Case Series / RNA extraction and cDNA synthesis
MSI1 mRNA levels were evaluated in a total of 59 pediatric patients (0-19 years) diagnosed with MBs in three Brazilian institutions that were previously classified as WNT (n=15), SHH (n=18), Grp3 (n=9), and Grp4 (n=17) [18], and in five non-neoplastic cerebellum, serving as controls. Total RNA was extracted from pediatric MB tissues and cell-lines using Trizol reagent (Invitrogen Inc, Carlsbad, USA) or AllPrep DNA/RNA/Protein Mini kit (QIAGEN, Hilden, Germany), following the manufacturers' specifications. RNA concentrations were determined by using the ND-1000 Spectrophotometer device (NanoDrop 1000 Technologies, Wilmington, DE, USA). The reverse transcription reaction for the synthesis of complementary DNA strand (cDNA) was performed using 500ng of total RNA and the High Capacity kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Additionally, expression data of an independent cohort of pediatric MBs (GSE85217, n=629) was downloaded from R2 Platform (Analysis and Visualization Platform – http://r2.amc.nl) [2] and used to validate MSI1 expression levels between the different molecular subgroups of MBs.
Cell lines and culture conditions
The pediatric MB cell lines D283 Med and USP-13-MED 4 [19, 20] (Grp3/4-MBs), and the Human Embryonic Kidney 293 cell (HEK93T) were used in this study. D283 Med (ATCC HTB-185) and HEK-293 (ATCC CRL-1573) cells were obtained from the American Type Culture Collection, and the USP 13-MED cell line was kindly provided by Prof. Dr. Oswaldo Keith Okamoto - Biosciences Institute of the University of São Paulo. The cell lines authentications were performed to validate the Short Tandem Repeat (STR) prolife. All cells were maintained in DMEM/F12 medium (Gibco™, Thermo Fisher®, Carlsbad, CA, USA), supplemented with 10% FBS, 100U/ml penicillin, 100 μg/ml streptomycin and kept in a humid atmosphere containing 5% CO2 at 37ºC.
Quantitative real-time PCR (qRT-PCR)
Relative mRNA expression levels were measured by quantitative PCR using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA- MSI1 Hs01045894_m1) in 59 pediatric MB samples, and in D283 Med and USP-13-MED cell lines. The reactions were performed on QuantStudioTM 12k Flex system (Applied Biosystems, Foster City, CA, USA) using two internal controls: GUSB (Beta Glucuronidase) (Hs4333767F_m1) and HPRT (hypoxanthine guanine phosphoribosyl transferase) (Hs4310809E_m1). The data were analyzed using the 2-ΔΔCT method and non-neoplastic cerebellum samples were used as calibrators [21].
Immunohistochemistry (IHC)
IHC staining was performed on formalin-fixed paraffin-embedded (FPFE) tissue sections (4 μm) of eight MB samples (WNT n=2; SHH n=1; Grp3 n=3 and Grp4 n=2) using the detection system EnVision™ polymer (Dako, Glostrup, Denmark), following the manufacturers’ recommendations. The antigen retrieval was carried out using citrate buffer (pH 6.0). The slides were incubated with anti-MSI1 antibody (dilution 1:70 cat. no. #5663, Cell Signaling Technology, Danvers, MA). Cerebral cortex was used as positive control [22]. The immunostaining was analyzed by a savvy neuropathologist, considering as positive cells those with cytoplasmic and/or nuclear staining for MSI1. Positive cells were scored according to the percentage of stained cells as following: Score (+) %, percentage of MSI1 positive tumor cells. + (< 25%), ++ (< 25-50%), +++ (< 50-75%) and ++++ (< 75-100%). Intensity of immunostaining (immunoreactivity) in tumor cells: Low (+), Strong (+ +). Representative cases were captured at ×40 magnification using Nikon ECLIPSE 80i.
Western blot (WB)
The wild type and transduced cells lines were lysed on ice in lysis buffer containing freshly added protease inhibitor cocktail (Roche Diagnostics, Branchburg, NJ, USA). Protein extracts (50μg) were size-fractionated by SDS-PAGE and proteins were immunoblotted with anti-MSI1 (dilution 1:1000, cat. no. #5663, Cell Signaling Technology, Danvers, MA). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All antibodies were diluted according to manufacturer’s instructions and HRP-conjugated goat anti-rabbit (Santa Cruz Biotechnology, Santa Cruz, CA) was used as a secondary antibody. The results were visualized using an enhanced chemiluminescence detection system (Bio-Rad Laboratories, Inc.), and the relative quantification of protein expression was determined using ImageJ® software (National Institutes of Health).
Immunofluorescence
For immunofluorescence staining, D283 Med and USP-13-MED cells were grown on glass coverslips and then fixed with 4% paraformaldehyde in PBS 1X for 15 minutes. After fixation, cells were permeabilized with 0.3% Triton X-100 at room temperature, washed with PBS 1X, and incubated with a blocking buffer (2% bovine serum albumin) for two hours. Samples were then incubated with primary antibody (1:500, Anti-Musashi-1 (D46A8) Rabbit mAb #5663, Cell Signaling Technology, Danvers, MA) in blocking buffer at 4oC overnight. Finally, cells were washed three times with PBS 1X and then stained with Alexa Fluor 647 anti-rabbit secondary antibodies (1:2500) diluted in blocking buffer containing Alexa Fluor™ 488 Phalloidin – ThermoFisher (1:40) for 1 hour, followed by three washes in PBS 1X. Coverslips were mounted in ProLong Gold Mounting Medium containing the nuclear stain 4′,6-diamidino2-phenylindole (DAPI) (Vectashield, Vector Laboratories, Burlingame, CA, EUA). Images were obtained using a laser scanning confocal microscope Leica DM2500 (LeicaBiosystems, Wetzlar, Germany) and the software LAS (Leica Biosystems, Wetzlar, Germany). Negative controls included incubation with secondary antibodies alone.
Lentivirus-mediated short hairpin RNA (shRNA) knockdown of gene expression
Silencing of MSI1 was performed using two shRNA vectors (pLV[shRNA]-EGFP:T2A:Bsd-U6>hMSI1[shRNA#1] and pLV[shRNA]-EGFP:T2A:Bsd-U6>hMSI1[shRNA#2]), and their respective control (pLV[shRNA]-EGFP:T2A:Bsd-U6>Scramble_shRNA#1) acquired from Vector Builder (https://en.vectorbuilder.com) containing a gene for blasticidin resistance. Plasmids were expanded in LB medium supplemented with 100μg/mL of Ampicillin; and purified using the QIAprep Spin Miniprep Kit protocol (Qiagen Company, Hilden, Germany, #Cat. 27104), following manufacturer's instructions. To analyze the yield and purity of the plasmids, the NanoDrop Spectrophotometer device (Thermo Scientific, DE, USA) was used. Lentiviral particles were produced by co-transfection of the trans-lentiviral packaging mix with a shRNA transfer vector into HEK 293T packaging cells (OpenBiosystems). For cell infection, viral supernatants were supplemented with 6μg/mL polybrene and incubated with cells for 24 hours. D283 Med transduced cells were selected with blasticidin (10 μg/mL) for 7 days.
Cell cycle analysis
For cell cycle analysis, 17.5x104 (D283 Med MSI1 knockdown and control shRNA Scramble) cells were plated in 6-well plates and kept in culture for 72h, without treatment. Then, cells were trypsinized and centrifuged at 1,200 rpm/5 min and washed with PBS 1X. Cells were fixed with 70% cold ethanol and incubated at -20o C overnight. Cells were then centrifuged at 800rpm/5 min, washed with PBS 1X, and incubated with 25 μL of RNAse A (10 ng/mL) at 37o C for 30 min. Next, centrifugation step was repeated and cells were stained with 100 μL of PI (50 μg/mL) shortly before acquisition by BD FACS Calibur TM flow cytometer (BD Biosciences, San Jose, CA, USA). The assay was performed in triplicate. Data were analyzed using FlowJo software.
Cell viability Assay
Cell viability was detected using CellTiter Glo® reagent (Promega) according to the manufacturer's recommendations. Briefly, 6x103 (D283 Med MSI1 knockdown and control shRNA Scramble) cells were plated in 96-well plates. The cells were treated with cisplatin at different concentrations (3µM, 5µM, 7µM and 10 µM) for 72h, and the results were obtained through SpectraMax® L Microplate Reader device. The assay was performed in triplicate. Furthermore, the concentration of cisplatin that inhibited 50% of cell viability (IC50) was determined by using the CalcuSyn Software (Biosoft, Cambridge, UK).
Apoptosis detection
In total 17.5x104 cells (D283 Med MSI1 knockdown and control shRNA Scramble) were plated and treatment for 72h with cisplatin at a dose of 4.1 µM. The detection of cell death was performed by labeling apoptotic cells with Annexin V (APC) (BD Biosciences Pharmingen, USA) and Propidium Iodide (PI). Cells were trypsinized and centrifuged at 1,200 rpm for 5 min at 4°C, washed with ice-cold PBS 1X and then resuspended in 200 µL of 1X binding buffer (BD Biosciences Pharmingen, USA) with 5 µL of annexin-V and 50 µL of a solution of PI (50 μM), and incubated for 15 minutes, protected from light, at room temperature. Cells were analyzed by BD FACSCalibur TM flow cytometer (BD Biosciences, San Jose, CA, USA). The experiments were carried out in triplicate. Data were analyzed using FlowJo 8.7 software.
Statistical analysis
Statistical analysis was performed using the software’s (Graph Prism 5.0 GraphPad Software, San Diego, CA USA) and SPSS 15.0 (SPSS Inc. Chicago, USA). Comparisons between two or more groups were carried out using Kruskal-Wallis and One-way-ANOVA, respectively. Receiver operating characteristic (ROC) curves were used to evaluate the discrimination of Grp3/Grp4-MBs from the other molecular subgroups according to MSI1 expression levels. The accuracy was determined by the area under the curve (AUC). A p-value ≤0.05 was considered as statistically significant.