Suppression of keratoepithelin and myocilin by small interfering RNA (an American Ophthalmological Society thesis).
OBJECTIVE
Mutations of keratoepithelin (KE) and myocilin (MYOC) have been linked to certain types of inherited corneal stromal dystrophy and open-angle glaucoma, respectively. In this study, the feasibility of using small interfering RNAs (siRNAs) to suppress the expression of keratoepithelin and myocilin and their capabilities to reduce the related cytotoxic effects caused by mutant myocilins were investigated.


METHODS
cDNAs of human KE gene and myocilin gene were amplified by polymerase chain reaction and subcloned into pEGFP-N1 to construct respective plasmids, KEpEGFP and MYOCpEGFP, to produce fluorescence-generating fusion proteins. Short hairpin RNAs (shRNAs) were generated from an RNA polymerase III promoter-driven vector (pH1-RNA). Transformed HEK293 and trabecular meshwork (TM) cells were cotransfected via liposomes with either KEpEGFP or MYOCpEGFP and respective shRNA-generating plasmids to evaluate the suppression efficacy of shRNAs. Suppression of KE-EGFP by KE-specific shRNAs was evaluated by fluorescence microscopy and Western blotting. Suppression of MYOC-EGFP by myocilin-specific shRNAs was quantified with UN-SCAN-IT software on digitized protein bands of Western blots. A BiP promoter-driven luciferase reporter assay was used to evaluate the stress response of TM cells induced by misfolded mutant myocilins.


RESULTS
Two KE-specific shRNAs that effectively suppressed the expression of KE-EGFP in HEK293 cells were identified. One shRNA (targeting the coding sequence starting at 1528bp of KE) reduced the expression of KE-EGFP approximately by 50%, whereas the other shRNA (targeting the 3'-UTR region of KE) suppressed greater than 80% of the expression. Cotransfection of MYOCpEGFP and various shRNA-generating plasmids targeting different regions of myocilin (containing amino acid residues R76, E352, K423, or N480 associated with inherited glaucoma) showed effective reduction of MYOC-EGFP, ranging from 78% to 90% on average. The activation of BiP gene (as a stress response induced by mutant myocilins) in transformed TM cells was significantly reduced when mutant myocilin proteins were suppressed by myocilin-specific shRNAs.


CONCLUSIONS
KE- or myocilin-specific shRNAs could effectively suppress the expression of recombinant KE or myocilin proteins and the related cytotoxicity of mutant myocilins. RNA interference may have future therapeutic implications in suppressing these genes.
Huang
Andrew J W
AJ
Department of Ophthalmology, University of Minnesota, Minneapolis, USA.
D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000172:deficiency* / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000172:deficiency* / Q000235:genetics*; D005136:Eye Proteins / Q000235:genetics*; D005784:Gene Amplification; D017930:Genes, Reporter; D006023:Glycoproteins / Q000172:deficiency* / Q000235:genetics*; D006801:Humans; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D016212:Transforming Growth Factor beta / Q000172:deficiency* / Q000235:genetics*; D000099222:betaIG-H3 Protein; D000099179:Myocilin
D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein; D000099179:Myocilin