- 學位論文
M1巨噬細胞對於p53突變型肺腺癌細胞的抑癌作用
The anti-tumor effect of M1 macrophage on mutant p53 in lung adenocarcinoma cells
摘要
腫瘤相關巨噬細胞分群在腫瘤微環境中扮演著關鍵的角色。其中，M1巨噬細胞(M1 Macrophage, M1)具有抑制腫瘤的能力。另一方面，轉錄因子p53的突變會使p53從原先的抑癌基因轉變成致癌基因，而這種突變亦廣泛地在人類腫瘤中被發現。實驗室先前研究已知M1會透過促進正常p53堆積，使帶有野生型p53的A549肺癌細胞經由自噬作用而凋亡，但對於具有突變型p53的腫瘤細胞是否有抑制效果仍未知。 首先我們利用p53缺失的非小細胞肺癌細胞株H1299，分別轉送突變型p53-R175H和p53-R273H，接著以THP-1單核球細胞株分化成的M0、M1巨噬細胞，個別收集巨噬細胞培養液(condition medium, CM)後，以CM培養H1299細胞進行後續實驗。我們發現M1 CM培養能夠降低突變型p53所促進的細胞遷移與增生能力，在克隆形成試驗中，M1 CM具有減少克隆數量的能力。而在帶有內生突變型p53的細胞株H1975(p53R273H)和CL1-0 (p53R248W)中，M1 CM對於細胞同樣具有抑制克隆形成的效果，並且我們發現突變型p53的mRNA和蛋白質表現皆在M1 CM培養後下降。另以p53靜默試驗減少表現細胞中突變型p53之後，也確認細胞生長能力會受到突變型p53表現減少而降低。在M1 CM組別中，我們發現突變型p53 mRNA有較高的降解率，同時也觀察到結合在p53 mRNA 3’UTR上維持mRNA穩定的調控蛋白－Wig-1表現亦在M1 CM培養組別中下降。為了找出M1 CM如何調控WIG-1，我們利用MetaCore分析M1 CM中的轉錄蛋白體(transcriptome) 並且找到IFN-β。以中和型IFN-β抗體添加在M1 CM中能有效使WIG-1和突變型p53所減少的表現回復，而以IFN-β培養細胞時，則能夠降低WIG-1和突變型p53的表現。此外，我們還發現在缺乏3’UTR的轉送突變型p53 H1299細胞株中，p53蛋白表現亦會受到M1 CM所抑制，同時伴隨著蛋白質半衰期下降。顯示了M1 巨噬細胞對於突變型p53有著多重的調控機制。 在本篇實驗中，我們發現M1巨噬細胞透過增加突變型p53 mRNA和蛋白質降解減少突變型p53的表現，最終抑制了突變型p53的促癌能力。而M1 CM其中的IFN-β能透過抑制WIG-1表現，加速了mRNA的降解率，最終導致p53表現減少、促癌能力下降。
並列摘要
Tumor-associated macrophages (TAMs) are critical factors for the plasticity in the tumor microenvironment. Among the TAMs subtypes, M1 macrophage (M1) has the anti-tumor capability. The well-known tumor suppressor p53 is frequently mutated in human cancers, leading to gain of the oncogenic functions. Our preliminary study showed that M1 macrophages are able to induce lung cancer cells apoptosis through stabilizing p53 protein. However, the effects of M1 macrophage on mutant p53 are still unclear. First, the mutant p53R175H and p53R273H were stably ectopic-expressed in H1299 cells, and then the cells were treated with conditioned medium (CM) from the THP-1-polarized M0 and M1 macrophages. In functional assays, M1 CM decreased the cell viability, cell proliferation, and both anchorage-independent and anchorage-dependent colony formations that were all elevated by mutant p53 in H1299 cells. On endogenous mutant p53 cells, M1 CM not only reduced the anchorage-dependent colony formation, but also decreased the mRNA and protein of mutant p53 in H1975(p53R273H) and CL1-0(p53R248W) cells. The cell growth abilities suppressed by silencing mutant p53 indicates the gain-of-function of mutant p53 in H1975 and CL1-0 cells. Furthermore, the mutant p53 mRNA showed higher decay rate in M1 CM than in M0 CM treatment. We noticed that M1 CM also reduced WIG-1 expression, whose functions as a p53 mRNA stabilizer via binding 3’UTR of mRNA. To identify by which ligand-induced signaling reduces WIG-1, the transcriptome of M1 macrophage was analyzed with MetaCore and IFN-β was identified. Indeed, neutralizing IFN-β could reverse the decrease of WIG-1 and p53 caused by M1 CM. Otherwise, IFN-β addition suppressed mutant p53 and WIG-1 expression. However, the expression of exogenous mutant p53 lacking 3’UTR is still reduced by M1 CM in H1299 cells. Here, we presented the data revealing that p53R175H protein has shorter half-life in M1 CM compared to M0 CM. Collectively, the results demonstrated the complex influences of M1 macrophages on mutant p53. This study showed that M1 macrophages suppress the oncogenic functions of mutant p53 by inhibiting p53 via accelerating p53 decay at mRNA and protein levels. Obviously, IFN-β in M1 CM is a key mediator which down-regulates WIG-1 expression, following accelerated the decay rate of mutant p53, finally decreased the mutant p53 expression and cell malignancy.
參考文獻
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