Abstract
Purpose :
To explore the effect of mitomycin C (MMC)-caused nuclear fibulin-1 (FBLN1) accumulation on gene expression in human corneal stromal cells (HCFs).
Methods :
HCFs were treated with 0.2 mg/ml MMC for 5 minutes and then cultivated in DMEM with 10% fetal bovine serum for 1 day. Original FBLN gene, directly isolated from primary HCFs, was cloned into a lentivirus-based pseudovirus plasmid with an enhanced green fluorescent protein (EGFP) fusion and expressed in HCFs as the FBLN1-overexpressed cells. FBLN1 distribution was determined by confocal microscope and cell nuclear/cytoplasmic fractionation. Gene expressions in transcription levels were detected by Agilent human microarray (50,599 genes). Gene changes by more than 2-fold increase or decrease in HCFs with MMC treatment and/or FBLN1 overexpression were classified into four different groups (Figure 1) and analyzed by gene ontology (GO) analysis in biological processes, cellular components, and molecular functions.
Results :
Of the selected 1,652 genes in MMC-treated HCFs, 898 genes (54.4%) were up-regulated and 754 genes (45.7%) were down-regulated. In contrast, there were 2,595 genes by more than 2-fold changes in FBLN1-overexpressed HCFs after MMC treatment, containing 1,109 up-regulated genes (42.7%) and 1,486 down-regulated genes (57.3%). However, FBLN1 overexpression only modulated 1,160 genes by more than 2-fold changes. Of them, 972 genes (83.8%) were up-regulated and only 188 genes (16.2%) were down-regulated. Furthermore, compared to mock cells (only 754 down-regulated genes, 45.7%), MMC significantly increased the ratio of down-regulated genes in FBLN1-overexpressed HCFs (1,063 of the 1,309 genes more than 2-fold change, 81.2%). The amount of nuclear FBLN1 significantly increased in FBLN1-overexpressed HCFs after MMC treatment. The GO analysis also indicated that FBLN1 overexpression played a critical role in gene expression process.
Conclusions :
High FBLN1 expression enriched the MMC-triggering nuclear translocation of FBLN1 to suppress the gene expressions in human corneal stromal cells after ocular surgery with topical MMC application.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.