Rationally Induced RNA:DNA G-Quadruplex Structures Elicit an Anticancer Effect by Inhibiting Endogenous eIF-4E Expression
Rationally Induced RNA:DNA G-Quadruplex Structures Elicit an Anticancer Effect by Inhibiting Endogenous eIF-4E Expression
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DOI：
10.1021/bi5008904
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发表时间：
2014-08-26
期刊：
影响因子：
2.9
通讯作者：
Basu, Soumitra
中科院分区：
文献类型：
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作者：
Bhattacharyya, Debmalya;Nguyen, Kim;Basu, Soumitra
RNA g -四重体（GQ）结构作为多种细胞过程的调节剂，包括翻译、mRNA前加工和mRNA靶向。我们在此报告一种利用RNA基因自然能力的策略。通过在目标mRNA上合理诱导GQ敲低内源基因表达来抑制翻译。我们之所以选择eIF-4E作为靶点，是因为它在多种癌症的翻译起始和过表达中起着关键作用，并期望下调eIF-4E可导致癌细胞的抗增殖。通过合理设计和部分修饰外源DNA序列，在eIF-4E mRNA的5′-非翻译区（UTR）和蛋白质编码区诱导靶向杂交（RNA:DNA） GQ结构，并测定其对eIF-4E表达的影响。通过生物物理和生化蛋白表达方法建立了稳定的诱导g -四重体的形成。从CD熔化计算的热力学参数表明，在生理上相关的盐浓度下，形成了稳定的诱导GQ。我们通过监测一个报告基因的靶向抑制，建立了诱导GQ形成的特异性和有效性。最重要的是，我们已经证明，在人类癌细胞中，在5'-UTR和eIF-4E mRNA的蛋白质编码区诱导GQ，分别可以抑制30%和60%的内源性蛋白表达。用GQ诱导寡核苷酸序列处理导致人类癌细胞活力呈剂量依赖性降低。上述概念为内源基因表达的靶向调控开辟了一种新的策略。
RNA G-quadruplex (GQ) structures act as regulators of a diverse array of cellular processes including translation, pre-mRNA processing, and mRNA targeting. We report here a strategy of harnessing the natural ability of RNA GQs. to inhibit translation by rationally inducing a GQ on a targeted mRNA to knockdown endogenous gene expression. We chose to target eIF-4E because of its key role in translation initiation and overexpression in multiple cancers and with the expectation that downregulation of eIF-4E would result in antiproliferation of cancer cells. Targeted hybrid (RNA:DNA) GQ structures were induced at the 5'-untranslated region (UTR) and the protein coding region of the eIF-4E mRNA by rationally designed and partially modified extraneous DNA sequences and their effect on eIF-4E expression was determined. The formation of a stable induced G-quadruplex was established by biophysical and biochemical Protein Expression methods. Thermodynamic parameters calculated from CD melting indicate formation of a stable induced GQ at a physiologically relevant salt concentration. We established the specificity and efficacy of the induced GQ formation by monitoring the targeted repression of a reporter gene. Most importantly we have demonstrated that inducing GQ in the 5'-UTR and the protein coding region of eIF-4E mRNA in human cancer cells results in 30% and 60% inhibition of the endogenous protein expression, respectively. Treating with the GQ inducing oligonucleotide sequences resulted in a decrease in the viability of human cancer cells in a dose-dependent manner. The above concept opens up a new strategy for targeted modulation of endogenous gene expression.