Suppression of keratoepithelin and myocilin by small interfering RNA (an American Ophthalmological Society thesis).
Suppression of keratoepithelin and myocilin by small interfering RNA (an American Ophthalmological Society thesis).
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发表时间：
2007
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通讯作者：
A. Huang
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作者：
A. Huang
角膜上皮素（keratoepithelin，KE）和肌球蛋白（myocilin，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。在这项研究中，使用小干扰RNA（siRNA）来抑制角膜上皮素和myocilin的表达和它们的能力，以减少突变myocilins引起的相关细胞毒性作用的可行性进行了研究。方法用PCR方法扩增人KE基因和myocilin基因的cDNA，亚克隆到pEGFP-N1质粒中，分别构建KepEGFP和MYOCpEGFP表达质粒。从RNA聚合酶III启动子驱动的载体（pH 1-RNA）产生短发夹RNA（shRNA）。通过脂质体将转化的HEK 293和小梁网（TM）细胞与KEpEGFP或MYOCpEGFP和各自的产生shRNA的质粒共转染，以评价shRNA的抑制功效。通过荧光显微镜和蛋白质印迹法评价KE特异性shRNA对KE-EGFP的抑制作用。用UN-SCAN-IT软件在Western印迹的数字化蛋白条带上定量肌球蛋白特异性shRNA对MYOC-EGFP的抑制。BiP启动子驱动的荧光素酶报告基因测定用于评估错误折叠的突变型肌青霉素诱导的TM细胞的应激反应。结果在HEK 293细胞中筛选到两个能有效抑制KE-EGFP表达的KE-shRNA。一种shRNA（靶向KE的1528 bp编码序列）使KE-EGFP的表达降低约50%，而另一种shRNA（靶向KE的3 '-UTR区域）抑制超过80%的表达。MYOCpEGFP和靶向肌球蛋白不同区域（含有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的质粒的共转染显示MYOC-EGFP的有效减少，平均范围为78%至90%。在转化的TM细胞中BiP基因的激活（作为由突变型肌细胞素诱导的应激反应）在突变型肌细胞素蛋白被肌细胞素特异性shRNA抑制时显著降低。结论KE或myocilin特异性shRNAs能有效抑制重组KE或myocilin蛋白的表达及突变myocilin的细胞毒性。RNA干扰可能在抑制这些基因方面具有未来的治疗意义。
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.