Suppression of keratoepithelin and myocilin by small interfering RNAs (siRNA) in vitro.
Suppression of keratoepithelin and myocilin by small interfering RNAs (siRNA) in vitro.
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发表时间：
2007-11
期刊：
影响因子：
2.2
通讯作者：
Ching Yuan;E. J. Zins;A. Clark;A. Huang
中科院分区：
文献类型：
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作者：
Ching Yuan;E. J. Zins;A. Clark;A. Huang
目的角化上皮蛋白(KE)和肌红蛋白(MYOC)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。我们研究了小干扰RNA(SiRNAs)在体外抑制KE和MYOC的表达以及突变的myocilins的相关细胞毒性的潜在用途。方法用聚合酶链式反应扩增人角化上皮蛋白(KE)基因和霉菌毒素(MYOC)基因的cDNAs，将其亚克隆到pEGFP-N1载体上，构建KEpEGFP和MYOCpEGFP融合蛋白。从RNA聚合酶III启动子驱动的载体(PH1-RNA)中扩增出短发夹状RNA(ShRNAs)。脂质体分别与KEpEGFP或MYOCpEGFP及各自的shRNA生成载体共转染HEK293细胞和小梁网(TM)细胞，以评价shRNAs的抑制效果。荧光显微镜和免疫印迹检测KE特异性shRNAs对KE-EGFP融合蛋白的抑制作用。用UN-Scan-IT软件对MYOC-EGFP融合蛋白的抑制用UN-Scan-IT软件进行定量。用Bip启动子驱动的荧光素酶报告基因检测错折叠突变体myocilins对TM细胞的应激反应。结果1个shRNA(靶向KE基因1,528个碱基的编码序列)可使HEK293细胞中KE-EGFP的表达降低约50%，而另一个shRNA(靶向KE基因3‘端非编码区)可抑制80%以上融合蛋白的表达。MYOCpEGFP与针对MYOC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)的不同shRNA生成载体共转染后，MYOC-EGFP融合蛋白的平均降幅为78%~90%。当突变的myoclin蛋白被myoclin特异性shRNAs抑制时，转化的TM细胞中Bip基因(一种由突变的myocilins诱导的细胞应激反应)的激活显著降低。结论KE特异性或MYOC特异性shRNAs在体外能有效抑制重组KE或myoclin蛋白的表达及突变型myocilins的细胞毒性。RNA干扰在抑制这些基因方面可能会有未来的治疗意义。
PURPOSE Mutations of keratoepithelin (KE) and myocilin (MYOC) have been linked to certain types of inherited corneal stromal dystrophy and open-angle glaucoma, respectively. 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.