Suppression of keratoepithelin and myocilin by small interfering RNAs (siRNA) in vitro.
OBJECTIVE
Mutations of keratoepithelin (KE) and myocilin (MYOC) have been linked to certain types of inherited corneal stromal dystrophy and open-angle glaucoma, respectively. We investigated the potential use of small interfering RNAs (siRNAs) to suppress the expression of KE and MYOC and the related cytotoxicity of mutant myocilins in vitro.


METHODS
cDNAs of the human keratoepithelin (KE) gene and myocilin (MYOC) 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 fusion protein by KE-specific shRNAs was evaluated by fluorescence microscopy and western blotting. Suppression of MYOC-EGFP fusion protein by myocilin-specific shRNAs was quantified with UN-SCAN-IT software on digitized protein bands of western blots. The cellular stress response of TM cells induced by misfolded mutant myocilins was evaluated with a BiP promoter-driven luciferase reporter assay.


RESULTS
One shRNA (targeting the coding sequence starting at 1,528 bp of KE) reduced the expression of KE-EGFP in HEK293 cells approximately by 50% whereas the other shRNA (targeting the 3'-UTR region of KE) suppressed more than 80% of the expression of fusion protein. Cotransfection of MYOCpEGFP and various shRNA-generating plasmids targeting different regions of MYOC (containing amino acid residues R76, E352, K423, or N480 associated with inherited glaucoma) showed effective reduction of MYOC-EGFP fusion protein, ranged from 78% to 90% on average. The activation of the BiP gene (a cellular 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-specific or MYOC-specific shRNAs effectively suppressed the expression of recombinant KE or myocilin proteins and the related cytotoxicity of mutant myocilins in vitro. RNA interference may have future therapeutic implications in suppressing these genes.
Yuan
Ching
C
Department of Ophthalmology, University of Minnesota, Minneapolis, MN, USA.
Zins
Emily J
EJ
Clark
Abbott F
AF
Huang
Andrew J W
AJ
D000818:Animals; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D016326:Extracellular Matrix Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D005136:Eye Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D005240:Feasibility Studies; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D006023:Glycoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics / Q000502:physiology; D006801:Humans; D008156:Luciferases / Q000235:genetics; D018832:Molecular Chaperones / Q000235:genetics / Q000502:physiology; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000494:pharmacology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000235:genetics*; D000099222:betaIG-H3 Protein; D000099179:Myocilin; D000091342:Endoplasmic Reticulum Chaperone BiP
D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D005456:Fluorescent Dyes; D006023:Glycoproteins; D049452:Green Fluorescent Proteins; D006360:Heat-Shock Proteins; D008156:Luciferases; D018832:Molecular Chaperones; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein; D000099179:Myocilin; C051684:GRP78 protein, rat; D000091342:Endoplasmic Reticulum Chaperone BiP; C485184:enhanced green fluorescent protein