derived miRNA markers, with diagnostic and prognostic significance and ultimately, hopefully also with therapeutic potential.

Acknowledgments

This project was supported by the Swiss Research Foundation Child and Cancer and by "Krebsliga Zürich".

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