MICRORNAS 128 AND 618 ARE POSSIBLE TARGETS OF PHOX2B TRANSCRIPTION FACTOR

PHOX2B is a homeodomain transcription factor whose expression is restricted to neurons that control the cardiovascular, digestive and respiratory systems. PHOX2B contains a sequence of 20 polyalanine in its C-terminal portion. An increase in Ala repeats number is associated with Congenital Central Hypoventilation Syndrome (CCHS), characterized by respiratory arrests during sleep and, occasionally, wakefulness and reduce the transcriptional activity of the protein. CCHS is also associated to Hirschprung disease and neuroblastoma along with symptoms of general autonomic nervous system dysfunction. Very little is known about the genes regulated by PHOX2B; thus identify PHOX2B target genes in order to get new insights into the molecular pathogenesis of the disease is mandatory. To this purpose, we carried out ChIP-Seq experiments from IMR-32 neuroblastoma cell line that revealed 8512 PHOX2B-binding enriched regions. Among the putative PHOX2B regulated genes, 40 microRNA (miRNA) genes were identified and, after a bioinformatics analysis, miR-128 and miR-618 were selected as possible PHOX2B transcription targets. These miRNAs are expressed in neuroblastoma cell lines. Overexpression of wild-type (wt) or polyalanine expanded (+7 Ala and +13 Ala) PHOX2B in neuroblastoma IMR-32 and SK(2)C cell lines leads to a 50% decrease of mature miR-128 and miR-618 in SK(2)C after wt PHOX2B overexpression, but no difference in miRNA levels in IMR-32. Treatment with retinoic acid, known to decrease PHOX2B levels in SK(2)C cells, leads to an increase in miR-128 and miR-618 levels. These miRNA genes are embedded in coding genes introns. Preliminary data seems to suggest that their transcription can be independent from their host gene. We are thus identifying their promoter regions using a luciferase assay in order to understand if PHOX2B directly regulates miRNAs at transcription level and if this mechanism could be altered in CCHS with PHOX2B polyalanine expansions.