The MYCN oncogene is a direct target of miR-34a - PubMed (original) (raw)
The MYCN oncogene is a direct target of miR-34a
J S Wei et al. Oncogene. 2008.
Abstract
Loss of 1p36 heterozygosity commonly occurs with MYCN amplification in neuroblastoma tumors, and both are associated with an aggressive phenotype. Database searches identified five microRNAs that map to the commonly deleted region of 1p36 and we hypothesized that the loss of one or more of these microRNAs contributes to the malignant phenotype of MYCN-amplified tumors. By bioinformatic analysis, we identified that three out of the five microRNAs target MYCN and of these miR-34a caused the most significant suppression of cell growth through increased apoptosis and decreased DNA synthesis in neuroblastoma cell lines with MYCN amplification. Quantitative RT-PCR showed that neuroblastoma tumors with 1p36 loss expressed lower level of miR-34a than those with normal copies of 1p36. Furthermore, we demonstrated that MYCN is a direct target of miR-34a. Finally, using a series of mRNA expression profiling experiments, we identified other potential direct targets of miR-34a, and pathway analysis demonstrated that miR-34a suppresses cell-cycle genes and induces several neural-related genes. This study demonstrates one important regulatory role of miR-34a in cell growth and MYCN suppression in neuroblastoma.
Figures
Figure 1
miR-34a inhibits the growth of neuroblastoma cells through apoptosis and suppressed DNA synthesis. (a) microRNAs in the 1p36 region. Five microRNAs were mapped between 0-10Mb corresponding to 1p36.22-1pter, which is commonly deleted in NB tumors, using the Sanger miRNA Registry (
http://microrna.sanger.ac.uk/sequences/index.shtml
, Release 10.0). The relative positions of them are depicted on the black bar which represents 0-10Mb of chromosome 1. Among these 5 microRNAs, three (miR-200b, 429 and 34a; marked with arrowheads) are predicted to target MYCN genes by the miRBase Targets available on the same website. Start and End mark the genomic coordinates for the stem-loop pre-microRNA sequences of these microRNAs (Human Genome Build 36.2). (b) miR-34a inhibits the growth of neuroblastoma cells. _MYCN_-amplified NB cell lines, IMR32 and LA-N-5, were transfected with synthetic miR-34a, miR-200b, miR-429, and mimic control, the growth was monitored using WST-1 assays for 4 days. The optical densities (OD) at 450nm corrected by OD650nm were plotted over time after background subtraction of the readings from the media-only wells. miR-34a clearly suppressed the growth of both IMR32 and LA-N-5, but miR-200b and -429 had little or no effect. (c) A representative flow cytometry experiment shows miR-34a inducing apoptosis and suppression of DNA synthesis. Flow cytometry was used to study cell death (7-AAD staining), apoptosis (caspase 3 staining) and DNA synthesis (BrdU staining) in IMR32 cells at 48 hours after transfection. Blue: mimic control transfection; Red: miR-34a transfection. The bar graph summarizes the results of flow cytometry. Cell death and apoptosis were increased by 29% and 113% respectively, whereas DNA synthesis was suppressed by 27% in the miR-34a transfected cells.
Figure 2
A box plot of miR-34a expression levels in 16 primary NB tumors of 1p36 normal (n=8) and 1p36 deletion (n=8). The expression levels of miR-34a was measured by real-time Taqman® RT-PCR assays, and represented as normalized log2 ratios between miR-34a and an internal control, RNU6B. The result shows that NB tumors with 1p36 deletion express less miR-34a than 1p36 normal tumors (p =0.038).
Figure 3
MYCN is a direct target of miR-34a. (a) Left panel: a Western blot shows that miR-34a suppressed the expression of MYCN protein at 48 hours after transfection in both IMR32 and LA-N-5 cells. Right panel: quantification of MYCN protein showed a suppression of 80% and 95% by miR-34a in IMR32 and LA-N-5 cells respectively after normalization using the levels of GAPDH protein. (b) Sequence alignment of miR-34a with the two binding sites on the MYCN 3’-UTR. The underlined sequences on MYCN 3’-UTR were mutated to make luciferase reporter constructs containing a single miR-34a binding mutation (pMIR-MYCN-MT1 or MT2) or a double mutation (pMIR-MYCN-MT1&2). The underlined italicized letters (GGCAGUG) in miR-34a denote the seed sequence of miR-34a (position 2-8 at the 5’ end) (Lim et al., 2005). (c) Luciferase activity assays demonstrated that miR-34a directly suppresses MYCN by targeting the MYCN 3’-UTR. Luciferase reporter constructs containing full length wild-type (WT) and mutant (MT1, MT2, and MT1&2) 3’-UTR of MYCN were introduced into SK-N-AS cells with microRNAs, and luciferase activity was measured at 24 hours after transfection. Mutation of either miR-34a binding sites (MT1 or MT2) resulted in a reduction of suppression of luciferase activities, whereas double mutation totally abolished the suppression by miR-34a.
Figure 4
(a) Summary of GSEA analysis. GSEA analysis was performed on the ranked genes according to the ratios of transcripts from mimic control and miR-34a transfected IMR32 cells at 48 hours. Nine gene sets with a _p_-value of <0.01 and a false discovery rate (FDR) of < 0.05 were considered significant. Of the 9 gene sets, 7 associate with the down-regulated genes by miR-34a, and 2 set associate with up-regulated genes. (b) One of the GSEA plots indicates significant enrichment of cell cycle genes in the transcripts suppressed by miR-34a (p < 0.001 and FDR = 0.007). Affymetrix U133 Plus 2 data for the duplicated experiments of IMR32 cells transfected with miR-34a and mimic control at 48h transfection were combined, and multiple probe sets for the same genes were averaged. Genes were ordered in their ranked ratios, and GSEA were performed using the GSEA tool at
. Plot (the green curve) shows the running sum of enrichment score (ES) for ranked genes comparing with the cell_cycle gene set. Red vertical line specifies the maximum ES score. Heat map shows the log2 ratio (miR-34a vs. control) of leading edge subset genes in their ranked order which contribute to the significance. A scale of the log2 ratios is shown on the right. Black lines indicate gene hits in the cell_cycle gene set; and the black arrowhead indicates where the log2 ratio=0.
Figure 5
Search for other direct targeted genes of miR-34a besides MYCN. (a) Identification of MYCN direct targets by induction of MYCN in NB cells. In order to delineate the genes directly targeted by miR-34a, but not through MYCN, we used a MYCN induction system to identify MYCN direct targets. MYCN-3 cells containing a tetracycline controlled MYCN expression construct were induced by tetracycline, and total protein was collected at 3, 6, 9, 12, and 24 hours. Left panel shows a Western blot for MYCN induction. Right panel: quantification of the Western blot demonstrates that MYCN protein is induced at least 6 fold after induction. MYCN expression is normalized by the non-induced (NI). (b) Venn diagram of the altered genes by miR-34a or MYCN induction. In order to identify the genes directly regulated by miR-34a but not through MYCN, gene expression profiling was performed on IMR32 cells transfected with miR-34a and its mimic control at 48 hours; or on MYCN-3 cells with MYCN induction at 12 hours compared to the non-induced (NI). Genes with a ratio of 33% difference was considered as changed. The numbers indicate the gene counts in the corresponding categories. (c) Enrichment analysis of miR-34a binding and E-box sequences shows the validity of our approach to identify the direct targets of miR-34a. Upper panel: a heat map of _p_-values for the enrichment of miR-34a binding sequence or E-box sequences in different categories. _P_-values were calculated for the enrichment of the complimentary sequences of miR-34a seed in 3’-UTRs, or the E-boxes (MYC binding motifs) in the 2.5 Kb promoter regions for gene groups A-C. A color scale of the _p_-values is shown on its right. Lower panel: a table of _p_-values of the enrichment analysis. Shaded cells indicate p<0.01.
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