Abstract
Purpose :
B lymphocyte-induced maturation protein 1 (BLIMP-1) is a transcription factor and, primarily, a key regulator of immune cells. Beyond the immune system, BLIMP-1 regulates many other pathways through its repression of c-Myc, including developmental pathways, as shown by BLIMP-1-/- embryonic lethality. BLIMP-1 regulation has also been heavily implicated in skin epithelial homeostasis and significant epithelial disruption is observed in the absence of BLIMP-1. It has been observed in presumptive corneal epithelial stem cells and more recently, in a Drosophila model, BLIMP-1 ablation leads to significant corneal lens defects. However, its mechanistic role in corneal epithelial maintenance is virtually unknown. To this end, we hypothesized that BLIMP-1 plays a role in human corneal epithelial homeostasis and differentiation, and tested the validity (or feasibility) of using a human corneal epithelial cell line for this purpose.
Methods :
We confirmed expression of BLIMP-1 in an immortalized human corneal epithelial cell line (hTCEpi). We used siRNA to knockdown its expression and sought to investigate the downstream effects of BLIMP-1 on different signalling cascades and corneal epithelial markers. After siRNA knockdown, cells were analysed using microscopy, scratch migration assay and immunohistochemistry. Bulk-RNASeq will be used to identify downstream targets of BLIMP-1 in corneal epithelial cells.
Results :
Initial results revealed that BLIMP-1 is expressed in hTCEpi cells, as shown by immunohistochemistry, RT-qPCR and Western blot. BLIMP-1 knockdown (87.2 ± 5.2% knockdown efficiency, n = 2), does not significantly alter cell viability or the epithelial phenotype as observed by microscopy, however epithelial cells display slightly reduced migration and proliferation as witnessed by scratch assay (5% increase in wound area 24 hours post-scratch in knocked down cells). Bulk RNASeq is currently underway to reveal any disruption of gene expression and signaling cascades that may have significant impacts in vivo.
Conclusions :
Here we show that BLIMP-1 is expressed robustly in the hTCEpi cell line. We achieved almost 90% knockdown of BLIMP1 expression in hTCEpi without significant impact on cell survival. BLIMP-1 may regulate epithelial cell migration as its knockdown delayed migration in scratch assays. Further gene expression analysis by bulk RNASeq may reveal further downstream effects of BLIMP-1 disruption.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.