Cisplatin and siRNA interference with structure and function of Wnt-5a mRNA:: design and in vitro evaluation of targeting AU-rich elements in the 3′UTR
Cisplatin and siRNA interference with structure and function of Wnt-5a mRNA:: design and in vitro evaluation of targeting AU-rich elements in the 3′UTR
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DOI：
10.1007/s00775-007-0327-6
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发表时间：
2008-03-01
影响因子：
3
通讯作者：
Elmroth, Sofi K. C.
中科院分区：
文献类型：
--
作者：
Haegerloef, Margareta;Papsai, Pal;Elmroth, Sofi K. C.
Wnt-5a 是一种分泌性糖蛋白，已被证明参与细胞粘附和运动的调节，这些过程对于癌细胞转移形成非常重要。我们在此提出一项初步研究，旨在评估小干扰 RNA (siRNA) 与顺铂组合是否可用于在体外条件下调节蛋白质表达水平。为此，选择对应于 Wnt-5a 3' 非翻译区的初始 260 个碱基的富含 AU 的区域作为靶标。通过监测瞬时表达的荧光素酶的活性，分析兔网织红细胞裂解物 (RRL) 和永生化非癌性乳腺上皮 (HB2) 细胞系中的蛋白质抑制水平，评估四种不同 siRNA 的效果。通过消化技术和聚丙烯酰胺凝胶电泳分析来跟踪 siRNA 与信使 RNA 靶标杂交的特异性和动力学。观察到特异性和温度依赖性杂交，4℃下半衰期约为 0.5 小时。对于 RRL 和 HB2，荧光素酶活性分别在微摩尔和纳摩尔范围内显着下调。此外，通过以选择性方式添加siRNA可以进一步增强由添加顺铂引起的蛋白质产生的下调。后一观察结果表明，联合使用顺铂和 siRNA 可能是降低治疗用顺铂浓度的一种方法。因此，可以最大限度地减少毒副作用，同时以高度特异性的方式靶向关键蛋白质。
Wnt-5a is a secreted glycoprotein which has been shown to be involved in the regulation of cell adhesion and motility, processes which are of importance in metastasis formation by cancer cells. We here present an initial study aiming at evaluating whether small interfering RNA (siRNA) in combination with cisplatin can be used to modulate protein expression levels under in vitro conditions. For this purpose, an AU-rich region corresponding to the initial 260 bases of the Wnt-5a 3' untranslated region was chosen as the target. The effect of four different siRNAs was evaluated by analysis of protein suppression levels in rabbit reticulocyte lysate (RRL) and an immortalized noncancerous mammary epithelial (HB2) cell line by monitoring the activity of transiently expressed luciferase. The specificity and kinetics for hybridization of the siRNA with the messenger RNA target were followed by digestion techniques and analysis by polyacrylamide gel electrophoresis. Specific and temperature-dependent hybridization was observed, with a half-life of approximately 0.5 h at 4 degrees C. Significant downregulation of luciferase activity was obtained in the micromolar and nanomolar range, for RRL and HB2, respectively. In addition, the downregulation of protein production caused by addition of cisplatin could be further potentiated by addition of siRNA in a selective manner. The latter observation suggests that combined use of cisplatin and siRNA could be a method to decrease therapeutically used cisplatin concentrations. Thus, toxic side effects could be minimized while key proteins are targeted in a highly specific manner.