Abstract 2111: SIP1/ZEB2 Targeting by the Nitric Oxide-dependent miR200 Family is Important for Early Mesodermic/Cardiovascular Commitment of Mouse Embryonic Stem Cells
INTRODUCTION: MicroRNAs (miRNA) are a class of small, non-coding RNA molecules recently emerged as important gene function regulators through their effects at post-transcriptional level. We recently found that nitric oxide (NO) treatment drives mouse embryonic stem cells (ES) toward an early expression of mesodermic/cardiovascular differentiation markers. No information, however, are available about the effect of NO on miRNAs expression neither about specific miRNA targets relevant to the NO-dependent differentiation process.
METHODS & RESULTS: A miRNA profiling was performed in ES cultured with or without leukemia inhibitory factor (LIF) and in the presence or absence of the NO donor DETANO (500 μM). In LIF-deprived ES the presence of NO determined a rapid expression of miRNA 200 family members, while no changes were observed with LIF or in absence of NO. Real-time PCR analysis confirmed that all miR-200 family members, namely mir 200a (5.31+/−0.71), 200b (4.33+/−1.2), 200c (5.9+/−2.74) and 429 (5.1+/−0.63) were up-regulated above control level by NO, but miR-141(1.4+/−1). This effect was also evident after forced endothelial Nitric Oxide Synthase (eNOS) expression or NO addition in the presence of LIF. Further evidences indicated that direct over-expression of mir200a, b, c and 429 alone or in combination reduced (3 fold decrease) the ES content of Smad interacting protein 1 (Sip1/ZEB2) driving cells toward a differentiated state. Similar results were obtained with the NO donor DETA/NO. Notably, the direct small-RNA interference of Sip1/ZEB2 induced cardiovascular differentiation markers similarly to NO donor.
CONCLUSIONS: Taken altogether our data identifies the mir200 family as responsive to NO treatment and provide evidences about the role of Sip1/ZEB2 in the regulation of the nitric oxide-dependent early cardiovascular ES differentiation.