Abstract
Purpose :
This study aimed to provide a comprehensive role of highly expressed microRNAs in mouse lens development and maintenance. The highly expressed microRNA miR26 was selected as a potential microRNA with significant biological importance. The potential role of the miR26 family was teased out using CRISPRcas9-mediated knockout and the next-generation sequencing platform.
Methods :
RNA sequencing was performed using newborn FVB/N mouse lens epithelium and lens fiber cells. Highly expressed microRNAs from the lens epithelium and fiber cells were identified and selected for knockout. CRISPR/Cas9 genome editing facilitated the deletion of all three members of the miR26 family (TKO) via zygote microinjection. The deletion was confirmed using sanger sequencing. A phenotypic study was done on the TKO mice at different stages. Furthermore, five-day-old (P5) lenses from TKO mice were isolated and used for total RNA extraction, followed by bulk RNA sequencing. Differentially expressed genes (DEGs) in the TKO lenses (compared to FVB/N) were identified using DESeq2. These DEGs were subjected to pathway enrichment analysis.
Results :
Analysis of sequencing data for newborn lens fiber and lens epithelium fractions identified miR26 as an abundant microRNA expressed in the murine lens. Deletion of one or two members of the miR26 family did not result in any lens defect. However, the deletion of all three members of the miR26 family resulted in nuclear cataracts that initiated at 4-weeks of age., The severity of these cataracts progressively increased, ultimately resulting in the rupture of the lens by 20 weeks of age. Furthermore, RNAseq of P5 lenses identified a total of 511 DEGs (log2foldchange >1.5 and padjust <0.05). Overrepresented pathways identified amongst the DEGs included extracellular matrix organization (Col11a1, Col18a1, Mmp9, Ptx3, Ccn1), collagen chain trimerization ( Col2a1, Slc16a19, Atp1a2, Fgf22), positive regulation of cell migration (Bmp2, Bmp4, Fgf1, Ackr3), and regulation of transmembrane receptor protein serine/threonine kinase signaling pathway (Bmp15, Fbn1, Tgfbr3, Sulf2).
Conclusions :
This miRNA-Seq study provides a classical example of genetic redundancy within the gene family. Loss of all three copies of miR26 is required for disrupting the miR26 function in the murine lens. The role of miR26 involves the maintenance of lens structure and function, rather than embryonic development.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.