探討缺氧誘導之長片段非編碼核糖核酸MTORT1在乳癌細胞中的調控機制與扮演的功能角色

Abstract

長片段非編碼核糖核酸(long non-coding RNA, lncRNA)最近被認為在癌症細胞中扮演著重要的角色，參與調控諸多遺傳途徑。其中，線粒體相關的lncRNA也被發現可調節粒線體功能和代謝。先前我們實驗室使用次世代定序(next generation sequencing)篩選乳癌細胞株MCF-7在常氧、缺氧、復氧的不同環境中，受氧氣調控的lncRNA，找到了受氧氣調控的粒線體lncRNA MTORT1。因此本研究目的即為探討MTORT1在乳癌細胞中的特性、可能扮演的功能角色以及參與調控的機制。首先、研究結果中發現，MTORT1主要分佈於線粒體中，相較於乳腺細胞，乳腺癌細胞中的內源性表達水平較低，並且發現在序列上擁有四個保守區域。此外，免疫螢光染色分析表明MTORT1可能在線粒體中可以轉譯出小分子蛋白。其次、關於MTORT1的功能角色方面，實驗結果顯示抑制MTORT1的表現可增強細胞增殖和遷移，表示在乳癌細胞中MTORT1扮演抑癌基因的角色。最後，我們使用RNA免疫沉澱分析法以及報導基因冷光活性分析法探討MTORT1是否能作為吸附微小RNA (miRNA)的海綿，實驗結果證實MTORT1藉由吸附miR-26a-5p，來調控CREB1和 STK4。總而言之，在這些結果中我們發現了線粒體lncRNA MTORT1的特性、扮演的功能角色以及參與調控的機制，包括可轉譯出小分子蛋白、作為吸附miRNA的海綿，並且能減緩乳腺癌的生長。

Long non-coding RNAs (lncRNAs) have been regarded to participate in multiple genetic pathways in cancer. Also, mitochondria-associated lncRNAs have been discovered to module mitochondrial function and metabolism. Previously, our lab identified oxygen responsive lncRNAs in breast cancer MCF-7 cells under normoxic, hypoxic and re-oxygenated conditions by using next generation sequencing technology. Among them, a novel mitochondrial lncRNA Mitochondrial Oxygen Responsive Transcript 1 (MTORT1) was chosen for further investigation. Therefore, the purpose of this study was to investigate the characterizations, function roles, and mechanisms of MTORT1 in breast cancer cells. First, the characterization of MTORT1 included four conserved regions, lower endogenous expression levels in breast cancer cells, and mainly distributed in mitochondria. In addition, immunofluorescence assays showed that MTORT1 might translate peptides in mitochondria. Second, functional assays revealed that knockdown of MTORT1 enhanced cell proliferation and migration, implicating the tumor suppressor role of this novel mitochondrial lncRNA. Finally, RNA immunoprecipitation and Luciferase reporter assays indicated that MTORT1 served as sponge of miR-26a-5p to up-regulate its target genes, CREB1 and STK4. In summary, our findings shed some light on the characterization, function, and regulatory mechanism of the novel hypoxia-induced mitochondrial lncRNA MTORT1, which could encode peptide, functioned as miRNA sponge, and inhibited breast cancer progression.