受缺氧所誘發的NDRG1-OT1_v4於乳癌MCF-7細胞中藉由泛素化調控路徑而促使NDRG1降解

Abstract

缺氧為固態癌症細胞中常見且重要的壓力因子，其可藉由調控諸多細胞內訊息傳遞路徑使細胞更加惡化。長鏈非編碼核糖核酸(long non-coding RNA; lncRNA)最近被發現在癌症細胞中扮演著重要的角色，其可藉由調節諸多基因表現的過程，使癌症細胞回應外在環境的刺激而造成細胞功能上的變化。然而，在乳癌細胞中是否有受外在氧氣濃度變化而影響表現的lncRNA還尚未釐清，因此本次研究目的即在探討乳癌細胞株MCF-7中受氧氣控制的lncRNA，並研究其相關機轉。為了尋找受氧氣控制的lncRNA，本實驗室使用高通量次世代定序(next generation sequencing)的技術來找尋。本實驗室所建立篩選受氧氣調控的lncRNA條件為從常氧到缺氧以及從缺氧回到復氧的狀態下表現量倍數大於3倍以及有顯著差異(P值<0.001)，而最後發現了472條受氧氣濃度變化而有反應的lncRNA。從受缺氧而誘發的5條倍數差異最高的lncRNA中，NDRG1-OT1被選中作為接下來的研究對象。而NDRG1-OT1的5個亞型中，第4號亞型在缺氧環境下的表現量差異最高，因此第4號亞型NDRG1-OT1(NDRG1-OT1_v4)作為接下來進一部探討的實驗對象。為了尋找NDRG1-OT1_v4調控的下游基因與可能參與的調控路徑，本實驗室於細胞中大量表現NDRG1-OT1_v4，並使用基因微陣列(microarray)的技術。本實驗室找到了108個受NDRG1-OT1_v4所調控的基因，而在5個表現差異最高的基因中，又以NDRG1表現量差異最為明顯。根據即時定量聚合酶連鎖反應(real-time polymerase chain reaction)以及西方點墨法的驗證，證明NDRG1-OT1_v4抑制NDRG1之mRNA與蛋白質的表現。接著本實驗使用了免疫沉澱法的方式，成功證明NDRG1-OT1_v4可藉由增加NDRG1的泛素化，促進NDRG1的降解，進而減少NDRG1蛋白質的表現。本次的實驗研究成功地在乳癌細胞中找到了新的表觀遺傳調控NDRG1的方式。

Hypoxia is a crucial factor that can lead to solid tumor aggressiveness by driving multiple signaling pathways. Long non-coding RNAs (lncRNAs) respond to several extrinsic stimuli, causing changes in cancer function by participating in multiple steps of gene expression. However, it is unclear whether lncRNAs respond to oxygen concentrations in breast cancer. Therefore, the aims of this study are to identify oxygen responsive lncRNAs, and to delineate their regulatory mechanisms in breast cancer MCF-7 cells. The expression profiling of lncRNAs in MCF-7 cells growing under normoxic, hypoxic, and re-oxygenated conditions was examined using next-generation sequencing technology. The criteria for selecting oxygen-responsive lncRNAs consisted of greater than 3-fold change, significant differences (P < 0.001), and opposite expression profiling between normoxia and hypoxia, as well as between hypoxia and re-oxygenation. Four hundred and seventy-two lncRNAs that met these rules were identified. Examining the top five differentially expressed lncRNAs in hypoxia, we selected NDRG1-OT1 for further study. Among the five isoforms of NDRG1-OT1, NDRG1-OT1_v4 was chosen for further investigation because it was the most responsive isoform to oxygen changes. We overexpressed NDRG1-OT1_v4 under normoxia and performed microarray analysis to identify 108 NDRG1-OT1_v4 regulated genes and their functions. Among these genes, we focused on the effect of NDRG1-OT1_v4 on NDRG1, and found that both NDRG1 mRNA and NDRG1 protein were inhibited by NDRG1-OT1_v4. Finally, we used co-immunoprecipitation and discovered that NDRG1-OT1_v4 destabilized NDRG1 by promoting ubiquitin-mediated proteolysis. Our findings revealed a new form of epigenetic regulation of NDRG1 by NDRG1-OT1_v4 in breast cancer.