MiR-1253 exerts tumor-suppressive effects in medulloblastoma via inhibition of CDK6 and CD276 (B7-H3)

Abstract

Of the four primary subgroups of medulloblastoma, the most frequent cytogenetic abnormality, i17q, distinguishes Groups 3 and 4 which carry the highest mortality; haploinsufficiency of 17p13.3 is a marker for particularly poor prognosis. At the terminal end of this locus lies miR-1253, a brain-enriched microRNA that regulates bone morphogenic proteins during cerebellar development. We hypothesized miR-1253 confers novel tumor-suppressive properties in medulloblastoma. Using two different cohorts of medulloblastoma samples, we first studied the expression and methylation profiles of miR-1253. We then explored the anti-tumorigenic properties of miR-1253, in parallel with a biochemical analysis of apoptosis and proliferation, and isolated oncogenic targets using high-throughput screening. Deregulation of miR-1253 expression was noted, both in medulloblastoma clinical samples and cell lines, by epigenetic silencing via hypermethylation; specific de-methylation of miR-1253 not only resulted in rapid recovery of expression but also a sharp decline in tumor cell proliferation and target gene expression. Expression restoration also led to a reduction in tumor cell virulence, concomitant with activation of apoptotic pathways, cell cycle arrest and reduction of markers of proliferation. We identified two oncogenic targets of miR-1253, CDK6 and CD276, whose silencing replicated the negative trophic effects of miR-1253. These data reveal novel tumor-suppressive properties for miR-1253, i.e., (i) loss of expression via epigenetic silencing; (ii) negative trophic effects on tumor aggressiveness; and (iii) downregulation of oncogenic targets.

Keywords: CD276; CDK6; chromosome 17p13.3; group 3 medulloblastoma; miR-1253.

Figure 1

MiR‐1253 expression deregulation in medulloblastoma. A. MiR‐1253 expression analysis by Nanostring applied to CSF from normal cerebellum (CB n = 14) and medulloblastoma (MB n = 15) patients (Alessandra et al, gse62381). B. Relative expression of miR‐1253 by real‐time reverse transcriptase PCR normalized to U6b expression in total medulloblastoma specimens (MB n = 28) compared to normal cerebellum (CB n = 16). C. Subgroup‐specific comparison (SHH n = 5, Grp3 n = 9, Grp4 n = 14) to normal cerebellum (ped CB n = 11, adult CB n = 5). D. Similar comparison in 7 well‐studied medulloblastoma cell lines compared to normal human astrocytes (NHA). E. In situ hybridization analysis of normal cerebellum (n = 13) vs. medulloblastoma (n = 21). Representative micrographs at 10× magnification; scale bars 400 μm. Data presented as mean ± SD and analyzed using (A) Mann‐Whitney U, (B) Student’s t‐test, (C and D) One‐way ANOVA and (E) Chi‐square test for contingency analysis; *P < 0.05, **P < 0.005 and ***P < 0.001 compared to control.

Figure 2

Epigenetic silencing of miR‐1253 by hypermethylation. A i‐iii. Subgroup‐specific miR‐1253 methylation (SHH n = 5, Grp3 n = 8, Grp4 n = 13) compared to normal cerebellum (Ped CB n = 10, Adult CB n = 5). miR‐1253 chr17 location: 2,651,372‐2,651,476. Relative expression of miR‐1253 (B) and its targets (C) in HDMB03 and D425 cells treated with 5‐AzaC (5 µM) for 96 h. D. Analysis of proliferation after pre‐treatment with miR‐1253 inhibitor for 24 h followed by demethylation with 5‐AzaC (5 µM) over a 96‐h period. Data presented as mean ± SD and analyzed using Student’s t‐test (or One‐way ANOVA for Aiii); *P < 0.05, **P < 0.005, and ***P < 0.001.

Figure 3

Negative trophic effects of miR‐1253 expression restoration in medulloblastoma tumor cells. Adherent medulloblastoma cell lines, DAOY and HDMB03, were transiently transfected with miR‐1253 mimic (50 or 100 nM) for up to 72 h and subsequent functional studies performed. A. Expression restoration of miR‐1253 at 72 h after transient transfection. B. Tumor cell proliferation examined over a 48‐h time frame with mimic alone and (C) with inhibitor + mimic (100 nM). D. Assessment of colony formation in transfected HDMB03 cells seeded in six‐well plates for nine days followed by methanol fixation and crystal violet staining. Representative images of cancer cell colonies presented at 10× magnification. E. Wound healing “scratch” assay with cell migration‐to‐wound closure monitored up to 48 h. For migration/invasion assays, transfected cells were reseeded in transwells coated with or without matrigel. Total number of migrating cells (F) or those invading through the matrigel layer (G) were quantified after overnight incubation. Data presented as mean ± SD from experiments done in triplicates and analyzed using One‐way ANOVA; *P < 0.05, **P < 0.005 and ***P < 0.001.

Figure 4

Effect of miR‐1253 expression restoration on cell cycle and apoptosis. DAOY and HDMB03 cells were transiently transfected with miR‐1253 mimic or scramble as described previously; biochemical analyses were conducted at 72 h. A. Western blotting expression analysis of markers of tumor cell proliferation with incremental changes in the ratio of phospho‐AKT to total AKT tabulated. B. Cell cycle analysis in HDMB03 cells by flow cytometry. C. Assessment of early vs. late apoptosis by flow cytometry, and (D) Western blotting analysis of standard makers of apoptosis with tabulated ratios between cleaved and total proteins in both cell lines. E. Given the high myc expression in HDMB03 cells, the ratio of phospho‐c‐Myc‐T58 vs. phospho‐c‐Myc‐S62 was further studied in HDMB03 cells. Data presented as mean ± SD from experiments done in triplicates and analyzed using Student’s t‐test; *P < 0.05, **P < 0.005 and ***P < 0.001.

Figure 5

Isolation of oncogenic targets of miR‐1253. A. Common miR‐1253 target genes (47) that sustain significant expression reduction (P < 0.05) with miR‐1253 treatment (in HDMB03 cells) when compared with three online miR target databases with heatmap expression patterns included (adult normal n = 4, pediatric normal n = 7, SHH n = 5, Grp3 n = 9, Grp4 n = 13). B. Survival (Cavali et al., gse85217) and expression analysis for CDK6 and CD276 using the R2 platform (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi). Nl CB, Roth et al 2008 (n = 9, gse3526); MB 1, Gilbertson et al. 2012 (n = 76, gse37418); MB 2, Pfister et al 2017 (n = 223); MB 3, Kool et al 2009 (n = 62, gse10327); MB4, Delattre et al 2012 (n = 57). C. mRNA transcript and protein levels for CDK6 and CD276 with miR‐1253 transfection in HDMB03 cells. D. Dual‐luciferase reporter assays confirming direct binding of miR‐1253 to 3′ UTR region of CDK6 and CD276 in HDMB03 cells. E, F. Subgroup‐specific comparison of CDK6 and CD276 expression between normal cerebellum (pedi CB n = 10, adult CB n = 5) and medulloblastoma (SHH n = 3, Grp3 n = 6, Grp4 n = 10) by immunohistochemistry. Representative micrographs at 10× magnification; scale bars 400 μm. Data presented as mean ± SD from experiments done in triplicates and analyzed using one‐way ANOVA; *P < 0.05, **P < 0.005, and ***P < 0.001.

Figure 6

Silencing CDK6 and CD276 and medulloblastoma tumor cell behavior. A. Effect of transfection of HDMB03 cells with si‐CDK6 or si‐CD276 (50 and 100 nM) on target gene expression at 72 h. B. Colony formation assessment in HDMB03 cells transfected with si‐CDK6 (50 and 100 nm). Transfected cells were reseeded in six‐well plates 24 h after transfection for nine days followed by methanol fixation and crystal violet staining. Stain was extracted and quantified at 590 nm. Representative images of cancer cell colonies presented at 10× magnification. To examine migratory potential, HDMB03 cells transfected with si‐CD276 (50 and 100 nM) were studied using the (C) wound healing “scratch” assay and the (D) trans‐well migration and invasion assays. Data presented as mean ± SD from experiments done in triplicate and analyzed using one‐way ANOVA; *P < 0.05, **P < 0.005 and ***P < 0.001.