Suppression of keratoepithelin and myocilin by small interfering RNAs (siRNA) in vitro.

摘要: Purpose: Mutations of keratoepithelin (KE) and myocilin (MYOC) have been linked to certain types inherited corneal stromal dystrophy open-angle glaucoma, respectively. We investigated the potential use small interfering RNAs (siRNAs) suppress expression KE MYOC related cytotoxicity mutant myocilins in vitro. Methods: cDNAs human gene were amplified by polymerase chain reaction subcloned into pEGFP-N1 construct respective plasmids, KEpEGFP MYOCpEGFP, produce fluorescence-generating fusion proteins. Short hairpin (shRNAs) generated from an RNA III promoter-driven vector (pH1-RNA). Transformed HEK293 trabecular meshwork (TM) cells cotransfected via liposomes with either or MYOCpEGFP shRNA-generating plasmids evaluate suppression efficacy shRNAs. Suppression KE-EGFP protein KE-specific shRNAs was evaluated fluorescence microscopy western blotting. MYOC-EGFP myocilin-specific quantified UN-SCAN-IT software on digitized bands blots. The cellular stress response TM induced misfolded a BiP luciferase reporter assay. Results: One shRNA (targeting coding sequence starting at 1,528 bp KE) reduced approximately 50% whereas other 3'-UTR region suppressed more than 80% protein. Cotransfection various targeting different regions (containing amino acid residues R76, E352, K423, N480 associated glaucoma) showed effective reduction protein, ranged 78% 90% average. activation (a myocilins) transformed significantly when proteins Conclusions: MYOC-specific effectively recombinant interference may future therapeutic implications suppressing these genes.