Abstract
Purpose :
Keratoconus (KC) is the most common corneal ectasia and a major cause of corneal transplantation worldwide. Recent studies have suggested the contribution of two missense mutations (N843S and S419A) in the phosphatase domain of PPIP5K2 to the etiology of KC. PPIP5K2 is a cell-signaling kinase/phosphatase highly expressed in human and mouse cornea tissues. We aimed to identify the potential target genes of PPIP5K2 using human cancer cell line HCT116, primary human corneal epithelial cells (HCEC), and human corneal stromal fibroblast (HCSF).
Methods :
First, differentially expressed (DE) genes were identified using Total RNA-Seq in HCT116 cells with stable PPIP5K2-knockdown (n=8). Data were analyzed by Ingenuity Pathway Analysis and WebGestalt’s gene ontology analysis. Second, we knocked down PPIP5K2 expression in HCEC (n=3) and HCSF (n=3) for 24 hours using Dicer-Substrate Short Interfering RNAs (DsiRNA) via electroporation. Third, we validated changes in expression of six DE genes (MGMT, NR2F1, BMP4, HIST1H2AI, CDK6 and FUT8) in HCEC and HCSF using droplet digital PCR (ddPCR).
Results :
We identified 81 genes with DE in HCT116 cells (|fold change| > 1.5, p<0.05). Pathway analyses implicated the enrichment of genes involved in protein degradation/synthesis, connective tissue disorders, cellular assembly and organization. The pathways affected include TGFβ-signaling and LPS/IL-1 mediated inhibition of RXR function, which have been suggested to be involved in KC pathogenesis. We successfully decreased the expression of PPIP5K2 in human HCEC and HCSF cells for more than 80%. Following the successful knockdown of PPIP5K2 expression in HCEC and HCSF, our ddPCR data confirmed that CDK6 and HIST1H2AI were downregulated in HCEC whereas MGMT, NR2F1 and FUT8 were upregulated in HCSF, similar to those discovered in HCT116 cells.
Conclusions :
We identified several target genes of PPIP5K2 in HCT116 cells and validated five target genes in primary HCEC and HCSF cells. In addition, we will perform Total RNA-Seq in PPIP5K2-knockdown corneal cells to identify DE genes and their pathways which may be more specific to corneal cells and contributing to KC.
This is a 2021 ARVO Annual Meeting abstract.