Ou Zhaoxing, Zeng Rui, Lin Yifan, Zhang Si, Alzogool Mohammad, Zeng Peng, Lan Yuqing
Department of Ophthalmology, Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China.
J Ophthalmol. 2020 Apr 15;2020:1848540. doi: 10.1155/2020/1848540. eCollection 2020.
To seek for a small interfering RNA (siRNA) sequence targeting a cynomolgus monkey inhibitor of nuclear factor kappa B (IB) that can specifically and effectively suppress IB gene expression of cynomolgus monkey ciliary muscle (CM) cells and trabecular meshwork (TM) cells in vitro and screen for optimal siRNA transfection concentration.
Three IB-specific double-stranded siRNAs were designed and synthesized. They were transfected into primarily cultured cynomolgus monkey CM cells and TM cells. The mRNA and protein levels of IB were examined by using real-time quantitative polymerase chain reaction (real-time PCR) and western blot to screen a pair of candidate valid sequences with the highest inhibitory rate. Both cells were transfected with Cy5-labeled nonspecific control-siRNA (NC-siRNA) of four different concentrations (10, 20, 50, and 100 nmol/L(nM)), and flow cytometry was used to assess transfection efficiency. Then, cells were transfected with the candidate valid IB -siRNA of the same four concentrations, and the cytotoxicity was detected by using Cell Counting Kit-8 (CCK8), and the inhibitory efficiency of IB was identified via real-time PCR to find out optimal siRNA transfection concentration.
The suppression effect of the siRNA targeting the GCACTTAGCCTCTATCCAT of IB gene was most obvious by in vitro screening. The inhibitory rate of IB was 82% for CM cells and 82% for TM cells on the mRNA level and 98% for CM cells and 93% for TM cells on the protein level, respectively. The results of flow cytometry showed that the transfection efficiency was the highest at 100 nM, which was 89.0% for CM cells and 48.2% for TM cells, respectively. The results of CCK8 showed that there was no statistically significant difference in cell viability after transfection of different concentrations of IB-siRNA. The results of real-time PCR indicated that there was no statistical difference in the inhibitory efficiency of IB after transfection of different concentrations of IB-siRNA.
It proves that the siRNA targeting the GCACTTAGCCTCTATCCAT of IB gene is the valid sequence to suppress cynomolgus monkey IB expression of CM cells and TM cells by RNAi. 10 nM is the optimal transfection concentration.
寻找一种靶向食蟹猴核因子κB抑制因子（IκB）的小干扰RNA（siRNA）序列，该序列能在体外特异性、有效地抑制食蟹猴睫状肌（CM）细胞和小梁网（TM）细胞的IκB基因表达，并筛选出最佳的siRNA转染浓度。
设计并合成3条IκB特异性双链siRNA。将它们转染到原代培养的食蟹猴CM细胞和TM细胞中。采用实时定量聚合酶链反应（实时PCR）和蛋白质免疫印迹法检测IκB的mRNA和蛋白质水平，以筛选出抑制率最高的一对候选有效序列。用4种不同浓度（10、20、50和100 nmol/L（nM））的Cy5标记的非特异性对照siRNA（NC-siRNA）转染两种细胞，采用流式细胞术评估转染效率。然后，用相同的4种浓度的候选有效IκB-siRNA转染细胞，采用细胞计数试剂盒-8（CCK8）检测细胞毒性，并通过实时PCR鉴定IκB的抑制效率，以找出最佳的siRNA转染浓度。
体外筛选结果显示，靶向IκB基因GCACTTAGCCTCTATCCAT的siRNA抑制效果最明显。在mRNA水平上，CM细胞和TM细胞的IκB抑制率分别为82%；在蛋白质水平上，CM细胞和TM细胞的IκB抑制率分别为98%和93%。流式细胞术结果显示，在100 nM时转染效率最高，CM细胞和TM细胞的转染效率分别为89.0%和48.2%。CCK8结果显示，转染不同浓度的IκB-siRNA后细胞活力无统计学差异。实时PCR结果表明，转染不同浓度的IκB-siRNA后IκB的抑制效率无统计学差异。
证明靶向IκB基因GCACTTAGCCTCTATCCAT的siRNA是通过RNA干扰抑制食蟹猴CM细胞和TM细胞IκB表达的有效序列。10 nM是最佳转染浓度。
