May 2003
Volume 44, Issue 13
ARVO Annual Meeting Abstract  |   May 2003
Suppressing the Expression of Keratoepithelin (BIGH3) in Cultured Cells by Small Interfering RNAs (siRNA)
Author Affiliations & Notes
  • C. Yuan
    Ophthalmology, Univ Minnesota, Minneapolis, MN, United States
  • A.J. Huang
    Ophthalmology, Univ Minnesota, Minneapolis, MN, United States
  • Footnotes
    Commercial Relationships  C. Yuan, None; A.J.W. Huang, None.
  • Footnotes
    Support  Minnesota Medical Foundation Research Grant 3180-9927-02
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 3850. doi:
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      C. Yuan, A.J. Huang; Suppressing the Expression of Keratoepithelin (BIGH3) in Cultured Cells by Small Interfering RNAs (siRNA) . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3850.

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

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Abstract: : Purpose: Keratoepithelin (KE) is the protein encoded by a recently identified gene, BIGH3. Mutations of the BIGH3 gene have been linked to several types of inherited corneal dystrophies in human. Current therapies are limited for corneal deposits or opacities and painful recurrent corneal erosions associated with these dystrophies. We hypothesize that the amyloid or non-amyloid corneal deposits are caused by the accumulation of mutant KEs. Therefore, prevention of these untoward deposits may alleviate the clinical morbidity. We investigated the feasibility of using RNA interference to suppress the expression of mutant KEs as a potential therapy to reduce the abnormal firbril/amyloid aggregations of KE-related dystrophies. Methods: siFinder (, a web-based program developed in our laboratory was used to design siRNAs. Oligonucleotides were synthesized and inserted into RNA polymerase III promoter-driven plasmids to generate 21-nt hairpin siRNAs targeting at various parts of KE coding and 3’-UTR regions. The resulting plasmids that generated KE-specific siRNAs were co-transfected with KEpEGFP into 293 cells via liposome. The suppression efficiency by these siRNAs was evaluated with fluorescence microscopy (by GFP signal), Western blots (by custom-made α-KE antibody) and Northern hybridization techniques. Results: We have identified at least two siRNAs specifically suppressing the expression of KE in cultured cells, of which one targeted at the coding region and the other one targeted at the 3’-UTR of KE mRNA. By fluorescence microscopy, significant reduction of the KE-EGFP fusion protein was observed. Quantification by Western and Northern blots showed the expression of KE-EGFP fusion protein and KE mRNA was reduced in parallel to a level of 50% in cultured cells. Conclusions: We have demonstrated that the siRNA-mediated RNA interference method could be used successfully to suppress the expression of KE in cultured cells. This may provide a novel therapeutic approach to ameliorate KE-related corneal dystrophies.

Keywords: cornea: epithelium • gene/expression • gene transfer/gene therapy 

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