受缺氧誘導因子調控的MALAT1藉由抑制微小RNA來促進乳癌細胞的惡性

Abstract

缺氧是腫瘤生長中的常見過程，可促進腫瘤侵襲性並與不良癒後緊密相關。長片段非編碼核糖核苷酸(long non-coding RNA, lncRNA）其長度大於200 鹼基對，且其無法轉譯成蛋白質，已知其調控的生理機制非常的廣泛且影響許多細胞的功能，其中可作為吸附微小RNA (microRNA, miRNA）的海綿。先前我們實驗室使用次世代定序技術（NGS）發現在MCF-7 乳癌細胞株中，lncRNAMALAT1 會被缺氧誘導表現，但MALAT1 在乳腺癌中的調控機制和功能仍不清楚。因此本研究的目的是探討MALAT1 是否可透過miRNA 調節乳腺癌細胞的功能。第一，以定量即時聚合酶鏈鎖反應檢測正常乳腺細胞MCF-10A 中以及乳 癌細胞株MCF-7 和MAD-MB-231 中MALAT1 內生性表現量。在不同氧濃度中以定量即時聚合酶鏈鎖反應檢測MCF-7 的MALAT1 表現量。接著，以定量即時聚合酶鏈鎖反應檢測過表現HIF-1A 或 HIF-2A 的MCF-7 中MALAT1 表現量。並且以冷光酶報導基因分析法檢測HIF-1α 或 HIF-2α 是否與MALAT1 啟動子之間的交互作用。第三，以核質分離檢測MALAT1 的分布情況，第四，細胞在MALAT1 基因靜默後，透過次世代定序檢測受到MALAT1 影響的miRNA，接著運用AGO2 蛋白質(miRNA 誘導的沉默複合體次單元)抗體進行RNA 免疫沉澱分析法。最後以細胞存活率分析以及傷口癒合測定細胞的增殖以及遷移能力。以流式細胞儀檢測乳癌細胞的細胞週期分布狀況。 由結果發現，MALAT1 表現量在MDA-MB-231 中高於MCF-7 以及MCF-10A。乳癌細胞在缺氧條件下會促使MALAT1 的表現量顯著性的上升，且缺氧誘導因子1α (HIF-1α) 或 HIF-2α 可以促進MALAT1 轉錄的表現量。透過核質分離檢測和螢光原位雜交發現MALAT1 分佈於細胞質。為了檢測受MALAT1 影響的miRNA，在缺氧的環境下將MALAT1 進行基因靜默並進行次世代定序。由NGS 結果發現五個明顯差異表達的miRNA，包括miR-3150a-3p、miR-4325、miR-378c、miR-3064-5p 和miR-7855-5p。探討MALAT1 是否能作為吸附 miRNA，利用AGO2 蛋白抗體進行RNA 免疫沉澱（RIP）分析法檢測，過度表現MALAT1 後，發現AGO2 結合MALAT1 的能力高於IgG，結果指出MALAT1 能作為吸附miRNA 的角色。最後，功能性檢測結果顯示MALAT1 可以促使乳癌細胞的遷移和增殖，且利用流式細胞儀檢測MALAT1 基因靜默的乳癌細胞株時，發現細胞週期G1 期的百分比顯著增加，表明MALAT1 沉默導致G1 停滯。因此，MALAT1 可能是抑制乳腺癌進展的目標候選藥物。

Hypoxia, a common process during tumor growth, can lead to tumor aggressiveness and is tightly associated with poor prognosis. Long noncoding RNAs (lncRNAs) are ribonucleotide (>200 base) with limited ability to translate proteins, and lncRNAs are known to affect many aspects of cellular functions. One of the regulatory mechanisms is functioned as microRNA (miRNA) sponge to modulate the biological functions. Previously, we used next-generation sequencing (NGS) technology to identify oxygen-responsive lncRNAs in breast cancer MCF7 cells, and identified MALAT1 in the top five up-regulated lncRNAs under hypoxia. However, the regulatory mechanism and functions of MALAT1 in breast cancer were still unclear. Therefore, the aim of this study is to explore whether MALAT1 can regulate the functions of breast cancer cells through miRNA. First, the endogenous expression levels of MALAT1 of MCF-10A, MCF-7, MDA-MB-231 cells were determined by quantitative RT-PCR. The expression levels of MALAT1 in MCF-7 grown in different oxygen concentrations were examined by quantitative RT-PCR. Next, expression levels of MALAT1 in MCF7 expressing HIF-1A or HIF-2A were examined by quantitative RT-PCR. Luciferase reporter assays were used to validate the transcriptionally interaction of HIF-1α or HIF-2α with MALAT1 promoter. Third, distribution of MALAT1 were examined by nuclear-cytoplasmic RNA fractionation assays. Fourth, to identify the miRNAs affected by MALAT1, next-generation sequencing was performed in MALAT1-knockdown cells, and followed by RNAbinding protein immunoprecipitation (RIP) assays using antibody against AGO2 protein, an essential component of the miRNA-induced silencing complex, and by quantitative RT-PCR. Lastly, cellular proliferation and migration of breast cancer cells were examined by MTT assays and wound healing assays. Cell cycle distribution of breast cancer cells were determined by flow cytometry. Regarding the results of this study, the expression levels of MALAT1 of MDAMB-231 cells were more than MCF-7 and MCF-10A. The expression levels of MALAT1 were significantly upregulated under hypoxia. Next, HIF-1α or HIF-2α could promote the transcriptional levels of MALAT1. Third, the nuclear and cytoplasm fraction assays and fluorescence in situ hybridization indicated that distributions of MALAT1 were located at cytoplasm. Fourth, five differentially expressed miRNAs, including miR-3150a-3p, miR-4325, miR-378c, miR-3064-5p and miR-7855-5p, were identified using NGS and validated by qPCR, when MALAT1 was knocked down under hypoxia. The binding between MALAT1 and AGO2 enhanced in cells overexpressing MALAT1, indicating MALAT1 may function as miRNA sponge. Lastly, functional assays revealed that MALAT1 could promote cellular migration and proliferation of breast cancer cells via miRNAs. When MALAT1 was knocked down in MDA-MB-231 cells, the percentage of G1 phase of cell cycle significant increased, indicating silence of MALAT1 resulted in G1 arrest. Therefore, MALAT1 may be the candidate of therapeutic target for inhibiting the progress of breast cancer.