RT Journal Article
SR Electronic
T1 Cell signaling pathways in human mutant PAX6 corneal cells: an in vitro model for aniridia-related keratopathy
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.03.19.436143
DO 10.1101/2021.03.19.436143
A1 Marta Słoniecka
A1 André Vicente
A1 Berit Byström
A1 Fátima Pedrosa Domellöf
YR 2021
UL http://biorxiv.org/content/early/2021/03/19/2021.03.19.436143.abstract
AB PURPOSE To establish an in vitro model of aniridia-related keratopathy (ARK) using CRISPR/Cas9 engineered human keratocytes with mutations in the PAX6 gene, and to study the Notch Homolog 1, Translocation-Associated (Notch1), sonic hedgehog (SHH), mammalian target of rapamycin (mTOR), and Wnt/β-catenin signaling pathways in the PAX6 mutant keratocytes.METHODS Primary human keratocytes were isolated from healthy corneas. Keratocytes were transduced with Cas9 lentiviral particles in order to create cells stably expressing Cas9 nuclease. Lentiviral particles carrying PAX6 sgRNA were transduced into the Cas9 keratocytes creating mutants. Analysis of signaling pathways was assessed by RT-qPCR for gene expression and western blot for protein expression.RESULTS Human keratocytes stably expressing Cas9 nuclease were created. Keratocytes carrying PAX6 gene mutation were successfully generated. PAX6 mutant keratocytes showed modified expression patterns of extracellular matrix components such as collagens and fibrotic markers. Analysis of the Notch1, SHH, mTOR, and Wnt/β-catenin signaling pathways in the PAX6 mutant keratocytes revealed altered gene and protein expression of the key players involved in these pathways.CONCLUSIONS A properly functioning PAX6 gene in keratocytes is crucial for the regulation of signaling pathways important for cell fate determination, proliferation, and inflammation. Pax6 mutation in the in vitro settings leads to changes in these pathways which resemble those found in corneas of patients with ARK.Competing Interest StatementThe authors have declared no competing interest.