探討長片段非編碼核糖核酸NDRG1-OT1在乳癌細胞中的調控機制與扮演的功能角色

Abstract

缺氧是在一個在腫瘤微環境中典型的特徵，其對於癌症進程有深切的影響，而且與較差的預後密切相關。長片段非編碼核糖核酸(long non-coding RNA, lncRNA)是非編碼基因體的一員，由於其在腫瘤生成中扮演許多不同的角色而逐漸受到重視。先前我們實驗室使用次世代定序找到了在乳癌細胞株MCF-7中會被缺氧誘導表現的lncRNA，NDRG1-OT1，但是對於NDRG1-OT1的調控機制以及功能仍尚未釐清。因此本研究目的即為探討NDRG1-OT1在乳癌細胞中的轉錄機制以及可能扮演的功能角色。NDRG1-OT1的表現量分析顯示其在不同乳癌細胞株處於缺氧時皆會上升。而在過表現或者敲低缺氧誘導因子1α (HIF-1α)時分別會上調以及下調NDRG1-OT1的表現量。冷光酶報導基因分析法以及染色質免疫沉澱分析法證實了HIF-1α會結合到NDRG1-OT1的啟動子(-1,773到-1,769鹼基對及-647到-643鹼基對)上來活化其轉錄作用。接下來，為了探討NDRG1-OT1是否能作為吸附微小RNA (miRNA)的海綿，生物資訊工具首先被用來預測可能有交互作用的miRNA，而預測到的miRNA的表現量分析以及RNA免疫沉澱分析法的結果說明了NDRG1-OT1並不能作為miRNA海綿。最後關於NDRG1-OT1的功能角色方面，在常氧及擬缺氧狀況下過表現NDRG1-OT1並不會影響細胞增生、細胞遷移以及細胞週期的分布。總而言之，在這些結果中我們發現一個新的轉錄機制，即當乳癌細胞在缺氧刺激下，HIF-1α會透過轉錄活化而促進NDRG1-OT1的表現量上升。

Hypoxia is a classic feature of tumor microenvironment, which has profound effects on cancer progression and is tightly associated with poor prognosis. Long non-coding RNAs (lncRNAs), a member of non-coding genome, have been increasingly investigated due to their diverse roles in tumorigenesis. Previously, our lab identified a hypoxia-induced lncRNA, NDRG1-OT1, in MCF-7 breast cancer cells using next-generation sequencing. However, the regulatory mechanism and functional roles of NDRG1-OT1 remain elusive. Therefore, the purpose of this study is to investigate the transcriptional mechanism and potential function roles of NDRG1-OT1 in breast cancer cells. Expression profiling of NDRG1-OT1 revealed that it was upregulated under hypoxia in different breast cancer cells. Over-expression and knockdown of HIF-1α up- and down-regulated NDRG1-OT1 respectively. Luciferase reporter assays and chromatin immunoprecipitation assays validated that HIF-1α transcriptionally activated NDRG1-OT1 by binding to its promoter (-1,773 to -1,769 and -647 to -643 bp). Next, to investigate whether NDRG1-OT1 could function as miRNA sponge, in silico analysis, expression profiling of predicted miRNAs, and RNA immunoprecipitation assays were performed, and the results implied that NDRG1-OT1 could not act as miRNA sponge. Lastly, regarding the function of NDRG1-OT1, ectopic expression of NDRG1-OT1 could not affect cell proliferation, migration, and cell cycle distribution under normoxia and hypoxia mimic conditions. In summary, our findings revealed a novel transcriptional mechanism of NDRG1-OT1 regulated by HIF-1α upon hypoxic condition in breast cancer cells.