Abstract 5162: MicroRNA regulation of epidermal lineage differentiation of NT2/D1 embryonal carcinoma cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Micro RNAs (miRNAs) are small non-coding RNAs that regulate gene expression by targeting the complementary mRNA sequence for post-transcriptional modification, or mRNA cleavage. MiRNAs regulate a wide range of biological processes such as nervous system and cardiac development, as well as stem cell maintenance and differentiation. However, their role in regulating cell fate choice still remains largely unexplored. Cell lines derived from human embryonal carcinoma (EC) comprise a valuable in vitro resource for study of molecular events regulating cell fate and lineage decisions. NT2/D1 EC cells differentiate into an epithelial/smooth muscle phenotype when induced with BMP-2 (Chadalavada R et al., Stem Cells 25: 771-8, 2007). We used this experimental system to discover miRNAs likely to regulate epidermal cell specification. In a time-course experiment treating NT2/D1 cells with BMP-2, we performed gene expression profiling (GEP) using Affymetrix HG-U133A+B arrays and miRNA expression profiling using Agilent Human V.2 arrays. A set of miRNAs were identified that were up- or down-regulated as cells differentiated towards the epidermal lineage. 53 miRNAs were specifically down-regulated (p<0.01), of which 36 had predicted target genes that were known to play a role in epidermal lineage development. In contrast, 24 miRNAs were specifically up-regulated (p<0.01), whose predicted target genes were mainly involved in suppression of nervous system and cardiac development. Our result also identified expression of several other miRNAs that have been reported to be highly expressed in murine skin epidermis such as mmu-miR-99b and mmu-miR-143 (Rui Yi et al., Nat. Genet. 38:356-362, 2006). Our main interest however was miRNAs that were down-regulated in the NT2/D1-BMP-2 time-course since they presumably targeted genes involved in epidermal development. Among the top 10 miRNAs that were down-regulated during the time-course, hsa-miR-18b and hsa-miR-518b was associated with the up-regulated expression of the highest number of their predicted targets in the time-course GEP (hsa-Mir18b: FZD6, JAG1, CDH13, DAG1, and ELF3; hsa-miR-518b: LAMC1, ATP2A2, SOX9, ACVR1B, and TCF12), known to function in the epidermal lineage. Other down-regulated miRNAs such as hsa-miR-141, hsa-miR-425, and hsa-miR-101 also have predicted target genes with roles in the epidermal lineage development, although the numbers of such target genes were less than those of hsa-miR-18b and hsa-miR-518b. Their predicted target genes were also up-regulated in the time-course GEP. To further validate their biological function, we are currently performing Antagomir or LNA-mediated silencing of these miRNAs to evaluate the phenotype changes and expression changes of their presumptive target genes with epidermal lineage-associated functions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5162.