Endocrine Abstracts

Introduction: Although current medical therapies for acromegaly are highly effective, 30% of patients are left with inadequately controlled growth hormone (GH) levels. To investigate a novel therapeutic strategy we have used RNA interference (RNAi) to target GH coding and promoter sequences in vitro and assessed the effect on gene expression and DNA methylation.

Method: A careful bioinformatics approach revealed only one area in the coding sequence that could be targeted. We also targeted areas of the promoter of rat GH with 3 custom designed short interfering RNAs (siRNAs) in cultured rat pituitary cells (GH3). Specifically, homology to the transcription factor binding sites was established. Transfection efficiency was established with scrambled fluorescent siRNAs using fluorescent activated cell sorting and confocal microscopy. Cells were incubated with the pooled siRNA complexes and cultured for 48 and 96 h. The histone deacetylase inhibitor Trichostatin A and the methyltransferase inhibitor 5-azacytidine were utilised to assess whether promoter-targeted siRNAs caused silencing by induction of DNA methylation and histone deacetylation. Real Time qPCR was used to measure the relative GH mRNA expression. Media GH levels were analysed using an ELISA.

Results: Transfection efficiency was measured at >90% on both FACS analysis and microscopy. siRNA to the exonic sequence was not effective. In contrast the pooled siRNA sequences demonstrated a 60% reduction in GH mRNA at 96 h.

GH levels were reduced by 50% relative to controls at 96 h.

Treatment with TSA for 48 h did not result in detectable rescue of GH mRNA levels however there was a 27% rescue of observed knockdown and 25% rescue of protein levels with AZA.

Conclusions: RNAi can be used to silence GH. This may have clinical significance in the treatment of Acromegaly. Knockdown observed may be attributed to de novo promoter methylation, but further investigation is required.