Musashi-1 regulates cell cycle and confers resistance to cisplatin treatment in Group 3/4 medulloblastomas cells

Groups (Grp) 3 and 4 are aggressive molecular subgroups of medulloblastoma (MB), with high rates of leptomeningeal dissemination. To date, there is still a paucity of biomarkers for these subtypes of MBs. In this study, we investigated the clinical significance and biological functions of Musashi-1 (MSI1) in Grp3 and Grp4-MBs. First, we assessed the expression profile of MSI1 in 59 primary MB samples (15-WNT, 18-SHH, 9-Grp3, and 17-Grp4 subgroups) by qRT-PCR. MSI1 mRNA expression levels were also validated in an additional public dataset of MBs (GSE85217). The ROC curve was used to validate the diagnostic standards of MSI1 expression. Next, the potential correlated cell-cycle genes were measured by RNA-Seq. Cell cycle, cell viability, and apoptosis were evaluated in a Grp3/Grp4 MB cell line after knockdown of MSI1 and cisplatin treatment. We identified an overexpression of MSI1 with a high accuracy to discriminate Grp3/Grp4-MBs from non-Grp3/Grp4-MBs. We identified that MSI1 knockdown not only triggered transcriptional changes in the cell-cycle pathway, but also affected G2/M phase in vitro, supporting the role of knockdown of MSI1 in cell-cycle arrest. Finally, MSI1 knockdown decreased cell viability and sensitized D283-Med cells to cisplatin treatment by enhancing cell apoptosis. Based on these findings, we suggest that MSI1 modulates cell-cycle progression and may play a role as biomarker for Grp3/Grp4-MBs. In addition, MSI1 knockdown combined with cisplatin may offer a potential strategy to be further explored in Grp3/Grp4-MBs.


Introduction
Medulloblastoma (MB, World Health Organization grade 4) is the most common malignant brain tumor in the pediatric population [1].MBs originate in the cerebellum and are classified into four different molecular subgroups: Wingless signaling activated (WNT), Sonic Hedgehog (SHH), Groups 3 (Grp3-MBs) and 4 (Grp4-MBs) [2].Grp3 accounts for 25% of all MB cases, representing the deadliest of all molecular subgroups, with a 5-year overall survival (OS) ranging from 42 to 66%, depending on Grp3 subtypes (alpha, beta, or gamma) [3].On the other hand, although accounting for 40% of MBs cases, Grp4-MBs remain poorly characterized with an OS of around 70% (intermediate prognosis).Intriguingly, both subgroups have a high propensity to metastasize [4,5].Currently, the treatment options for MBs are based on multimodal strategies that include maximal safe tumor resection, chemotherapy, radiotherapy, and immunotherapy [6].However, surviving patients often suffer from severe treatment-related side effects, including permanent cognitive and motor disabilities, particularly infants and children of pre-scholar age [7].In this context, it is of note the scantiness of effective targeted-therapies for Grp3/Grp4-MBs that could provide better OS, aligned with an improvement in patients' life quality.In fact, this lack of new actionable biomarkers depicts a challenge in the daily clinical practice due to the limited understanding of tumorigenesis and the inconclusive molecular stratification for Grp3/Grp4-MBs [1].
Musashi-1 (MSI1) is an evolutionarily highly conserved gene and a member of the RNA-binding proteins (RBP) family, which was first identified in Drosophila sp. in 1994.Interestingly, this group of proteins received this name in honor of Miyamoto Musashi, a great swordsman of Eastern culture [8].MSI1 is crucial in the initiation of the development of the central nervous system (CNS) and is vastly expressed in neural progenitor cells of vertebrates, being characterized as a neuronal stem cell (SC) marker [9,10].MSI1 acts through its direct interaction with the 3'-UTR (3′ untranslated region) in several mRNA targets at a posttranscriptional level, inducing the increase or silencing the expression of these genes.For this reason, MSI1 is recognized as a translational regulator of cell fate, and maintenance of the stem cell state [11].Dysregulations in MSI1 expression can lead to cellular dysfunctions promoting tissue instability, as well as tumorigenesis [7,12].Additionally, MSI1 has been reported to regulate cell cycle, chemoresistance, proliferation, and cell death in several tumors, including tumors of the CNS, such as gliomas, glioblastomas, and astrocytomas [12].Thus, in recent years, the role of MSI1 in cancer has gained increasing interest [13].
Herein, we identified an overexpression of MSI1 in Grp3/Grp4-MBs in a Brazilian cohort and in an independent dataset (GSE85217) of pediatric MBs.Besides, we also observed a significant role of MSI1 expression in the discrimination between Grp3/Grp4-MBs from non-Grp3/Grp4-MBs.Knockdown of MSI1 by short-hairpin (sh) RNA in D283 Med cell-line (a Grp3/Grp4-MB cellline) promoted cell-cycle interruption in the G1/S transition and, consequently, decreased the number of cells in the G2/M phase, repressed cell growth, and sensitized these cells to cisplatin treatment by enhancing cell apoptosis.Overall, our study provides new insights of MSI1 as a potential target involved in Grp3/Grp4-MBs carcinogenesis.

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) [14], 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 manufacturer's specifications.RNA concentrations were determined 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 500 ng 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) [3] 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 [15,16] (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% of Fetal Bovine Serum (FBS), MaxSpec Catalog number: A4766801, Gibco™, Thermo Fisher ® ), with 100U/ml penicillin and 100 μg/ml streptomycin, and kept in a humid atmosphere containing 5% CO2 at 37 °C.

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 Quant-Studio™ 12 k Flex system (Applied Biosystems, Foster City, CA, USA) using two internal controls: GUSB (Beta Glucuronidase) (Hs4333767F_m1) and hypoxanthine guanine phosphoribosyl transferase (HPRT) (Hs4310809E_m1).The data were analyzed using the 2 −ΔΔCT method and nonneoplastic cerebellum samples were used as calibrators [17].

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 (50 μg) were size-fractionated by SDS-PAGE and proteins were immunoblotted with anti-MSI1 (dilution 1:1000, cat.no.#5663, Cell Signaling Technology, Danvers, MA).And the anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (dilution 1:10.000,cat.no.#2118, Cell Signaling Technology, Danvers, MA).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).

Cell cycle analysis
For cell-cycle analysis, 17.5 × 10 4 (D283-Med MSI1 knockdown and control shRNA Scramble) cells were plated in 6-well plates and kept in culture for 72 h, without treatment.Then, cells were trypsinized and centrifuged at 1200 rpm/5 min and washed with PBS 1X.Cells were fixed with 70% cold ethanol and incubated at -20 °C overnight.Cells were then centrifuged at 800 rpm/5 min, washed with PBS 1X, and incubated with 25 μL of RNAse A (10 ng/ mL) at 37 °C for 30 min.Next, the centrifugation step was repeated and cells were stained with 100 μL of PI (50 μg/ mL) shortly before acquisition by BD FACS Calibur™ 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, 6 × 10 3 (D283-Med MSI1 knockdown and control shRNA Scramble) cells were plated in 96-well plates.The cells were treated with cisplatin at different concentrations (3 µM, 5 µM, 7 µM, and 10 µM) for 72 h, and the results were obtained using the SpectraMax ® L Microplate Reader device.The assay was performed in triplicate.Furthermore, the concentration of cisplatin that inhibited 50% of cell viability (IC 50 ) was determined using the CalcuSyn Software (Biosoft, Cambridge, UK).

Apoptosis detection
In total, 17.5 × 10 4 cells (D283-Med MSI1 knockdown and control shRNA Scramble) were plated and treated for 72 h 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 min, protected from light, at room temperature.Cells were analyzed by BD FACSCalibur ™ flow cytometer (BD Biosciences, San Jose, CA, USA).The experiments were carried out in triplicate.Data were analyzed using FlowJo 8.7 software.

RNA-sequencing (RNA-seq) analysis
RNA samples of D283-Med cells with MSI1 knocked-down (shRNA_MSI1#2) vs. control cells (shRNA_Scramble) were obtained as previously described by Martins-da-Silva [18], and sequenced on Illumina HiSeq 2500.Samples were processed for RNA-seq according to the manufacturer's instructions (Illumina, San Diego, CA, USA).Approximately 4 million paired-end reads were mapped against the human reference genome (GRCh38/hg38) using HISTA2 [19] to generate read alignments for each sample.After transcripts were assembled, gene-level counts were obtained using featureCounts (version 2.0.1).Differentially expressed genes (DEGs) were identified using the DESeq2 package [20] (considering an adjusted p value < 0.05 and log2 foldchange (log2FC) > or < − 0.3 as a threshold.RNA-seq data were deposited in the Gene Expression Omnibus (GEO) repository under the accession code GSE226733.Volcano plot and KEGG's pathways enrichment analysis were performed using the SRplot webtool (https:// www.bioin forma tics.com.cn/ en, accessed on 10 April 2023).The expression levels of DEGs associated with cell cycle were plotted using the Complex heatmap package [21].

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.

MSI1 is overexpressed in Grp3 and Grp4-MBs subtypes: analysis of an in-house cohort
To examine the expression profile of MSI1 in pediatric MBs, we determined its mRNA expression levels in a Brazilian cohort of 59 MB samples (WNT: n = 15; SHH: n = 18; Grp3: n = 9 and Grp4: n = 17) and five non-neoplastic cerebellums.Furthermore, we explored MSI1 expression level between different pediatric MB subgroups using an additional and independent dataset (GSE85217: WNT n = 51; SHH n = 146; Grp3 n = 131 and Grp4 n = 300).Interestingly, an overexpression of MSI1 was observed in MBs tissues when compared to normal cerebellum (p < 0.05, Fig. 1B).Within MB molecular subtypes, MSI1 expression was higher in Grp3 and Grp4-MBs (p < 0.001 and p < 0.0001, respectively) when compared to WNT and SHH-MBs subtypes in our cohort (Fig. 1B).The overexpression of MSI1 in Grp3 and Grp4-MBs was also observed in the GSE85217 dataset (Fig. 1C).
Once we observed that MSI1 was overexpressed in both Grp3 and Grp4-MBs, we further evaluated its potential as a predictive biomarker for MB subgroups.Receiver-operating characteristic (ROC) analysis was employed to examine the discrimination accuracy of the MSI1 as a diagnostic marker for Grp3/Grp4-MBs.As shown in Fig. 1D, the area under the ROC curve was 0.856 (p < 0.001), suggesting a significant high accuracy in discriminating between Grp3/ Grp4-MBs from non-Grp3/Grp4-MBs.

MSI1 is overexpressed in D283-Med: a Grp3/ Grp4-MBs' cell line
The mRNA and protein levels of MSI1 were evaluated in two MB cell lines classified as Grp3/Grp4-MBs: D283-Med and USP-13-MED [15,16].D283-Med cells showed an increased expression of MSI1 in contrast to USP-13-MED (p < 0.01) (Fig. 2A).At protein level, D283-Med cell line consistently presented a higher expression of MSI1 when compared to USP-13-MED cells when analyzed by both western blot and immunofluorescence (Figs.2B, C).As shown in Fig. 2C, MSI1 was strongly stained in the cytoplasm and nuclei, while in the USP-13-MED, a weak MSI1 staining was observed in the cytoplasm, with a dot pattern staining in apical cell extensions of cell-to-cell contact (Figs.2D, E).Based on these findings, we selected the D283-Med cell line to explore the functional roles of MSI1 into Grp3/Grp4-MBs cells.
The knockdown of MSI1 was performed in D283-Med cells using lentiviral transduction with two different vectors: shRNA_MSI1#1 (constructed to the Exon 4 region) and shRNA_MSI1#2 (directed to Exon 8 of MSI1 gene structure), as well as an empty vector (pure/empty vector-shRNA_ Scramble) as a control.The qRT-PCR analysis showed a reduction of approximately 60% in shRNA_MSI1#1 and 85% of MSI1 mRNA levels in shRNA_MSI1#2 (p < 0.001) cells relative to control (shRNA_Scramble; Fig. 3A).Similarly, the western blot analysis of transduced cells revealed a significant reduction of MSI1 protein levels in both shRNA_MSI1#1 (p < 0.05) and shRNA_MSI1#2 (p < 0.001) cells (Fig. 3B, C).However, the silencing of MSI1 was more pronounced and significant in the cells transduced with the second clone (shRNA_MSI1#2), which were used for the functional assays.

MSI1 knockdown alters genes implicated in cell cycle control and inhibits cell-cycle progression
To gain insight into the role of knockdown of MSI1 on Grp3/ Grp4-MBs, we performed an RNA-seq to obtain the differential gene expression profile between the shRNA_Scramble and shRNA_MSI1#2.MSI1 knockdown efficiency in RNA-seq is shown in Supplementary Fig. 1.We found a total of 543 DEGs significantly upregulated (Fig. 4A 2, log2 Fold change < − 0.3; p value adjusted < 0.05) in D283-Med shRNA_MSI1#2 compared to D283-Med shRNA_Scramble.KEGG enrichment analysis demonstrated that pathways involved in ribosome, DNA replication, cell cycle, and p53 signaling were enriched by the upregulated signature (Fig. 4B, bar plots in red), while genes downregulated were enriched by lysosome, protein processing in endoplasmic reticulum, and HIF-1 signaling pathway (Fig. 4B, bar plots in blue).Among the DEGs, we observed relevant genes associated with cell-cycle progression, such as CCND3, TP53, CDC7, CDK1, MDM2, MCM2, MCM4, and MCM5 (Fig. 4C, D), suggesting that low MSI1 gene in D283-Med cell-line expression induces expression of subsets of genes belonging cell-cycle mechanisms.
Consistently with our RNA-seq findings, MSI1 is known to be associated with cell-cycle dysfunction [22].To verify whether genetic silencing of MSI1 alters cell cycle in Grp3/Grp4-MB cells, we examined the distribution of cells in different cell-cycle phases.Interestingly, MSI1 knockdown decreased the number of cells in G2/M phase in D283-Med shRNA_MSI1#2 (p < 0.001) compared to control (shRNA_Scramble; Fig. 5A, B).Thus, altogether these results revealed that MSI1 not only triggered transcriptional changes in the cell-cycle pathway, but also affected G2/M phase in vitro, supporting the role of knockdown of MSI1 in cell-cycle arrest.

MSI1 knockdown decreases chemoresistance to cisplatin and increases cell death by apoptosis
To explore whether MSI1 was involved in resistance to cisplatin treatment, frequently present in MBs [23,24], we evaluated and compared whether MSI1 expression is involved in viability and chemoresistance of Grp3/Grp4-MBs cancer cells.As shown in Fig. 5C, D283-Med shRNA_MSI1#2 cells were more sensitive to chemotherapy compared to shRNA_Scramble (p < 0.05) when treated with different concentrations (3, 5, 7, and 10 µM) of cisplatin for 72 h.MSI1-knocked-down cells exhibited a 50% inhibitory effect (IC 50 ) with a concentration of 4.1 µM compared to IC50 of 10.98 µM of the control shRNA_Scramble (Fig. 5C and Supplementary Table 3).However, MSI1 silencing itself had no effect on apoptosis (Fig. 5C), which highlights its role in cell-cycle control.
To further investigate the mechanism by which MSI1 knockdown decreases cisplatin resistance, we evaluated the role of MSI1 silencing in triggering cell apoptosis.Annexin V/PI-positive cells were counted as an index of cell death, at the time of 72 h after treatment with 4.1 µM of cisplatin.As expected, cisplatin treatment induced a significant increase in the apoptosis rate in the shRNA_Scramble (30%, p < 0.01) and shRNA_MSI1#2 (63%, p < 0.001) compared to untreated cells (Figs. 5D, E).Collectively, our findings suggest that MSI1 knockdown represses D283 Med cells' progression whereas it sensitizes these cells to cisplatin by enhancing apoptosis of Grp3/Grp4-MBs' cancer cells.

Discussion
Currently, the paucity of specific target therapies associated with an increased tumor resistance to current chemotherapeutic regimens are key clinical challenges of Grp3/ Grp4-MBs treatment [1].In this study, we investigated the expression profile and functional roles of MSI1, a marker and a regulator of neural stem cells [25,26], in Grp3/Grp4-MBs.MSI1 is recognized as a key regulator of neural stem cell proliferation and maintenance within the Dentate Gyrus, a part of the hippocampal formation in the temporal lobe of the brain and the subventricular zone [27].Of note, any dysregulation of MSI1 expression is associated with tissue instability and can lead to tumorigenesis [7,12].
Recent insights into molecular subtype MBs' origins hypothesized that during early cerebellar development, the Grp3-MBs arise from progenitor cells of the outer granular layer, while Grp4-MBs originate from cells of the upper rhombic lip (uRL) [4,28].Herein, we demonstrated that MSI1 is upregulated in MB tissues compared to normal cerebellum and highlighted its overexpression specifically in Grp3/Grp4-MBs, in contrast to the other MBs subtypes.Thus, our results suggest that the overexpression of MSI1 may have a potential role in tumor progression/aggressiveness in this specific setting of MBs.
Consistent with our results, the overexpression of MSI1 was found in different pediatric brain tumors, such as glioblastomas and gliomas [29,30].VO Dat T. et al. [31] have shown that increased levels of MSI1 in MBs tissues were associated with poor overall and progression-free survival.On the other hand, using ROC analysis, we demonstrated a very high correlation of MSI1 expression for identifying Grp3/Grp4 cases of MBs, indicating that MSI1 may be a potential biomarker and could be further explored as a therapeutic target not only for Grp3-MBs, but possibly to Grp4-MBs, as well.To our knowledge, the present study is the first to depict MSI1 as a potential biomarker for Grp3/ Grp4-MBs.
Given that cancer is the result of multiple genetic alterations, there are many transcriptional and post-transcriptional mechanisms modulated by RBPs that are involved in key cellular processes, such as cell proliferation and cell fate [32].However, the role and the underlying molecular mechanisms in which the overexpression of MSI1 could lead to Grp3/Grp4-MBs oncogenesis remain unexplored.Surprisingly, we identified that MSI1 knockdown not only triggered transcriptional changes in the cell-cycle pathway, but also affected G2/M phase in vitro, supporting the role of knockdown of MSI1 in cell-cycle arrest.Of note, cellcycle arrest is one important mechanism by which cancer cells growth may be suppressed [33].Interestingly, MSI1 has been proposed to act as a repressor of the translation of mRNAs encoding inhibitors of cell-cycle progression [27,34].In addition, the previous reports have shown that MSI1 modulates endometrial carcinoma cell-cycle progression by p21 WAF1/CIP1 regulation [35].Also, the knockdown of MSI1 induced cell-cycle arrest at the G1/S phase in hepatoma cells in vitro [36], as well as inhibited cell-cycle progression by targeting p21 and p27 in human osteosarcoma cells [37].Recently, a robust study has demonstrated that many of the main target genes of MSI1 in glioblastoma are involved in cell-cycle control, highlighting the cyclin-dependent kinases: CDK2, CDK6, CCNA1, CCNA2, as well as p21, and p27 [22].
Another critical aspect of our study was to investigate whether MSI1 is involved in the progression, drug resistance, and induction of programmed cell death in Grp3/Grp4-MBs carcinogenesis.Interestingly, we found that the knockdown of MSI1 in Grp3/Grp4-MBs' cell lines decreased cell viability and enhanced cell death by apoptosis in response to cisplatin treatment.Although more studies are clearly necessary to unravel the mechanistic relationship between these phenomena, in agreement with our findings, it has been reported that MSI1 is involved in drug resistance of colorectal cancer cells [38], glioblastoma [29], as well as gastric cancer [39].
Detailed understanding on how MSI1 may contribute to the processes of drug resistance and cell death in cancer remains elusive to date.Thereby, one of the main goals of this research was to investigate MB cell-line behavior following MSI1 silencing in association to cisplatin.More recently, new compounds were described to be able to disrupt MSI1 regulatory functions.These novel MSI1 inhibitors may be interesting drugs to be tested in animal models, in association to classical chemotherapeutic agents (i.e., cisplatin) for Grp3/4 MB, to define if this association may recapitulate in vivo tumor arrest and decrease of cisplatin chemoresistance in this setting.

Conclusions
Taken together, our results demonstrated that MSI1 is overexpressed in Grp3/Grp4-MBs when compared to non-Grp3/ Grp4 tumors.In addition, our findings provide, for the first time, experimental evidence, indicating that MSI1 knockdown in association to cisplatin enhances tumor apoptosis by promoting cell-cycle disruption and ultimately decreasing cisplatin chemoresistance.This in vitro evidence may support further investigation on the potential role of combining MSI1 inhibitors in association with chemotherapy for Grp3/ Grp4-MBs.

Fig. 1
Fig. 1 Clinicopathological features and MSI1 expression overview in a Brazilian cohort of MBs.A Summary of clinical features and molecular characteristics of tumor samples from 59 pediatric patients: molecular classification (WNT-MBs, SHH-MBs, Grp3-MBs, and Grp4-MBs); gender (female or male); age at diagnosis (below or above 3 years); tumor resection (gross total resection-GTR or subtotal resection-STR); tumor metastasis (absent or present); risk stratification (average or high); tumor relapse (yes or no); patient status and outcome (alive or tumor relapse/death); disease status (alive; death related to MB or others).B MSI1 relative expression in MB molecular subgroups and normal cerebellum of an in-house Brazil-

Fig. 2
Fig. 2 MSI1 expression in Grp3/Grp4-MBs' cell lines.A MSI1 relative expression in D283-Med and USP-13-MED cell lines by RT-qPCR.The graph shows the mean ± standard deviation of three independent experiments (*p < 0.01).B Relative MSI1 protein quantification.C Representative image of MSI1 protein levels in pediatric MB cell lines D283-Med and USP-13-MED determined by Western blot.GAPDH protein was used as an endogenous control.The relative quantification of protein expression was determined by ImageJ software.Representative photomicrographs of MSI1 immunofluorescence staining (magenta) showing a strong intensity for the nuclear and cytoplasmic staining in D283-Med (D); and a lower cytoplasmic and weak nuclear staining in USP-13-MED cells (E).Photomicrographs generated by confocal laser scanning microscopy

Fig. 3
Fig. 3 Validation of MSI1 lentiviral knockdown in D283-Med cells.A Quantitative RT-PCR showing relative expression of MSI1 in D283-Med cells transduced with two different shRNA (D283 Med shRNA_MSI1#1, D283 Med shRNA_MSI1#2, and D283 Med Scramble as control).B, C Western blot and protein relative quantification showing reduced levels of MSI1 in D283 Med shRNA_