Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
Br J Cancer. 2013 Feb 5;108(2):450-60. doi: 10.1038/bjc.2012.564. Epub 2013 Jan 8.
When designing therapeutic short-interfering RNAs (siRNAs), off-target effects (OTEs) are usually predicted by computational quantification of messenger RNAs (mRNAs) that contain matches to the siRNA seed sequence in their 3' UTRs. It is assumed that the higher the number of predicted transcriptional OTEs, the greater the size of the actual OTE signature and the more detrimental the phenotypic consequences in target-negative cells.
We tested this general assumption by investigating the OTEs of potential therapeutic siRNAs targeting the human papillomavirus (HPV) type-16 E7 oncogene. We studied HPV-negative squamous epithelial cells, from normal cervix (NCx) and skin (HaCaT), which would be vulnerable to 'bystander' OTEs following transfection in vivo.
We observed no correlation between the number of computationally predicted OTEs and the actual number of seed-dependent OTEs (P=0.76). On average only 20.5% of actual transcriptional OTEs were seed-dependent (i.e., predicted). The unpredicted OTEs included stimulation of innate immune pathways, as well as indirect (downstream) effects of other OTEs, which affected important cancer-associated pathways. Although most significant OTEs observed were seen in both NCx and HaCaT cells, only 0-5.9% of differentially expressed genes overlapped between the two cell types.
These data do not support the assumption that actual OTEs correlate well with predicted OTEs.
在设计治疗性短干扰 RNA（siRNA）时，通常通过计算 3'UTR 中与 siRNA 种子序列匹配的信使 RNA（mRNA）的数量来预测脱靶效应（OTE）。人们认为，预测转录 OTE 的数量越多，实际 OTE 特征的大小就越大，对非靶细胞的表型后果就越不利。
我们通过研究针对人乳头瘤病毒（HPV）16 型 E7 癌基因的潜在治疗性 siRNA 的 OTE 来检验这一普遍假设。我们研究了 HPV 阴性的鳞状上皮细胞，来自正常宫颈（NCx）和皮肤（HaCaT），这些细胞在体内转染后容易受到“旁观者”OTE 的影响。
我们没有观察到计算预测的 OTE 数量与实际种子依赖性 OTE 数量之间存在相关性（P=0.76）。平均而言，只有 20.5%的实际转录 OTE 是种子依赖性的（即预测的）。未预测的 OTE 包括先天免疫途径的刺激，以及其他 OTE 的间接（下游）效应，这些效应影响了重要的癌症相关途径。尽管观察到的大多数显著 OTE 都出现在 NCx 和 HaCaT 细胞中，但两种细胞类型之间只有 0-5.9%的差异表达基因重叠。
这些数据不支持实际 OTE 与预测 OTE 相关性良好的假设。
