Article Figures & Data
Figures
- Figure 1.
Summary heat map of differentially expressed miRNA loci. A, heat map summarizing the patterns of expression for 32 miRNA loci that were differentially expressed in neuroblastoma subtypes. Yellow, low-stage hyperdiploid tumors with favorable histopathology; pink, high-stage 11q− tumors with unfavorable histopathology; blue, high-stage MNA tumors with unfavorable histopathology; red, high expression; green, low expression. MNA tumors form a distinct cluster characterized by low expression levels of many miRNA loci. The 11q− tumors formed two separate clusters, which could not always be perfectly distinguished from the low-stage hyperdiploid tumors. Interestingly, the same clustering patterns were observed based on mRNA expression profiling ( 5, 6). B, alterations in miRNA expression following ATRA exposure or siRNA inhibition of MYCN. The fold change in expression of miRNA loci following exposure of SK-N-BE cells to ATRA or Kelly cells to a MYCN siRNA was determined by the same quantitative real-time PCR-based approach used in the expression profiling of primary tumors. Red, increase in expression (>1.5-fold); green, decrease (>1.5-fold) relative to normal controls.
- Figure 2.
Induction of differentiation in neuroblastoma cells with ATRA. Untreated (A) and ATRA-treated (B; 5 μmol/L) SK-N-BE cells. The cells treated with ATRA had reduced proliferation and displayed neurite-like processes.
- Figure 3.
siRNA inhibition of MYCN mRNA. Quantitative RT-PCR analysis of MYCN expression in Kelly cells following transfections with a scrambled control oligonucleotide (left column) and commercially available MYCN siRNAs (middle and right columns). Bars, SD.
- Figure 4.
Analysis of neuroblastoma cell lines transfected with pre-miR-184. A, expression of miR-184 in Kelly cells transfected with a scrambled oligonucleotide [normal control (NC)] and with pre-miR-184 assessed by quantitative PCR. Virtually no miR-184 is present in Kelly cells. Expression of endogenous RNU66 (bottom) shows that equal amounts of PCR product were loaded onto the gel. B, analysis of cell viability using the MTT assay. Y axis, arbitrary units of measuring the accumulation of formazan dye in metabolically active cells; X axis, units of time in days. Measurements were taken at days 1 to 6 after transfection of Kelly (black lines) and SK-N-AS (red lines) with pre-miR-184 molecules or with scrambled control oligonucleotides. miR-184 slowed down cell proliferation in both cell lines but had a more dramatic effect in the MNA Kelly cell line. C, FACS analysis of Kelly cells transfected with scrambled oligonucleotide (left) or pre-miR-184 (right). The number of apoptotic cells increased in the Kelly cells overexpressing miR-184 (2.7-fold), and there was a substantial number of cells arrested at G1 (54% versus 75.5%). D, analysis of caspase-3/caspase-7 activity in Kelly and SK-N-AS cells transfected with scrambled oligonucleotide (white column) or pre-miR-184 (black column). Both cell lines show an increase in apoptotic cells using the caspase-3/caspase-7 assay. Consistent with the MTT cell viability assay, the increase was more dramatic in the MNA Kelly cell line than in the MYCN single-copy cell line SK-N-AS. Bars, SD.
Tables
- Table 1.
Characteristics of primary tumors used in study
Tumor sample Subgroup INSS Histopathology * T-203 HYP 2b Favorable T-238 HYP 1 Favorable T-269 HYP 2a Favorable T-1251 HYP 1 Favorable T-1253 HYP 1 Favorable T-1354 HYP 1 Favorable T-296 HYP 2a Unfavorable T-347 HYP 1 Favorable T-25 HYP 1 Favorable T-1 HYP 2 Favorable T-36 HYP 2a Favorable T-136 11q− 4 Unfavorable T-283 11q− 4 Unfavorable T-1100 11q− 4 Unfavorable T-1194 11q− 4 Unfavorable T-1220 11q− 4 Unfavorable T-1352 11q− 4 Unfavorable T-1359 11q− 4 Unfavorable T-1012 11q− 4 Unfavorable T-355 11q− 4 Favorable T-1200 11q− 4 Favorable T-31 11q− 4 Unfavorable T-48 11q− 4 Unfavorable T-433 MNA 4 Unfavorable T-493 MNA 4 Unfavorable T-1009 MNA 4 Unfavorable T-1034 MNA 4 Unfavorable T-1198 MNA 4 Unfavorable T-1266 MNA 4 Unfavorable T-1068 MNA 4 Unfavorable T-1109 MNA 4 Unfavorable T-14 MNA 4 Unfavorable T-39 MNA 4 Unfavorable T-53 MNA 2 Unfavorable T-1406 MNA 2a Unfavorable -
Abbreviations: HYP, hyperdiploid; 11q−, loss of 11q; INSS, International Neuroblastoma Staging System.
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↵* Histopathology classification using Shimada system based on patient age at diagnosis, degree of differentiation, and mitosis-karyorrhexis index.
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- Table 2.
Differential expression of miRNAs in neuroblastoma subtypes
miRNA Chromosome map MNA/hyperdiploid * 11q−/hyperdiploid * MNA/11q− * let-7a 22q13/9q22/11q24 2.4× let-7b 22q13 3.99× miR-27b 9q22.32 0.48× 0.36× miR-30b 8q24.22 0.44× miR-30c 1p34.2/6q13 0.44× miR-30e 1p34.2 0.24× miR-92 13q31.3/Xq26.2 3.64× miR-107 10q23.31 0.32× miR-129 7q32.1/11p11.2 0.08× miR-137 1p21.3 0.19× miR-141 12p13.31 2.09× 0.26× miR-146a 5q33.3 0.25× miR-149 2q37.3 0.26× miR-150 19q13.33 0.34× miR-181a 9q33.3 2.4× 3.75× miR-181b 9q33.3/1q31.3 4.05× 8.85× miR-184 15q25.1 2.2× 0.42× miR-186 1q31.1 0.38× miR-187 18q12.2 2.92× miR-189 9q22.32 0.27× 0.39× miR-190 15q22.2 0.10× miR-200a 1p36.33 0.26× miR-200c 12p13.31 0.45× miR-216 2p16.1 0.27× miR-301 17q23.2 0.37× miR-302a 4q25 0.39× miR-323 14q32.31 0.21× 0.27× miR-324-5p 17p13.1 0.22× miR-326 11q13.4 0.35× 0.62× miR-330 19q13.32 0.29× miR-331 12q22 0.40× 0.63× miR-335 7q32.2 0.45× -
↵* Fold difference in mean expression between the tumor subtypes being compared (i.e., MNA versus hyperdiploid, 11q− versus hyperdiploid, and MNA versus 11q−. P values obtained from one-way ANOVA ranged from <0.01 to <0.05, with 31 comparisons having P values of <0.01 and 10 comparisons having P values between 0.01 and <0.05. All comparisons were statistically significant when the Tukey-Kramer correction for multiple comparisons was applied.
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Volume 67, Issue 3
