探討第三型拓樸異構酶α亞型與HDAC4在p53介導的腫瘤抑制中的交互作用

Abstract

癌症是經由種種因素的誘導與累積所造成的疾病，基因變異與表觀遺傳變異皆是造成癌症的原因。人類拓樸異構酶作為調控DNA拓樸結構問題的重要一員，是參與DNA 複製、重組及轉錄調節的重要酵素。實驗室先前研究發現，人類第三型拓樸異構酶alpha亞型 (hTop3α)與p53 具有交互關係，並經由蛋白質與啟動子的結合調控，促進p53與p21的表現來抑制腫瘤生成。組織蛋白去乙醯酶是表觀遺傳學中重要的調控因子，透過去乙醯化的酵素功能，可使染色質纏繞變緊密、抑制基因的轉錄表現。組織蛋白去乙醯酶4 (HDAC4)具有調節轉錄及細胞生長的能力，已被證實是p53抑癌途徑的新重要成員，藉由抑制p21進而促進大腸癌的發展。hTop3α與HDAC4在癌症發展中皆扮演著重要角色，故本篇我們想要探討hTop3α和HDAC4在p53介導的腫瘤抑制中的相互作用。首先，透過免疫共沉澱實驗探討hTop3α和HDAC4兩者是否具有交互作用，發現hTop3α可與HDAC4能形成複合體，並從定量即時聚合酶鏈鎖反應結果發現hTop3α缺失會導致HDAC4、p53、p21的基因表現量下降；相反的，當hTop3α過表現時，會使HDAC4、p53、p21的基因、蛋白質表現量上升。此外，我們也藉由軟瓊脂培養基探討hTop3α對腫瘤生成的影響，發現hTop3α缺失會使細胞群落數增加。最後，我們利用p53缺失的細胞進行相關實驗，發現hTop3α對HDAC4的調控並不受p53的影響。此外，hTop3α對p53及HDAC4都有正向的調控，然而p53與HDAC4在p21的表現有不同的影響，從實驗結果可以發現p53帶來的正向調控較HDAC4所帶來的負向調控更為顯著。綜合以上，我們觀察到hTop3α對HDAC4具有正調控，且在p53缺失的細胞中更為顯著。

Cancer is a disease caused by the induction and accumulation of various factors. Both genetic variation and epigenetic variation are the causes of cancer. Human topoisomerases, as an important member of regulating DNA topological problems, are important enzymes involved in DNA replication, recombination and transcriptional regulation. Previous research in our lab found that human topoisomerase III alpha (hTop3α) has physical interaction with p53, which enhances the recruitment of p53 to p21 promoter binding site and promotes the expression of p53 and p21 to inhibit tumorigenesis. Histone deacetylases are important regulatory factor in epigenetics. Through the enzyme function of deacetylation, chromatin structure can be tightened and the transcription performance of genes can be suppressed. Histone deacetylase 4 (HDAC4) has the ability to regulate transcription and cell growth. It has been demonstrated to be a new important member of the p53 tumor suppressor pathway, which promotes the development of colorectal cancer by inhibiting p21. Both hTop3α and HDAC4 play an important role in cancer development, so we want to explore the interaction between hTop3α and HDAC4 in p53-mediated tumor suppression pathway. First, we explored whether the hTop3α and HDAC4 have interaction through co-immunoprecipitation experiment and found that hTop3α can form a complex with HDAC4, thereby may regulate its expression or function. From the quantitative real-time polymerase chain reaction results, we found that hTop3α defect leaded to the decrease gene expression of HDAC4, p53, p21; conversely, when hTop3α was overexpressed, the gene and protein expression of HDAC4, p53, and p21 increased. Furthermore, we explored the effect of hTop3α on tumorigenesis by soft agar assay, and found that the hTop3α defect will increase the colony numbers. We also used p53-deficient cells to perform experiments and found that hTop3α-regulated HDAC4 is p53-independent. In addition, hTop3α can positively regulate p53 and HDAC4, but p53 and HDAC4 have oppositely effects on p21 expression, and the p21 expression showed that the positively regulation by p53 was stronger than the negatively regulation by HDAC4 in hTop3α-mediated manner. In summary, we observed that hTop3α positively regulates HDAC4, and is more significant in p53-deficient cells.