抑制多基因表現之微核醣核酸整合性設計平台

Abstract

藉由siRNA抑制單一基因的表現已經成為生物醫學研究的常用工具，然而利用siRNA同時去調控大量的基因表現仍是生醫研究之一大挑戰。近年來，研究指出miRNA可藉由依附在多基因三端未轉譯區段來抑之多基因表現，對於使用單一微小RNA同時調控大量基因之表現帶來一線曙光。本研究提出一個新的演算法以進行指定基因調控之人造miRNA之序列設計。 為證明本方法之效果，我們設計了多組miRNA進行基因調控。我們在A549細胞株上進行實驗，利用蛋白質免疫印跡(immunobloting以)及觀察細胞遷移狀態證實，其中一組人造miRNA對基因EGFR及AURKA有調控效果，另一組人造miRNA對CEP55及PBK基因有抑制效果。此外，我們利用miRNA目標接合點位軟體對生物體及人造miRNA進行隨機抑制之基因數量進行評估，發現人造miRNA之隨機抑制基因遠小於生物體內之miRNA影響之基因數量。 本演算法之設計乃基於miRNA與目標基因預測之數項基本條件，並試圖提升人造miRNA對目標基因的抑制能力降低隨機影響之基因數量，雖然現階段人造miRNA對單一基因的影響力不若siRNA對單一基因的抑制能力強，但人造miRNA對多基因同時之抑制能力，其總和之影響力將遠超過單一siRNA之影響力。

Targeting single gene via small interfering RNAs (siRNAs) is a powerful tool to reduce the expression of a single gene in human cells, but its cellular processes often affect sets of genes acting in concert. And from the manipulation of cell behavior with the siRNAs approach, simultaneously suppressing multiple target genes remain a challenging task. Because endogenous miRNAs can translationally repress multiple genes by binding on 3’UTR via partially complementary, we designed a novel computational algorithm and miRNA replicate to simulate the mechanism of miRNA that can target manually-assigned genes. miRNA replicates were designed to suppress genes group of CEP55/FLJ10540 and PBK/TOPK and genes group of AURKA and EGFR. Immunoblotting and migration analyses indicated that the designed miRNA replicates could suppress the protein expression of these target genes and inhibited simultaneously the migration ability of A549 lung cancer cells, but the designed miRNA was not as effective as individual siRNA. The off-target effect of miRNA replicate was also observed by using three target site prediction tools: miRanda, TargetScanS, and PITA. The predicted target sites of miRNA replicate in all human genes is fewer than that of endogenous miRNA and siRNAs. The algorithm of miRNA replicate design was based on several important criteria of miRNA target site prediction. Two important goals of this designation are to maximize the inhibition efficacy of miRNA replicates in target genes and to minimize the off-targeting effects. Currently, miRNA replicate could suppress partial expression of target genes; miRNA replicate was shown by immunobloting experiments. Although the suppression power for a single target gene is not as powerful as siRNA does, the synergy of suppressing multiple genes by a miRNA replicate might be more powerful than the synergy of suppressing a gene by a single siRNA.