靶向bcl-2基因不对称siRNA的干扰效果及其脱靶效应研究
摘要
自从RNA干扰(RNAi)现象发现以来,小干扰RNA (Small interfering RNA, siRNA)就被广泛应用于科研领域,成为一个研究热点。siRNA在RNAi过程中以一种序列特异性的方式高效沉默靶基因的表达。通常研究中所采用的化学合成siRNA一般由19-21个碱基对的长度相等的两条链组成,且每条链的3’-末端带有2个核苷酸的凸出。这种传统结构的siRNA有引起脱靶效应的报道,给其进一步的临床应用带来了新的研究课题。近来有研究表明,两条链长度不相等的不对称siRNA (asymmetric siRNA, asiRNA)同样能有效沉默靶基因的表达,并且asiRNA还能显著减少脱靶效应的产生。我们在研究中针对bcl-2基因设计了一系列双链配对区长度为13-17个碱基对的asiRNA,研究了其体外干扰效果及脱靶效应。结果表明,双链区长度为17个碱基对的asiRNA (17bp asiRNA)能够有效沉默bcl-2基因的表达,并能降低肿瘤细胞的活性。进一步的研究表明,与传统结构的siRNA相比,17bp asiRNA能有效减少脱靶效应的产生。另外靶向bcl-2基因的17bp asiRNA还能有效逆转肿瘤细胞的多药耐药性。
化学合成siRNA的不稳定性和缺乏有效的递送系统是其体内系统应用所面临的两大主要问题。化学修饰是提高siRNA稳定性的一条有效途径,许多研究报道经过化学修饰的siRNA稳定性有了显著提高。为了提高17bpasiRNA的稳定性,我们也对其进行了一系列的化学修饰,包括硫代磷酸修饰,2’-O甲基修饰、2’-氟代修饰和末端胆固醇连接。通过对各个修饰片段干扰效果的比较发现,各种化学修饰均不同程度地降低了17bp asiRNA的干扰能力。在所有化学修饰片段中,其中一种修饰方案的17bp asiRNA-M2的干扰效果最好,并且血清稳定性实验表明其稳定性较不修饰的17bp asiRNA有了明显提高。为了进一步将asiRNA用于体内实验的研究,我们制备了由聚乙二醇2000-二硬酯酰磷脂酰乙醇胺(PEG-PE)、二油基磷脂酰丝氨酸(DOPS)和重组人内皮抑素(rhEndo statin)组成的复合脂质胶束用于asiRNA的体内递送。将17bp asiRNA-M2与复合脂质胶束混合形成复合物,并对复合物的表征、血清稳定性作了研究。结果表明,复合脂质胶束能与17bp asiRNA-M2形成带正电荷的纳米微粒,并能在血清中有效保护17bp asiRNA-M2短时间内不被降解。体内分布及药效实验表明,经尾静脉注入体内的复合脂质胶束递送的17bp asiRNA-M2能在实验动物体内较长时间的稳定存在,有效抑制小鼠多种肿瘤模型的生长,并且急性毒性实验表明,其对小鼠没有明显的毒副作用。
综上所述,本研究发现：(1)对称结构的双链对于siRNA的干扰效果并不是必需的,不对称结构的siRNA同样能有效地沉默靶基因的表达。(2)与传统siRNA相比,靶向bcl-2基因的17bp asiRNA能有效减少脱靶效应的产生。(3)靶向bcl-2基因的17bp asiRNA能有效逆转肿瘤细胞的多药耐药性。(4)化学修饰的17bp asiRNA-M2在保持一定干扰效果的基础上,血清稳定性得到了显著提高。(5)17bp asiRNA-M2经PEG-PE、DOPS和rhEndostatin组成的复合脂质递送到实验动物体内,能在体内稳定存在并发挥抑瘤效果。我们的实验结果为RNAi中干扰片段的最优化设计及siRNA在肿瘤治疗的应用提供了基础和依据。
Small interfering RNAs (siRNAs) are valuable reagents for efficient gene silencing in a sequence-specific manner via the RNA interference (RNAi) pathway. The current synthetic siRNA structure consists of symmetrical duplexes of19-21base pairs (bp) with2nucleotide (nt)3'overhangs. However, this siRNA structure triggers several non-specific effects posing challenges to the application of RNAi therapeutics in clinical practice. Recent studies have shown that the asymmetric siRNAs (asiRNAs) were capable of effectively silencing target gene expressions and caused less off-target effects. In the present study, we synthesized a series of asiRNAs ranging from the13-17bp duplex region (13-17bp asiRNAs) targeting bcl-2gene, and evaluated their silencing ability and off-target effects in vitro. The results showed that17bp asiRNA had potent activity in downregulating bcl-2gene expression and inhibiting tumor proliferation in vitro. Importantly, this asiRNA structure significantly reduced off-target effects compared with conventional siRNA. Furthermore,17bp asiRNA targeting bcl-2gene could reverse multidrug resistance of tumor cells.
The poor stability and inefficient delivery of siRNA are two major obstacles for the therapeutic application of siRNA following systemic administration. Chemical modification is an effective method for improving the stability of siRNA and several studies have reported that some siRNAs with chemical modifications show durable activities in RNAi assays. To improve the stability of the17bp asiRNA, we introduced a series of chemically modification to17bp asiRNAs, including phosphorothioate,2'-O-methylation (2'-O-Me),2'-fluoro-uridine (2'-FU) and cholesterol conjunction. The comparison of silencing effect suggested that chemical modifications reduced the silencing activity of17bp asiRNA. The17bp asiRNA-M2, one of the modified17bp asiRNAs, exhibited the best gene silencing effect among all the chemically modified asiRNAs and its serum stability was significantly increased compared with17bp asiRNA. To further use asiRNA in the in vivo experiments, a proteolipid micelle was preparation to deliver asiRNA. The proteolipid micelle (termed PDE) was composed of polyethylene glycol-phosphatidylethanolamine (PEG-PE), phospholipid dioleylphosphatidylserine (DOPS) and rh-endostatin. The PDE was mixed with17bp asiRNA-M2to form17bp asiRNA-M2/PDE complexes, and its characterization and serum stability were studied. The results showed that the PDE micelle and17bp asiRNA-M2could form nano-size micelles with positive charge, and PDE micelle could protect17bp asiRNA-M2from nuclease in serum. The in vivo experiments suggested that17bp asiRNA/PDE complex could stably exist in mice for a long time and it effectively suppressed the growth of several tumor models in mice. The acute toxicity experiments showed that17bp asiRNA-M2/PDE complexes had no toxic effects.
In conclusion, our study indicates that symmetrical duplex is not necessary for the silencing effect of siRNA, and asymmetric siRNAs can also effectively silence target gene while reducing off-target effects. The chemically modified17bp asiRNA-M2maintained silencing effect andr the serum stability of it was increased. When17bp asiRNA-M2was delivered by PDE micelle it is very stable and significantly inhibited tumor growth in vivo. Our results provide foundation for siRNA design and siRNA application in cancer therapy in the future.
化学合成siRNA的不稳定性和缺乏有效的递送系统是其体内系统应用所面临的两大主要问题。化学修饰是提高siRNA稳定性的一条有效途径,许多研究报道经过化学修饰的siRNA稳定性有了显著提高。为了提高17bpasiRNA的稳定性,我们也对其进行了一系列的化学修饰,包括硫代磷酸修饰,2’-O甲基修饰、2’-氟代修饰和末端胆固醇连接。通过对各个修饰片段干扰效果的比较发现,各种化学修饰均不同程度地降低了17bp asiRNA的干扰能力。在所有化学修饰片段中,其中一种修饰方案的17bp asiRNA-M2的干扰效果最好,并且血清稳定性实验表明其稳定性较不修饰的17bp asiRNA有了明显提高。为了进一步将asiRNA用于体内实验的研究,我们制备了由聚乙二醇2000-二硬酯酰磷脂酰乙醇胺(PEG-PE)、二油基磷脂酰丝氨酸(DOPS)和重组人内皮抑素(rhEndo statin)组成的复合脂质胶束用于asiRNA的体内递送。将17bp asiRNA-M2与复合脂质胶束混合形成复合物,并对复合物的表征、血清稳定性作了研究。结果表明,复合脂质胶束能与17bp asiRNA-M2形成带正电荷的纳米微粒,并能在血清中有效保护17bp asiRNA-M2短时间内不被降解。体内分布及药效实验表明,经尾静脉注入体内的复合脂质胶束递送的17bp asiRNA-M2能在实验动物体内较长时间的稳定存在,有效抑制小鼠多种肿瘤模型的生长,并且急性毒性实验表明,其对小鼠没有明显的毒副作用。
综上所述,本研究发现：(1)对称结构的双链对于siRNA的干扰效果并不是必需的,不对称结构的siRNA同样能有效地沉默靶基因的表达。(2)与传统siRNA相比,靶向bcl-2基因的17bp asiRNA能有效减少脱靶效应的产生。(3)靶向bcl-2基因的17bp asiRNA能有效逆转肿瘤细胞的多药耐药性。(4)化学修饰的17bp asiRNA-M2在保持一定干扰效果的基础上,血清稳定性得到了显著提高。(5)17bp asiRNA-M2经PEG-PE、DOPS和rhEndostatin组成的复合脂质递送到实验动物体内,能在体内稳定存在并发挥抑瘤效果。我们的实验结果为RNAi中干扰片段的最优化设计及siRNA在肿瘤治疗的应用提供了基础和依据。
Small interfering RNAs (siRNAs) are valuable reagents for efficient gene silencing in a sequence-specific manner via the RNA interference (RNAi) pathway. The current synthetic siRNA structure consists of symmetrical duplexes of19-21base pairs (bp) with2nucleotide (nt)3'overhangs. However, this siRNA structure triggers several non-specific effects posing challenges to the application of RNAi therapeutics in clinical practice. Recent studies have shown that the asymmetric siRNAs (asiRNAs) were capable of effectively silencing target gene expressions and caused less off-target effects. In the present study, we synthesized a series of asiRNAs ranging from the13-17bp duplex region (13-17bp asiRNAs) targeting bcl-2gene, and evaluated their silencing ability and off-target effects in vitro. The results showed that17bp asiRNA had potent activity in downregulating bcl-2gene expression and inhibiting tumor proliferation in vitro. Importantly, this asiRNA structure significantly reduced off-target effects compared with conventional siRNA. Furthermore,17bp asiRNA targeting bcl-2gene could reverse multidrug resistance of tumor cells.
The poor stability and inefficient delivery of siRNA are two major obstacles for the therapeutic application of siRNA following systemic administration. Chemical modification is an effective method for improving the stability of siRNA and several studies have reported that some siRNAs with chemical modifications show durable activities in RNAi assays. To improve the stability of the17bp asiRNA, we introduced a series of chemically modification to17bp asiRNAs, including phosphorothioate,2'-O-methylation (2'-O-Me),2'-fluoro-uridine (2'-FU) and cholesterol conjunction. The comparison of silencing effect suggested that chemical modifications reduced the silencing activity of17bp asiRNA. The17bp asiRNA-M2, one of the modified17bp asiRNAs, exhibited the best gene silencing effect among all the chemically modified asiRNAs and its serum stability was significantly increased compared with17bp asiRNA. To further use asiRNA in the in vivo experiments, a proteolipid micelle was preparation to deliver asiRNA. The proteolipid micelle (termed PDE) was composed of polyethylene glycol-phosphatidylethanolamine (PEG-PE), phospholipid dioleylphosphatidylserine (DOPS) and rh-endostatin. The PDE was mixed with17bp asiRNA-M2to form17bp asiRNA-M2/PDE complexes, and its characterization and serum stability were studied. The results showed that the PDE micelle and17bp asiRNA-M2could form nano-size micelles with positive charge, and PDE micelle could protect17bp asiRNA-M2from nuclease in serum. The in vivo experiments suggested that17bp asiRNA/PDE complex could stably exist in mice for a long time and it effectively suppressed the growth of several tumor models in mice. The acute toxicity experiments showed that17bp asiRNA-M2/PDE complexes had no toxic effects.
In conclusion, our study indicates that symmetrical duplex is not necessary for the silencing effect of siRNA, and asymmetric siRNAs can also effectively silence target gene while reducing off-target effects. The chemically modified17bp asiRNA-M2maintained silencing effect andr the serum stability of it was increased. When17bp asiRNA-M2was delivered by PDE micelle it is very stable and significantly inhibited tumor growth in vivo. Our results provide foundation for siRNA design and siRNA application in cancer therapy in the future.
引文
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