探討氯化鎳透過活性氧誘導上皮-間質型態轉換與細胞自噬之表觀遺傳機制

Epigenetic mechanisms of nickel chloride-induced epithelial-mesenchymal transition and autophagy via reactive oxygen species

吳芝嫻(Chih-Hsien Wu)

指導教授 ： 柯俊良

中山醫學大學/醫學院/醫學研究所/博士(2014年)

https://doi.org/10.6834/CSMU.2014.00066

摘要

背景：上皮-間質型態轉換(EMT)被認為是腫瘤轉移與肺纖維化的重要過程，鎳化合物被歸類為致癌物且能促使組織纖維化。我們過去研究發現氯化鎳會誘導人類支氣管上皮細胞進行EMT，然而，氯化鎳誘導EMT過程與癌化的機制目前仍不清楚，本研究主要目的為釐清其中機轉。
材料與方法：運用Western blot分析經氯化鎳處理後的細胞EMT標記表現量之變化，及以甲基化專一性聚合酶連鎖反應進行定量分析與亞硫酸鹽修飾DNA後定序檢測E-cadherin啟動子甲基化狀況。利用不同活性氧專一性偵測劑配合流式細胞儀分析鎳所產生的活性氧種類。氯化鎳處理的細胞以Agilent SurePrint G3 Human V2 GE 8×60K微陣列檢測mRNA與HmiOA4.1微陣列檢測miRNA表現量的變化，最後用Gene ontology (GO)、MetaCore與Kyto Ecyclopedia Genes and Genomes (KEGG)軟體分析miRNA與mRNA表現差異的交互關係和相關途徑。
結果：使用抗氧化劑N-乙醯基半胱氨酸(NAC)與shRNA抑制 HIF-1α表現皆可以有效的逆轉氯化鎳誘導的EMT現象與HIF-1α表現。鎳誘導活性氧生成的種類主要為氫氧自由基。以染色質免疫沉澱 (ChIP) 分析發現氯化鎳促進 HIF-1α、Snail及Slug結合至E-cadherin 啟動子上。由甲基化專一性聚合酶連鎖反應進行定量分析與亞硫酸鹽修飾DNA後的定序結果發現，氯化鎳透過活性氧誘導E-cadherin啟動子高度甲基化。此外，氯化鎳誘使活化態caspase-3及LC3-II表現量增加，又使聚集型態的JC-1轉變成單體型態。以shRNA抑制自噬作用基因LC3A表現，則促進氯化鎳誘導細胞凋亡。微陣列結果顯示，經氯化鎳處理的細胞相較於控制組，其增加1.74倍的miRNAs有18個及減少1.74倍的miRNAs有28個；而增加2倍的mRNAs有2131個及減少2倍的mRNAs有3039個。TGF-β抑制劑(SB525334)可部分減少氯化鎳誘導的miR-4417表現，當miR-4417過度表現或以shRNA抑制miR-4417的標靶基因TAB2時，皆可提高fibronectin表現量。另外，在異種移植實驗中發現，灌食氯化鎳能促進經由尾靜脈注射BEAS-2B細胞的裸鼠，其肺部生成腫瘤。
結論：氯化鎳透過產生活性氧來促使E-cadherin啟動子高度甲基化導致喪失了E-cadherin與藉由增加miR-4417進而誘導 fibronectin表現，誘導細胞進行上皮-間質型態之轉換。此外，氯化鎳也會造成細胞自噬與細胞凋亡作用。本研究提出氯化鎳在促進肺纖維化及腫瘤形成過程的細胞分子機制之影響。

關鍵字

氯化鎳 ； 上皮-間質型態轉換 ； 活性氧 ； 表觀遺傳 ； 細胞自噬

並列摘要

Backgrounds: Epithelial-mesenchymal transition (EMT) has been considered an event in the pathogenesis of tumor metastasis and lung fibrosis. Nickel compounds are classified as carcinogens and have been shown to be associated with tissue fibrosis. Our previous study reported that nickel chloride (NiCl2) induces EMT. However, the mechanisms of Ni-induced EMT process and carcinogenesis are still unclear. The aim of this study was to investigate the mechanisms of Ni-induced EMT and tumorigenesis.
Materials and Methods: NiCl2-induced EMT markers expressions were analyzed by Western blot. The quantitative real-time methylation-specific PCR (QMSP) and bisulfite sequencing were used to examine the promoter methylation of E-cadherin. The types of reactive oxygen species (ROS) generation by NiCl2 were stained by specific detecting reagents and analyzed by flow cytometry. The mRNA and miRNA profiles in NiCl2-treated cells were established using Agilent SurePrint G3 Human V2 GE 8×60K and HmiOA4.1 microarrays, respectively. The differentially expressed miRNAs and mRNAs related miRNA-gene network have been analyzed by Gene ontology (GO), MetaCore, and Kyto Ecyclopedia Genes and Genomes (KEGG).
Results: The potent antioxidant N-Acetyl cysteine (NAC) and HIF-1α silence by shRNA reversed NiCl2-elicited EMT and HIF-1α expression. The major type of Ni-induced ROS formation is hydroxyl radical. The results of chromatin immunoprecipitation (ChIP) assay have shown that HIF-1α, Snail and Slug bind to E-cadherin promoter. Promoter hypermethylation of E-cadherin, determined by QMSP and bisulfite sequencing, was also induced by NiCl2 via ROS generation. In addition, NiCl2 up-regulated the active form of caspase-3 and the expression of LC3-II. Treatment with NiCl2 converted JC-1 from aggregate form to monomer form. Silencing of autophagy-related gene, LC3A, promotes NiCl2-induced apoptosis. Comparison to control group, the microarrays threshold identified 18 up-regulated and 28 down-regulated miRNAs with fold changes of ≥ 1.74 meanwhile 2431 up-regulated and 3039 down-regulated mRNAs with fold changes of ≥ 2 in expression. SB525334, a TGF-β inhibitor, partially inhibited the up-regulation of miR-4417 by NiCl2. Overexpression of miR-4417 and silencing of the miR-4417-targeted gene TAB2 induced the expression of fibronectin. Moreover, oral administration of nickel promoted lung tumor growth in nude mice that had received BEAS-2B cells by intravenously injection.
Conclusion: NiCl2 induces loss of E-cadherin through its promoter hypermethylation and up-regulation of fibronectin via induction of miR-4417 via ROS generation. In addition, NiCl2 promotes autophagy and apoptosis. This results shed new light on the contribution of NiCl2 to fibrogenesis and carcinogenesis.

並列關鍵字

Nickel chloride ； epithelial-mesenchymal transition ； reactive oxygen species ； epigenetic ； autophagy

參考文獻

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