Downregulation of 14q32 microRNAs in Primary Human Desmoplastic Medulloblastoma

Abstract

Medulloblastoma (MB) is one of the most common pediatric cancers, likely originating from abnormal development of cerebellar progenitor neurons. MicroRNA (miRNA) has been shown to play an important role in the development of the central nervous system. Microarray analysis was used to investigate miRNA expression in desmoplastic MB from patients diagnosed at a young age (1 or 2 years old). Normal fetal or newborn cerebellum was used as control. A total of 84 differentially expressed miRNAs (64 downregulated and 20 upregulated) were found. Most downregulated miRNAs (32/64) were found to belong to the cluster of miRNAs at the 14q32 locus, suggesting that this miRNA locus is regulated as a module in MB. Possible mechanisms of 14q32 miRNAs downregulation were investigated by the analysis of publicly available gene expression data sets. First, expression of estrogen-related receptor-γ (ESRRG), a reported positive transcriptional regulator of some 14q32 miRNAs, was found downregulated in desmoplastic MB. Second, expression of the parentally imprinted gene MEG3 was lower in MB in comparison to normal cerebellum, suggesting a possible epigenetic silencing of the 14q32 locus. miR-129-5p (11p11.2/7q32.1), miR-206 (6p12.2), and miR-323-3p (14q32.2), were chosen for functional studies in DAOY cells. Overexpression of miR-129-5p using mimics decreased DAOY proliferation. No effect was found with miR-206 or miR-323 mimics.

Keywords: 14q32 miRNA cluster; ESRRG; desmoplastic medulloblastoma; miR-129-5p; miRNA profile.

Figure 1

Hierarchical clustering analysis of medulloblastoma and normal cerebellum. Unsupervised hierarchical cluster analysis representing the 84 miRNAs expressed in medulloblastoma samples versus normal cerebellum. 14q32 miRNAs are depicted by a left vertical line. -st, star.

Figure 2

Ingenuity pathway analysis networks (IPA). (A) Network 1; (B) Network 2; both were constructed with deregulated miRNAs in medulloblastoma.

Figure 3

Validation of miR-206, 129-5p, 323-3p, and 495 downregulation by reverse transcriptase-quantitative PCR. Expression levels of miR-206, 129-5p, 323-3p, and 495 were performed in MB, normal fetal/newborn cerebellum and four different MB cell lines. Expression of miR-495 was not performed in D341 cell line. ○, DAOY; ▾, Meb-Med-8A; x, D283Med; ♢, D341. Comparisons of MB versus normal cerebellum were done by the Mann–Whitney test.

Figure 4

Effect of miR-206, 129-5p, and 323-3p overexpression in DAOY cells proliferation. DAOY cells were transiently transfected with miRNAs mimic and replated after 20 h at a 1,500 cells/well density. Cell proliferation was evaluated by the MTS assay at 4, 28, and 52 h after re-plating, thus after 24, 48, and 72 h post-transfection. Results are representative of three independent experiments. Mean ± SE are shown; **P < 0.01 according to two-tailed t-test; NC, negative control miRNA mimic (scrambled).

Figure A1

Expression of OTX2 and PTCH1 in the different primary medulloblastomas (MB), medulloblastoma cell lines (named), and normal cerebellum (C). Total RNA (100 ng) was reverse transcribed and gene expression was measured by qPCR, in triplicates, using the SYBR method. Primers are available upon request to the authors. Values were normalized to the HPRT endogenous control gene. Relative expression values were calculated using the 2^−ΔΔCt method.

Figure A2

Transfection efficiency of miR-206, miR-129-5p, and miR-323-3p in DAOY cells as evaluated by RT-qPCR. Cells transfected with the scrambled mimic were included as negative control (NC). Values were normalized to the RUN6B endogenous control RNA. Relative expression values were calculated using the 2^−ΔΔCt method. Bars represent mean ± SD. Comparisons were performed by the Mann–Whitney test.

Figure A3

Apoptosis analysis of DAOY cells at 24 and 48 h post-transfection with miR-206, 129-5p, 323-3p, or negative control mimics, as evaluated by Annexin V and propidium iodide staining and FACS analysis. DAOY cells grown in serum-free RPMI-1640 medium. The percentage of necrotic (Q1), late apoptotic (Q2), viable (Q3), and early apoptotic (Q4) cells are shown in the corresponding quadrants. NC, negative control (scrambled mimic).

Figure A4

Gene expression graph for different microarray probes of interest. Data from 64 primary human MB samples accessible through GEO Series accession number GSE28245 (38) in NCBI’s Gene Expression Omnibus. (A) probe sets for ESRRG. (B) Probe sets for MEG3. Samples were grouped according to the molecular MB subgroups into WNT, SHH, C, and D. SHH desmoplastic was separated from SHH classic.