雙股RNA經由MDA-5在石斑魚細胞中對於mROS及細胞自噬的調控作用

Regulation of dsRNA on MDA-5-dependent induction of mROS and autophagy in grouper cells

洪焌瑋(Chun-Wei Hung)

指導教授 ： 齊肖琪

國立臺灣大學/生命科學院/生命科學系/碩士(2015年)

https://doi.org/10.6342/NTU.2015.02695

摘要

魚類野田病毒(Nodavirus)是正意單股RNA病毒，是造成養殖魚類病毒性神經壞死症(VNN)的病原體，在宿主細胞內複製時，會產生雙股核糖核甘酸(dsRNA)的中間產物。野田病毒會誘發宿主細胞先天性免疫反應，然而，目前未知石斑魚黑色素瘤分化相關抗原5(MDA-5)是否和其在哺乳類系統中有一樣角色，即擔任辨認雙股RNA之受體並引發先天性免疫反應。本研究中，使用dsRNA類似物Poly I:C來模擬RNA病毒在細胞內複製時產生的雙股RNA中間產物，並以對NNV具有感受性的石斑魚腦部細胞株(cGB cell)作為測試轉染Poly I:C的研究系統。結果顯示，Poly I:C轉染cGB細胞可以刺激MDA-5、IRF-3、IRF-7及Mx基因表現，並造成粒線體過氧化物(mROS)和自噬體(autophagosome)表現量顯著上升。一旦MDA-5被siRNA先行抑制，轉染Poly I:C後的Mx基因表現以及細胞自噬體和mROS都顯著性下降，證明Poly I:C可以透過活化MDA-5路徑引發第一型干擾素、細胞自噬作用以及mROS的產生。當cGB細胞先處理mROS促進劑Rotenone，然後轉染Poly I:C，Mx基因表現量會比只轉染Poly I:C組高3倍，因此認為mROS也許有協同Poly I:C之作用去加強Mx基因表現。單單處理Rotenone造成細胞自噬體量上升，說明mROS的上升可刺激細胞自噬體的表現；當細胞先處理mROS抑制劑然後轉染Poly I:C，細胞自噬體表現量下降。當以5 mM 3-MA抑制細胞自噬體的形成，則mROS表現量會顯著上升，因此推測，細胞自噬體可能經由清除損壞粒線體來降低mROS量。本研究是第一篇在魚類細胞株中證明MDA-5可以擔任雙股RNA之辨認受體，並且mROS量升高時可以協同Poly I:C增強第一型干擾素之表現，以及細胞自噬作用可以降低mROS在細胞內的累積量達到負回饋調控。

關鍵字

模式辨認受體 ； 先天性免疫 ； 黑色素瘤分化相關抗原5 ； 第一型干擾素 ； 粒線體過氧化物 ； 細胞自噬作用

並列摘要

Fish nodavirus, the causative agent of viral nervous necrosis (VNN) of cultured marine fish, is a positive-sense single-stranded RNA virus, and will produce d.s.RNA intermediate during its replication. Fish nodavirus can stimulate innate immune responses in host cells; however, it is still unknown if melanoma different-associated protein 5 (MDA-5) can act as a d.s.RNA recognition receptor and activate intracellular innate immunity in grouper as its role in mammalian cells. In this study, poly I:C was used as an analog of viral d.s.RNA intermediate, and was transfected into a grouper brain cell line cGB which is susceptible to nervous necrosis virus. The results indicated that Poly I:C transfection could stimulate the expression of MDA-5, IRF3, IRF7 and Mx genes, and significantly increased the levels of mitochondrial oxidative substance (mROS) and autophagosomes. When MDA-5 was pre-downregulated by siRNA, the levels of Mx gene expression, mROS and autophagosomes stimulated by Poly I:C transfection all significantly decreased, indicating that d.s. RNA can induce type I interferon response, mROS and autophagy through activating MDA-5 pathway. When cGB cells were pretreated with mROS enhancer rotenone and then transfected with Poly I:C, Mx gene expression level became 3 folds higher than that in only Poly I:C-transfected cells. It is thus suggested that high mROS may synergetically enhance Poly I:C-induced Mx gene expression. Rotenone alone could upregulate autophagosome level in the treated cells, revealing that increasing of mROS could stimulate the formation of autophagosomes. When cGB cells were pretreated with mROS inhibitor and then transfected with Poly I:C, the autophagosome level decreased; furthermore, when the formation of autophagosome was inhibited by 5 mM 3MA, mROS level significantly increased, suggesting that autophagosomes may remove damaged mitochondria and downregulate mROS. This is the first report to demonstrate that MDA-5 could act as a d.s.RNA recognition receptor in fish cell line, and high mROS could synergetically enhance the Poly I:C-induced type I IFN response, and autophagy exhibited negative feedback regulation of mROS.

並列關鍵字

Pattern recognition receptor (PRRs) ； innate immunity ； melanoma different-associated protein 5 (MDA-5) ； type I IFN ； Mx ； mitochondria ROS (mROS) ； autophagy

參考文獻

王任翔 (2009)。魚類神經壞死症病毒與細胞自噬的交互作用機制。國立台灣大

陳子翔 (2014)。鹽度對龍膽石斑先天免疫、鈉鉀氯共轉運蛋白及神經壞死病毒

A.M., C., J.F., D. (1996) A receptor for the selective uptake and degradation of proteins by lysosomes. Science 273, 501-503.

Ablasser, A., Bauernfeind, F., Hartmann, G., Latz, E., Fitzgerald, K.A., Hornung, V. (2009) RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat Immunol 10, 1065-1072.

Akira, S., Uematsu, S., Takeuchi, O. (2006) Pathogen Recognition and Innate Immunity. Cell 124, 783-801.