非对称性小RNA干扰技术介导的USP22基因沉默的研究

Effect of the specific asymmerytic structure interfering RNA on ubiquitin specific peptidase 22 silencing in bladder cancer EJ cells

目的 观察非对称小干扰RNA（aiRNA）对膀胱癌EJ细胞泛素特异肽酶22（USP22）基因的沉默效率及特异性的影响.方法 设计并合成USP22基因的小干扰RNA（siRNA）及aiRNA,利用脂质体Translipid转染EJ细胞,采用实时荧光定量聚合酶链反应（PCR）检测转染前后EJ细胞USP22 mRNA的表达水平变化,比较USP22 siRNA与USP22 aiRNA对USP22基因沉默效率及特异性的差异.结果 浓度为50 nmol/L的15/21 USP22 aiRNA能明显抑制EJ细胞中USP22基因mRNA的表达,转染48 h后USP22 mRNA的表达水平下降到最低[（8.30±1.68）%,P＜0.05],且其沉默效率和特异性明显优于USP22 siRNA.结论 以非对称小RNA干扰技术为基础设计的USP22aiRNA能够有效沉默USP22基因,并且降低了非特异性的脱靶效应,减少了受RNA干扰中的＂饱和机制＂及＂竞争机制＂的影响,弥补了传统siRNA的一些不足.

Objective To explore the effect of ubiquitin specific peptidase 22 （USP22） asymmetric structure interfering RNA （aiRNA） on USP22 gene silencing in bladder cancer EJ cells. Methods Control small interfering RNA （siRNA）, the USP22-specific siRNA, and aiRNAs were designed and synthesized. The EJ cells were treated with control siRNA, USP22 siRNA or aiRNAs. The expression levels of USP22 mRNA were detected by using real-time reverse transcription-polymerase chain reaction （ RT-PCR）, and the effects of the siRNA- and aiRNA-mediated silence of the target USP22 gene expression were compared. Results In contrast to USP22 siRNA, the 50 nM 15/21 USP22 aiRNA displayed the strongest inhibitory activity among these inhibitory RNAs tested. After EJ cells were treated with 15/21 USP22 aiRNA for 48 h, the expression levels of USP22 mRNA were reduced to the lowest levels[（8.30 ±1.68）% ,P〈0.05]. Conclusion The USP22 gene silencing mediated by 15/21 USP22 aiRNA was efficient, durable and reduced off-target silencing and nonspecific effects. The technology of asymmetric structure iRNA may be a novel approach for the gene research.