June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Mitomycin C Suppresses Gene Splicing in Corneal Fibroblasts
Author Affiliations & Notes
  • Tsan-Chi Chen
    Department of Ophthalmology, Far Eastern Memorial Hospital, New Taipei City, Taiwan
  • Shu-Wen Chang
    Department of Ophthalmology, Far Eastern Memorial Hospital, New Taipei City, Taiwan
    Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan
  • Chih-Chieh Lee
    Department of Ophthalmology, Far Eastern Memorial Hospital, New Taipei City, Taiwan
  • Han-Fang Teng
    Department of Ophthalmology, Far Eastern Memorial Hospital, New Taipei City, Taiwan
  • Footnotes
    Commercial Relationships Tsan-Chi Chen, None; Shu-Wen Chang, None; Chih-Chieh Lee, None; Han-Fang Teng, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 5245. doi:https://doi.org/
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      Tsan-Chi Chen, Shu-Wen Chang, Chih-Chieh Lee, Han-Fang Teng; Mitomycin C Suppresses Gene Splicing in Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5245. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

To investigate the molecular mechanism of how mitomycin C (MMC) modulates the gene expression via accumulation of Fibulin-1 (FBLN1) in nucleus of human corneal fibroblasts (HCFs ).

 
Methods
 

HCFs were treated with MMC at 0.2 mg/ml for 5 minutes. FBLN1 and survival motor neuron 1 (SMN1) were cloned in the lentivirus-based expression vectors with green fluorescent protein (GFP) and red fluorescent protein (RFP), respectively, and expressed in HCFs by pseudovirus infection system. Distribution of FBLN1 and SMN1 was observed by confocal microscopy after immunofluorescent staining for their endogenous expression or overexpression of GFP-tagged FBLN1 and RFP-tagged SMN1. Expression and association of FBLN1 and SMN1 in cytosol and nucleus was assayed by co-immunoprecipitation and immunoblotting after nuclear extraction. Expression level of pre-mRNA and spliced mRNA was determined by real-time PCR.

 
Results
 

MMC reduced cytosolic FBLN1 expression, but enhanced nuclear FBLN1 expression in HCFs. The fluorescent images showed that FBLN1 accumulated in nucleus of MMC-treated HCFs and co-localizes with SMN1 (Figure 1). However, the co-immunoprecipitation results revealed that FBLN1 might indirectly bind to SMN1. Silence of FBLN1 in MMC-treated HCFs resulted in decreasing both accumulation in nuclear FBLN1 and the pre-mRNA level of focal adhesion kinase (FAK), but increasing the spliced mRNA level of FAK.

 
Conclusions
 

Our findings first showed that FBLN1 translocated into cell nucleus after MMC treatment. FBLN1 might play a novel role as a suppressor in SMN1-dependent pre-mRNA splicing.

 
 
Figure 1. Colocalization of FBLN1 and SMN1 in nucleus of MMC-treated HCFs. HCFs at 3 days post MMC treatment were coimmunostained with DAPI (blue) for cell nucleus and the specific antibodies for FBLN1 (red) and SMN1 (green), respectively.
 
Figure 1. Colocalization of FBLN1 and SMN1 in nucleus of MMC-treated HCFs. HCFs at 3 days post MMC treatment were coimmunostained with DAPI (blue) for cell nucleus and the specific antibodies for FBLN1 (red) and SMN1 (green), respectively.
 
Keywords: 480 cornea: basic science • 503 drug toxicity/drug effects • 533 gene/expression  
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