探討微核醣核酸miR-26a辨認之標的與基因交互作用的可能性

Abstract

基因的表達可以通過各種複雜的機制調控。而微型核醣核酸(microRNAs)也參與在其中。微型核醣核酸可以透過抑制蛋白質轉譯以及誘導信使核醣核酸(messenger RNAs)的降解來調控基因的表現。微型核醣核酸表現的失調已經與許多疾病有關連，例如癌症以及葡萄糖跟脂質代謝的缺陷。而最近微型核醣核酸miR-26也被證實可能對hepatocellular carcinoma (HCC)肝細胞癌是一個腫瘤抑制基因。 基因交互作用(Genetic interaction)的分析對於連結基因之間的功能是一個很有力的工具。在我們的研究中，我們嘗試去找尋與微型核醣核酸miR-26a有交互作用的基因進而去影響人類肝癌細胞的存活率。 首先我們引入一個miR-26a海綿，這是一個建構在綠色螢光蛋白的3’ 不轉譯的區域且有著重複的miR-26a結合的外來序列，而這些海綿會與目標基因競爭miR-26a的結合。而實際上我們也觀察到這些綠色螢光蛋白在人類肝癌細胞中確實會受到miR-26a 的結合而受到抑制。我們也使用的流式細胞儀去確認綠色螢光蛋白受到抑制的這個事實。 之後我們利用real time Polymerase Chain Reaction (qPCR)觀察在有miR26a海綿的Huh7細胞中miR26a目標基因的表現情況，而這些目標基因是透過網路上的資料庫所取得。我們發現只有14%左右的目標基因與結果相符，但是在我們利用從大量表現miR-26a 的細胞株的微陣列(microarray)資料中得到的目標基因，利用qPCR觀察這些目標基因，我們發現有達到35.5%左右的目標基因與結果相符合。這也表示了利用微陣列所得到的這些目標基因在未來找尋微型核醣核酸是一個很有力的方法。

Gene expression can be modulated by various elaborate mechanisms. Among these microRNA (miRNA) is of particular interest. MiRNAs regulate gene expression by inhibiting translation or inducing degradation of target messenger RNAs (mRNAs) in multicellular organisms. Dysregulated miRNA expression has been linked to many diseases, such as cancers and defect of glucose and lipid metabolism. For example, miR-26a has recently been identified as a tumor suppressor gene of mouse hepatocellular carcinoma (HCC). Genetic interaction analysis is a powerful tool for establishing function linkages between genes. In our research, we attempt to look for some genes which have interactions with miR-26a to influence cell viability in human hepatocellular carcinoma cells. First we introduced a miR-26a “sponge” which is an exogenous construct containing repeated binding sequences of miR-26a integrated into the 3’ untranslated region (3’ UTR) of a reporter gene encoding green fluorescence protein (GFP), and sponge competes miR-26a binding with endogenous target gene. We actually observed the GFP expression was silenced in HepG2 cells expressing the sponge that binds to miR26a.We also measured GFP fluorescence intensity by flow cytometry to confirm the result. Next we examined the expression of miR-26a target genes by quantitative Polymerase Chain Reaction (qPCR). Only 14% of in silico predicted targets were repressed by miR-26a overexpression and stabilized by miR-26a sponge in Huh7 cells. Interestingly, in silico targets with genetic interactions to miR-26a are much more likely to be bona fide targets (over 35%, p-value<0.05), suggesting an integrative approach to efficiently identify miRNA target genes.