- 學位論文
探討MTA2調控人類子宮頸癌轉移之分子機制
Molecular mechanisms of the metastasis associated protein 2 in cell metastasis of human cervical cancer cells
摘要
Metastasis Associated 1 Family Member 2 (MTA2)是Mi2/NuRD複合體的組成物，主要功能為基因去乙醯化修飾並調控基因轉錄過程。研究顯示MTA2異常表達於癌組織並與癌細胞增殖、轉移和侵襲相關，但子宮頸癌的MTA2生物學功能仍尚未清楚。為了釐清子宮頸癌MTA2生物學功能以及相關調控機轉，透過組織晶片以及TCGA資料庫證實MTA2大量表達於子宮頸癌組織，且與腫瘤惡化程度呈現正相關。後續透過RNAi系統篩選穩定抑制MTA2表現之細胞株發現，抑制MTA2表達降低子宮頸癌細胞爬行和侵襲能力，但不影響癌細胞的生長。蛋白酶晶片結果發現抑制MTA2導致MMP-12表達量下降以及KLK10表現量上升。研究結果顯示抑制MTA2會誘導ASK1/MEK3/p38以及YB1蛋白磷酸化，並促使p-YB1進入細胞核內與AP-1結合，使AP-1無法調控MMP-12的轉錄活性；當阻斷p38訊息傳路徑則抑制YB1磷酸化並回復細胞轉移能力。另一方面，microRNA定序分析發現，抑制MTA2後導致miR-7表達量增加，透過 Targetscan資料庫比對出miR-7會結合上Sp1的3’-UTR並影響Sp1轉譯過程。此外，抑制MTA2表達後造成Sp1無法結合KLK10的5’-flank region進行負向調節KLK10表達。綜合以上研究證實，MTA2扮演重要調控子宮頸癌轉移的角色，可藉由標靶MTA2來減緩腫瘤的轉移，期待MTA2成為新穎標靶基因，應用於臨床子宮頸癌轉移治療。
並列摘要
Metastasis Associated 1 Family Member 2 (MTA2) is a central components of the Mi-2/NuRD complex, which possesses both nucleosome remodeling and histone deacetylase activities. Recent studies have indicated that MTA2 was associated with cells proliferation and metastasis in many cancer cells. The present study investigated the effect of MTA2 knockdown in cell proliferation and metastasis of human cervical cancer cells and the specific mechanism. Herein, we showed that MTA2 expression was correlated significantly with tumor differentiation and Gleason's grade in cervical cancer tissue. Lentivirus mediated short hairpin RNA (shRNA) was used to knockdown MTA2 expression in cervical cancer cell lines. In vitro migration and invasion assay demonstrated MTA2 depletion inhibited cells metastasis ability, but not affect of the cells proliferation. In addition, MTA2 knockdown decreased MMP-12 protein levels and increased KLK10 in cervical cancer cells, as determined by proteinase microarray analysis. Furthermore, western blot analysis indicated ASK1, MEK3/6, and p38 signaling pathways were activated, then induced p-YB1 (phosphorylated of Y box-binding protein 1) nuclear translocation, significantly inhibited AP-1 activity binding to the MMP-12 promoter, and trans-suppressed the expression of MMP-12. In vivo studies using tail intravenous injection in mice models indicated that MTA2 knockdown significantly inhibited metastasis to lung. In addtion, MTA2 depletion increased mirR-7 expression, which targets Sp1, by MicroRNA Sequencing analysis. Mechanistic investigations suggested that MTA2 knockdown inhibited Sp1 expression and interaction with the KLK10 5’-flank region via regulating miR-7 expression. Moreover, the protein levels of Sp1 were up-regulated and down-regulated of KLK10 after transfection with miR-7 inhibitor that reversed cell metastasis and invasion in cervical cancer cells. In conclusion, our findings suggest that MTA2 is important for tumor metastasis through knockdown of MTA2 will regulate p38/YB1/MMP-12 signaling pathway and induce miR-7 expression by targeting Sp1 mediated KLK10 expression.
參考文獻
延伸閱讀
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