American Association for Cancer Research
15417786mcr190187-sup-217356_2_supp_5812267_pyr7f7.pdf (1.11 MB)

Supplementary Figure Legends from ELF4 Is a Target of miR-124 and Promotes Neuroblastoma Proliferation and Undifferentiated State

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journal contribution
posted on 2023-04-03, 16:41 authored by Adam Kosti, Liqin Du, Haridha Shivram, Mei Qiao, Suzanne Burns, Juan Gabriel Garcia, Alexander Pertsemlidis, Vishwanath R. Iyer, Erzsebet Kokovay, Luiz O.F. Penalva

Figure S1. Knockdown efficiency 48 h after reverse transfection. Figure S2. miR-124 directly binds ELF4 and regulates ELF4 expression. Figure S3. ELF4 ectopic expression antagonizes the effect of miR-124 on differentiation of KELLY cells. Figure S4. ELF4 ectopic expression antagonizes the effect of miR-124 on differentiation of SK-N-DZ cells. Figure S5. High ELF4 expression correlates with poor prognosis of neuroblastoma patients. Figure S6. Synergistic effect of ALK and ELF4 inhibition on differentiation and proliferation of KELLY cells. Figure S7. Validation of RNA-seq Results.





13-Cis-retinoic acid (RA) is typically used in postremission maintenance therapy in patients with neuroblastoma. However, side effects and recurrence are often observed. We investigated the use of miRNAs as a strategy to replace RA as promoters of differentiation. miR-124 was identified as the top candidate in a functional screen. Genomic target analysis indicated that repression of a network of transcription factors (TF) could be mediating most of miR-124's effect in driving differentiation. To advance miR-124 mimic use in therapy and better define its mechanism of action, a high-throughput siRNA morphologic screen focusing on its TF targets was conducted and ELF4 was identified as a leading candidate for miR-124 repression. By altering its expression levels, we showed that ELF4 maintains neuroblastoma in an undifferentiated state and promotes proliferation. Moreover, ELF4 transgenic expression was able to counteract the neurogenic effect of miR-124 in neuroblastoma cells. With RNA sequencing, we established the main role of ELF4 to be regulation of cell-cycle progression, specifically through the DREAM complex. Interestingly, several cell-cycle genes activated by ELF4 are repressed by miR-124, suggesting that they might form a TF–miRNA regulatory loop. Finally, we showed that high ELF4 expression is often observed in neuroblastomas and is associated with poor survival. miR-124 induces neuroblastoma differentiation partially through the downregulation of TF ELF4, which drives neuroblastoma proliferation and its undifferentiated phenotype.