以視覺化分析跨平台資料庫探究人類乳癌轉移基因之調控網絡

乳癌在台灣及全球婦女癌症中分別占死亡原因的第一位及第二位，乳癌的死亡率亦隨著腫瘤轉移的發生而增加。在乳癌病患中，腫瘤雌激素受體為陰性(ER-negative)者有較差預後和較高轉移發生率。本研究利用跨平台巨量資料分析 (meta-analysis)方法，匯集353片具有雌激素受體檢測之原位癌及遠處轉移腫瘤的乳癌組織基因晶片，從中找出調控腫瘤轉移之目標基因及其調控路徑。首先，使用weighted gene co-expression network analysis (WGCNA)建立具有轉移特徵的基因模組以代表轉移基因組，再使用Cytoscape將轉移基因組視覺化，建構出腫瘤轉移之基因網絡圖及基因調控路徑。接著，利用人類乳癌MCF-7細胞(雌激素受體陽性)和MDA-MB-231細胞(雌激素受體陰性)驗證目標基因對於癌細胞啟始轉移之細胞移動和侵襲能力的影響。由基因網絡分析結果顯示，雌激素受體陽性乳癌和雌激素受體陰性乳癌具有相同調控轉移之目標基因TNFα (tumor necrosis factor alpha)、PRKCA (protein kinase C alpha)、ICAM1 (intercellular adhesion molecule 1)及VWF (Von Willebrand factor)。在細胞實驗中，利用PRKCA siRNA下調PRKCA表現量後，會伴隨著ICAM1 和VWF的表現量減少，顯示PRKCA會正向調控ICAM1和VWF。此外，利用VWF siRNA降低VWF表現量後，雌激素受體陰性細胞(30.4%, 46.8%)之細胞移動和細胞侵襲能力削弱程度更勝於雌激素受體陽性細胞(8.6%, 30.4%)。另在雌激素受體陰性乳癌網絡分析中得到VWF可透過BMP4 (Bone morphogenetic protein 4)來調控其表現量。在細胞實驗中，利用BMP4 siRNA下調BMP4表現量後，雌激素受體陰性乳癌細胞會伴隨著ICAM1 和VWF的表現量減少，顯示BMP4在雌激素受體陰性乳癌細胞會正向調控ICAM1和VWF。由以上實驗結果顯示，VWF可透過TNFα-PRKCA-ICAM1-VWF路徑以及BMP4-ICAM1-VWF 路徑來影響細胞移動和細胞侵襲能力，因此，抑制VWF可能可作為帶有雌激素受體陰性反應之乳癌病患，降低腫瘤轉移的治療方法。

關鍵字

乳癌；轉移；基因網絡；雌激素受體； PRKCA ； VWF ； BMP4

並列摘要

Breast cancer (BC) is the first and second female leading cause of death respectively in Taiwan and worldwide. Metastasis is a process that cancer cells migrate from one organ to another enhancing the mortality in BC patients. The molecular mechanism is still unclear that BC patients with estrogen receptor (ER)-negative have been observed poorer prognosis and higher incidence of metastasis than ER-positive patients. This study conducted a meta-analysis in 353 primary and metastatic breast tumor microarray datasets of ER-positive and ER-negative patients to identify the potential target genes and their regulatory pathways in the progression of metastasis. This study used weighted gene co-expression network analysis (WGCNA) and Cytoscape to explore metastasis-specific module genes, gene network and regulatory pathways. Two human BC cell lines, MCF-7 (ER-positive) cells and MDA-MB-231 (ER-negative) cells, were used to validate the regulation pattern of these target genes in the aspect of cell migration and invasion to initiate BC metastasis. The results of the gene network analysis showed four target genes to regulate the progression of metastasis in ER-positive and ER-negative breast tumors, including TNFα (tumor necrosis factor alpha), PRKCA (protein kinase C alpha), ICAM1 (intercellular adhesion molecule 1) and VWF (Von Willebrand factor). With the treatment of siPRKCA, this study demonstrated that the reduction of PRKCA attenuated ICAM1 and VWF expression in both ER-positive and ER-negative BC cells. Moreover, the attenuation of VWF by siVWF obviously decreased cell migration and invasion in ER-negative (30.4%, 46.8%) more than in ER-positive BC cells (8.6%, 30.4%). The further results of the ER-negative BC network analysis found that VWF was also regulated by BMP4 (Bone morphogenetic protein 4) in BMP4-ICAM1-VWF pathway. With the treatment of siBMP4, this study indicated that the reduction of BMP4 attenuated ICAM1 and VWF expression in ER-negative BC cells only. This study found TNFα-PRKCA-ICAM1-VWF and BMP4-ICAM1-VWF eventually contributed to cell migration and invasion, and VWF attenuation would be a new therapeutic way to retard metastasis particularly in ER-negative BC patients.

並列關鍵字

breast cancers ； metastasis ； gene network ； estrogen receptor ； PRKCA ； VWF ； BMP4

參考文獻

1. Breast Cancer Facts & Figures 2013-2014. American Cancer Society.

2. McGuire, W.L., Breast cancer prognostic factors: evaluation guidelines. J Natl Cancer Inst, 1991. 83(3): p. 154-5.

3. Perou, C.M., et al., Molecular portraits of human breast tumours. Nature, 2000. 406(6797): p. 747-52.

4. Reis-Filho, J.S. and L. Pusztai, Gene expression profiling in breast cancer: classification, prognostication, and prediction. Lancet, 2011. 378(9805): p. 1812-23.

5. Reis-Filho, J.S. and L. Pusztai, Gene expression profiling in breast cancer: classification, prognostication, and prediction. The Lancet. 378(9805): p. 1812-1823.

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以視覺化分析跨平台資料庫探究人類乳癌轉移基因之調控網絡

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