人類RNA靜默複合體(RISC)組成蛋白質參與癌化進程之功能性探討

Functional Analysis of RCK/p54, a Human RISC Component, in Cancer Progression.

陳妍君(Yen-Chun Chen)

指導教授 ： 朱家瑩

國立臺灣大學/生命科學院/動物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.11200

摘要

RCK/p54是DEAD-box蛋白質的一種，在轉錄後調控(post-transcriptional regulation)基因表現扮演重要角色。在酵母菌、非洲爪蟾、以及果蠅中的研究顯示出RCK/p54的同源基因會參與基因的轉譯抑制(translational repression)進而調控基因表現。在人類miRNA路徑中，RCK/p54會與Ago2及其他蛋白質形成靜默蛋白複合體(miRNA-induced silencing complex)，抑制目標RNA轉譯或是將其儲存。在人類大腸癌研究發現RCK/p54大量表現於癌細胞中。除此之外，RCK/p54也高度表現在C型肝炎引起的肝癌細胞中。然而，RCK/p54在癌症中所扮演的角色及其機制皆不甚清楚。為了探討RCK/p54於癌症中的所扮演的角色及其機制，我們利用次世代定序以及定量蛋白質體分析與RCK/p54蛋白質結合的RNA，及降低RCK/p54表現量後所造成的下游RNA及蛋白質表現量變化。研究結果顯示癌細胞的轉移能力會隨著RCK/p54過量表現而隨之提高。此外，藉由比對RCK/p54蛋白質所結合的RNA與蛋白質體的資料，我們發現RCK/p54可能直接經由轉譯抑制調控NUDT21以及GPRC5A兩個基因的蛋白表現。經由次世代定序分析去除RCK/p54時HeLa細胞的基因表現變化圖譜，發現當細胞去除RCK/p54時，MAGEE1的表現量上升，顯示其表現可能間接由RCK/p54調控。本研究顯示細胞轉移程度與RCK/p54表現量成正相關，並且可能是經由調控NUDT21、GPRC5A以及MAGEE1進而影響癌症細胞的惡性化。

關鍵字

轉錄後調控 ； RCK/p54 ； 轉錄抑制 ； 癌症 ； 惡性化

並列摘要

RCK/p54 is a member of DEAD-box protein family, and it plays a role in post-transcriptional regulation. Previous studies of RCK/p54 homologues in yeast, Xenopus and Drosophila showed that RCK/p54 participated in regulation of translational repression and moderates RNA stability. RCK/p54 is also involved in miRNA pathway. It interacts with Ago2 in miRNA-induced silencing complex (miRISC), which represses mRNA translation or stores target mRNA in processing body (P-body). Several studies in cancer biology showed that RCK/p54 is highly expressed in tumors. However, the detail mechanism of RCK/p54 in regulation of cancer progression is unrevealed. In this study, we aim to identify the role of RCK/p54 in cancer metastasis. We found out that migration of cancer cell was increased in cells overexpressing RCK/p54. Comparing the deep sequencing result of RCK/p54-associated mRNA and the change of proteomic profile in RCK/p54- depleted cell, we found that RCK/p54 may directly regulate NUDT21 and GPRC5A. MAGEE1 mRNA was increased in RCK/p54-depleted cell, but did not associate with RCK/p54, suggesting that MAGEE1 is a potential downstream target of RCK/p54. These findings suggest that RCK/p54 protein regulates cancer metastasis through various pathways and the potential targets or downstream of RCK/p54 were identified.

並列關鍵字

post-translational repression ； RCK/p54 ； translational repression ； cancer ； cell migration

參考文獻

Akao, Y., Yoshida, H., Matsumoto, K., Matsui, T., Hogetu, K., Tanaka, N., and Usukura, J. (2003). A tumour-associated DEAD-box protein, rck/p54 exhibits RNA unwinding activity toward c-myc RNAs in vitro. Genes to cells : devoted to molecular & cellular mechanisms 8, 671-676.

Bergkessel, M., and Reese, J.C. (2004). An essential role for the Saccharomyces cerevisiae DEAD-box helicase DHH1 in G1/S DNA-damage checkpoint recovery. Genetics 167, 21-33.

Boersema, P.J., Raijmakers, R., Lemeer, S., Mohammed, S., and Heck, A.J. (2009). Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nature protocols 4, 484-494.

Caruthers, J.M., and McKay, D.B. (2002). Helicase structure and mechanism. Current opinion in structural biology 12, 123-133.

Chang, L.C., and Lee, F.J. (2012). The RNA helicase Dhh1p cooperates with Rbp1p to promote porin mRNA decay via its non-conserved C-terminal domain. Nucleic acids research 40, 1331-1344.