小干扰RNA对角膜上皮素和肌纤蛋白的抑制作用（美国眼科学会论文）
Suppression of keratoepithelin and myocilin by small interfering RNA (an American Ophthalmological Society thesis).
作者信息
Huang Andrew J W
机构信息
Department of Ophthalmology, University of Minnesota, Minneapolis, USA.
出版信息
Trans Am Ophthalmol Soc. 2007;105:365-78.
PURPOSE
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.
目的
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有关。在本研究中，研究了使用小干扰RNA（siRNA）抑制角蛋白和肌纤蛋白表达的可行性，以及它们降低突变型肌纤蛋白引起的相关细胞毒性作用的能力。
方法
通过聚合酶链反应扩增人KE基因和肌纤蛋白基因的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以及各自的产生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的抑制作用。使用UN-SCAN-IT软件对蛋白质印迹数字化蛋白条带上的肌纤蛋白特异性shRNA对MYOC-EGFP的抑制作用进行定量。使用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细胞的应激反应。
结果
鉴定出两种可有效抑制HEK293细胞中KE-EGFP表达的KE特异性shRNA。一种shRNA（靶向KE 1528bp处开始的编码序列）使KE-EGFP的表达降低了约50%，而另一种shRNA（靶向KE的3'-UTR区域）抑制了超过80%的表达。将MYOCpEGFP与靶向肌纤蛋白不同区域（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有效降低，平均降低幅度为78%至90%。当通过肌纤蛋白特异性shRNA抑制突变型肌纤蛋白时，转化的TM细胞中BiP基因的激活（作为突变型肌纤蛋白诱导的应激反应）显著降低。
结论
KE或肌纤蛋白特异性shRNA可有效抑制重组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。
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