Abstract
Purpose :
An expanded CTG trinucleotide repeat (TNR) in the TCF4 gene is the most common genetic association with FECD. A proposed mechanism for development of FECD in corneal endothelial cells (CEC) is RNA toxicity, characterized by expansion of the TCF4 TNR region, formation of nuclear RNA foci, dysregulation of muscleblind-like splicing regulators (e.g., MBNL1), and mis-spliced mRNA transcripts. Metformin has been reported to upregulate MBNL1 expression. Therefore, we examined the effect of metformin treatment on RNA foci and MBNL1 mis-splicing in primary CEC lines established from FECD patients.
Methods :
Confluent matured control and FECD primary CEC lines were treated with 5-50 mM metformin. At various timepoints, cells were fixed in paraformaldehyde, incubated with a labeled CAG probe, imaged, and RNA foci were quantified using ZEN software (Zeiss). With similarly treated CEC lines and timepoints, total RNA was isolated, and cDNA generated. Expression and alternative splicing patterns of MBNL1 were evaluated by PCR.
Results :
Treatment with metformin for 48 h (n=3) showed no reduction in RNA foci at 5 mM (p=0.78), 14% reduction with 20 mM (p<0.01), and 24% reduction with 50 mM (p=0.09). Treatment for 7 days (n=2) with 20 mM showed 59% RNA foci reduction compared to untreated cells (p<0.01). Following cessation of treatment for 7 days, the number of RNA foci returned to baseline levels (p=0.62). MBNL1 expression increased 2.2 ± 0.5, 2.7 ± 1.1, and 2.7 ± 1.4 fold at 24 h, 48 h, and 7 days of treatment with 20 mM metformin. Cessation of metformin treatment returned MBNL1 expression levels to baseline (1.0 ± 0.3 fold). Compared to control CEC lines, PCR splicing analysis of MBNL1 transcripts in FECD CEC lines showed an increase in exon 5 inclusion which increased further in a dose-dependent manner during metformin treatment. Splicing patterns were not altered in control CEC lines with metformin exposure.
Conclusions :
Metformin reversibly reduces the number of RNA foci in primary FECD CEC lines. This foci reduction coincides with an increase in MBNL1 RNA expression and changes in MBNL1 splicing patterns.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.