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