靶向人乙型肝炎病毒siRNA的构效关系研究

A quantitative structure-activity relationship study of siRNA targeting HBV

目的:在体外模型HepG2.2.15细胞中研究靶向人乙型肝炎病毒(HBV)小分子干扰RNA(siRNA)的构效关系。方法:靶向HBV基因不同位置合成21条siRNA,分析多条siRNA在HBV 8个基因型(共120条序列)中的保守性情况以及siRNA的二级结构特点。利用乙肝表面抗原(HBsAg)ELISA定量方法与荧光定量PCR方法检测siRNA在不同浓度下(1,10,100 nmoL/L)对HepG2.2.15细胞中乙肝病毒HBsAg和mRNA表达的抑制情况。SPSS进行定量构效关系(QSAR)分析。结果:半数(13/21)的siRNA以浓度依赖模式抑制靶基因的表达(P<0.05)。其中537,672,1263,1658等序列活性显著高于阳性对照序列。QSAR分析显示siRNA基因型同源性(P<0.01)、S/P和X区(P<0.05)与siRNA活性有紧密关系;重叠基因[S/P/前基因组(pregenome)mRNA]区域局部靶mRNA二级结构保守性、发卡环、多茎环和茎的碱基数目与siRNA活性相关(R=0.994,P=0.009)。结论:siRNA的靶点序列、siRNA本身的一级序列与二级结构对其抑制靶基因的活性有显著影响,具有明显的构效关系。优选siRNA可能作为治疗多基因型乙肝感染的候选序列。

Objective：To study the structure-activity relationship （SAR） of small molecular interference RNA （siRNA） targeting hepatitis B virus （HBV） in hepG2.2.15 cell. Methods：Twenty-one siRNAs were designed targeting different regions of the HBV. Genotype homology within HBV genomes was identified through plentiful computational analysis, and secondary structure of siRNA was analyzed. The in-vitro inhibitory effect of siRNAs at 1, 10, and 100 nmol/L on the expression of S/P/pregenomic mRNA and hepatitis B surface antigen （HBsAg） was determined quantitatively in HepG2.2.15 cells. Multiple linear regression analysis was performed for quantitative structure-activity relationship （QSAR） study. Results： About half （ 13/21 ） of the siRNAs showed significant concentration-dependent silencing effects （P 〈 0.05 ）. The activity of si537, si672, si1263 and si1658 was much better than that of the positive controls. Statistic results showed that the genotypic homology （P 〈 0.01 ）, ORF S/P and X （P 〈 0.05 ） had close correlation with the efficacy of siRNAs. Within the local conservative secondary structures in overlapping regions （S/P/pregenome mRNA）, the numbers of nucleotides in the hairpin, malti-branch, and stem structure were correlated with the siRNA efficacy （R = 0. 994, P = 0. 009）. Conclusion： The primary and secondary structure characteristics of siRNA are associated with enhanced antiviral activity. QSAR study is useful for the optimal siRNA design, and the excellent siRNAs could be candidates for effective inhibition of multiple subtypes of HBV.