微核醣核酸誘導鼻咽癌細胞株細胞凋亡之機制

The mechanism of microRNA inducing apoptosis in nasopharyngeal carcinoma cell lines

吳承德(Cheng-Der Wu)

指導教授 ： 林欽塘

國立臺灣大學/醫學院/病理學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.03358

摘要

微核醣核酸 (microRNA) 是細胞內自行轉錄產生長度約 21 個核苷酸的非編碼核醣核酸 (non-coding RNA)。 microRNA 的功能是負向調控其它基因的表現及在各種細胞功能中扮演重要的角色。微核醣核酸-1 (miR-1) 常被使用在 microRNA 實驗中當正對照組。先前在我們其它 microRNA 的實驗中，當 miR-1 加到鼻咽癌細胞株 NPC-TW01 中當作正對照組，在曠時攝影顯微鏡觀察下出現很典型的細胞凋亡 (apoptosis) 現象。進一步用Annexin V、TUNEL 染色及caspase assay證實 miR-1會引發NPC-TW01 鼻咽癌細胞株的細胞凋亡。 測試其它細胞株，發現 miR-1也會讓 HeLa、 Cal-27、 KYSE-30及NPC-TW06等原本細胞內就低度表現miR-1的細胞株產生細胞凋亡， 但 miR-1不會讓 HepG2、 SW620、 HEK-293T、 SAS 等原本細胞內就高度表現miR-1的細胞株產生細胞凋亡。 為了解 miR-1誘導細胞凋亡的分子機制，我們找出 miR-1直接調控並且參與細胞凋亡作用的基因。方法是首先將miR-1送入NPC-TW01 與 HeLa 細胞中，並用 cDNA 表現微陣列(cDNA expression microarray) 篩選會被 miR-1抑制表現的基因，這些基因再到 microRNA 資料庫去篩選可能是 miR-1直接作用的標的基因 (target gene)，最後再進到細胞訊息傳遞資料庫(Ingenuity pathway database) 篩選與細胞凋亡有關的基因。總共有C5、CARD8、FAIM、GRIN2A、TP63及PTMA等6個基因符合條件。再利用Q-RT-PCR及冷光報導基因載體 (luciferase reporter vector) 方法證實miR-1會直接調控PTMA基因。因 miR-1會抑制PTMA的表現且會誘導細胞凋亡，所以我們使用PTMA siRNA代替 miR-1來抑制 PTMA的表現，並觀察PTMA siRNA 抑制PTMA表現後是否也會誘導細胞凋亡。結果單獨抑制PTMA的表現並不會誘導細胞凋亡，但會加速細胞凋亡誘導劑 (apoptotic inducer) 處理過的細胞進行細胞凋亡作用。此外我們使用細胞凋亡抗體陣列 (apoptosis antibody array) 發現miR-1會誘導一些促細胞凋亡蛋白 (pro-apoptotic protein) 的表現，及抑制抗細胞凋亡蛋白 (anti-apoptotic protein) 的表現。 總結本研究證明 miR-1會誘導鼻咽癌及其它癌細胞株進行細胞凋亡。這是一個新的microRNA誘導細胞凋亡的模式。它的機制是miR-1透過直接調控PTMA mRNA及間接調控其它細胞凋亡蛋白方式來進行。 PTMA siRNA會加速被細胞凋亡誘導劑處理過的細胞進行細胞凋亡，因有些細胞凋亡誘導劑 (例如Actinomycin D, camptothecin 及 etoposide) 在臨床上也是癌症化療藥物，所以PTMA siRNA在癌症治療上有當成化療輔助劑的潛在應用價值。 此外我們研究NPC-TW01與NPC-TW01N1細胞的腫瘤生成能力差異時，找到miR-486在兩個細胞間表現有差別。將miR-486送入原本細胞內很少表現miR-486的NPC-TW01N1細胞內，結果會降低NPC-TW01N1細胞的增殖速率及引起細胞凋亡。

關鍵字

鼻咽癌 ； 微核醣核酸 ； 前胸腺素α ； 細胞凋亡 ； 微核醣核酸-1 ； 微核醣核酸-486

並列摘要

MicroRNAs are endogenous non-coding single-strand RNAs, approximately 21 nucleotides in size. MicroRNAs function as negative regulators of gene expression and play important roles in biological processes. MicroRNA-1 (miR-1) has been used as a positive control in some microRNA experiments. Previously in our other microRNA experiment, when miR-1 was transfected into nasopharyngeal carcinoma (NPC-TW01) cells as a positive control, to our surprise, a typical apoptotic process was observed in NPC cells by time-lapse microscopy. Further observations using Annexin V staining, TUNEL staining, caspase assay all confirmed that miR-1 could induce NPC cell apoptosis. Transfection of miR-1 into other cancer cell lines, such as the HeLa, Cal-27, KYSE30, and NPC-TW06 which express low levels of endogenous miR-1, also induced apoptosis, but certain another cancer cell lines, such as SW620, HepG2, SAS, and PC-13 which express high levels of endogenous miR-1, did not show apoptosis. In order to clear the molecular mechanism of miR-1 inducing apoptosis, and identify the miR-1 directly regulated genes that involve apoptosis, cDNA expression microarray analysis of miR-1 transfected NPC-TW01 and HeLa cells were performed. All the down regulated genes were analyzed through microRNA database and Ingenuity pathway database. Six candidate miR-1 regulated genes, C5, CARD8, FAIM, GRIN2A, TP63, and PTMA were found. Using Q-RT-PCR analysis and luciferase reporter assay demonstrate that miR-1 could directly target PTMA mRNA. MiR-1 can induce cell apoptosis and directly down regulate PTMA mRNA so we used PTMA siRNA in place of miR-1 to down regulate PTMA mRNA and observed the apoptotic effect. We found that down regulated PTMA alone could not induce apoptosis, but PTMA siRNA could accelerate the apoptotic process when cells were treated with apoptotic inducers. Furthermore, using apoptosis antibody array, we found that miR-1 could up regulated some pro-apoptotic protein expressions and down-regulated anti-apoptotic protein expressions. We conclude that miR-1 could induce apoptosis in NPC and some other cancer cell lines. This is a novel model of microRNA-induced cell apoptosis. The mechanism is through miR-1 direct targeting of the apoptotic inhibitor PTMA mRNA and through indirect regulation of other apoptotic protein expressions. The PTMA siRNA can accelerate the apoptotic process when cells are treated with apoptotic inducers. The apoptotic inducers: actinomycin D, camptothecin and etoposide are also as chemotherapy drugs in clinical cancer therapy so PTMA siRNA may have potential applications as an adjuvant in cancer chemotherapy. In addition, we investigate the tumorigenesis variation between NPC-TW01 and NPC-TW01N1 and found miR-486 is alteration expression. Transfection of miR-486 into NPC-TW01N1 which expression low levels of endogenous miR-486 can decrease cell proliferation rate and induce cell apoptosis.

並列關鍵字

Nasopharyngeal carcinoma (NPC) ； microRNA ； PTMA (prothymosin alpha ； ProTalpha) ； apoptosis ； miR-1 ； miR-486

參考文獻

1. Kwak PB, Iwasaki S, Tomari Y: The microRNA pathway and cancer. Cancer Sci 2010, 101:2309-2315.

2. Li M, Li J, Ding X, He M, Cheng SY: microRNA and cancer. AAPS J 2010, 12:309-317.

3. Lima RT, Busacca S, Almeida GM, Gaudino G, Fennell DA, Vasconcelos MH: MicroRNA regulation of core apoptosis pathways in cancer. Eur J Cancer 2011, 47:163-174.

4. Zimmerman AL, Wu S: MicroRNAs, cancer and cancer stem cells. Cancer Lett 2011, 300:10-19.

5. Huang Y, Shen XJ, Zou Q, Wang SP, Tang SM, Zhang GZ: Biological functions of microRNAs: a review. J Physiol Biochem 2011, 67:129-139.