Yuan Ching, Zins Emily J, Clark Abbott F, Huang Andrew J W
Department of Ophthalmology, University of Minnesota, Minneapolis, MN, USA.
Mol Vis. 2007 Nov 7;13:2083-95.
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.
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.
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.
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.
角蛋白（KE）和肌纤蛋白（MYOC）的突变分别与某些类型的遗传性角膜基质营养不良和开角型青光眼相关。我们研究了小干扰RNA（siRNA）在体外抑制KE和MYOC表达以及突变型肌纤蛋白相关细胞毒性的潜在用途。
通过聚合酶链反应扩增人角蛋白（KE）基因和肌纤蛋白（MYOC）基因的cDNA，并亚克隆到pEGFP-N1中构建各自的质粒KEpEGFP和MYOCpEGFP，以产生产生荧光的融合蛋白。短发夹RNA（shRNA）由RNA聚合酶III启动子驱动的载体（pH1-RNA）产生。将转化的HEK293细胞和小梁网（TM）细胞通过脂质体与KEpEGFP或MYOCpEGFP以及各自产生shRNA的质粒共转染，以评估shRNA的抑制效果。通过荧光显微镜和蛋白质印迹法评估KE特异性shRNA对KE-EGFP融合蛋白的抑制作用。用UN-SCAN-IT软件对蛋白质印迹数字化蛋白条带进行定量，以评估肌纤蛋白特异性shRNA对MYOC-EGFP融合蛋白的抑制作用。用BiP启动子驱动的荧光素酶报告基因测定法评估错误折叠的突变型肌纤蛋白诱导的TM细胞的细胞应激反应。
一种shRNA（靶向KE编码序列起始于1528bp处）使HEK293细胞中KE-EGFP的表达降低了约50%，而另一种shRNA（靶向KE的3'-UTR区域）抑制了融合蛋白表达的80%以上。将MYOCpEGFP与靶向MYOC不同区域（包含与遗传性青光眼相关的氨基酸残基R76、E352、K423或N480）的各种产生shRNA的质粒共转染，结果显示MYOC-EGFP融合蛋白有效减少，平均减少幅度为78%至90%。当突变型肌纤蛋白特异性shRNA抑制突变型肌纤蛋白时，转化的TM细胞中BiP基因的激活（由突变型肌纤蛋白诱导的细胞应激反应）显著降低。
KE特异性或MYOC特异性shRNA在体外有效抑制重组KE或肌纤蛋白的表达以及突变型肌纤蛋白的相关细胞毒性。RNA干扰在抑制这些基因方面可能具有未来的治疗意义。
