Endocrine Abstracts

Background/aims: The transcription factor neurogenin 3 (NGN3) is required for pancreatic islet cell specification from multipotent progenitor cells; yet we have limited understanding of the downstream genetic program it directly initiates. A 3 Mb centromeric region on chromosome 20p contains a number of genes activated downstream of NGN3 including INSM1 and NKX2.2. We proposed that NGN3 alters the expression of other genes within this region as potential downstream targets.

Methods: In-silico analysis of the 3 Mb genomic region containing INSM1 and NKX2.2 was carried out to identify potential target genes and search for putative NGN3 binding elements (E-boxes). An engineered human pancreatic ductal cell line, PANC1, with inducible NGN3 expression (Piper Hanley et al. J Endo, 2010) was used to up-regulate NGN3 following which quantitative RT-PCR (qRT-PCR) analysed gene expression.

Results: NGN3 was induced in the PANC1 cell line by infection with recombinant adenoviral vectors expressing the human NGN3 following which eight evolutionary conserved genes were identified by qRT-PCR to be significantly altered in expression. AF147354, MIR3192, LINC00261, and THBD were down-regulated by at least 0.27-fold by NGN3 whereas INSM1, RIN2, C20orf74, and PLK1S1 were up-regulated by at least 0.35-fold, making them all potential NGN3 targets. Evolutionary conserved elements consistent with putative E-boxes were identified upstream of AF147354, LINC00261, INSM1, RIN2, C20orf74, and PLK1S1.

Discussion: The thrombomodulin gene (THBD) and polo-like kinase 1 substrate 1 gene (PLK1S1) were the two genes most significantly affected by NGN3 induction, with a 1.2- and 0.5-fold change respectively. PLK1S1 up-regulation may be important in stabilising and strengthening the expanded pericentriolar region of replicated chromosomes; whilst THBD down-regulation may enhance cell migration, an important aspect of islet formation during human pancreas development. In summary, both PLK1S1 and THBD could be novel targets of NGN3 and may play a role in islet cell development.