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研究生： 陳貝瑜
Chen, Pei-Yu.
論文名稱： PD-L1之基因及表觀基因調控於幹細胞分化與肺癌細胞進程之研究
Genetic and Epigenetic Regulation of PD-L1 in Stem Cell Differentiation and Lung Cancer Cell Progression
指導教授： 周裕珽
Chou, Yu-Ting
口試委員: 蕭世欣
Hsiao, Shih-Hsin
林賜恩
Lin, Sey-En
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 生物科技研究所
Biotechnology
論文出版年： 2018
畢業學年度： 106
語文別： 英文
論文頁數： 59
中文關鍵詞： 表觀遺傳調控 、細胞分化 、肺癌細胞異質性 、肺癌進程
外文關鍵詞： PDL1, epigenetic regulation, stem cell differentiation, lung cancer cell heterogeneity, TGFBI, lung cancer progression
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    • 儘管PD-L1在免疫療法中已被當成生物指標與治療標的，該基因在細胞分化及癌症中受表觀基因及基因調控尚不明確。在這份研究中，我們發現在幹細胞分化及重編程（reprogramming）中，PD-L1會被表觀基因的H3K27乙醯化（H3K27 acetylation, H3K27ac）調控其表現量。此外，H3K27ac亦影響了PD-L1在肺癌細胞中的表現量異質性。臨床資料揭示大多數肺癌腫瘤有PD-L1的拷貝數變異刪除（copy number variation deletion），且從肺癌細胞中剔除PD-L1表現的實驗結果暗示了PD-L1在肺癌中扮演抑制癌細胞生長的角色。藉由分析PD-L1剔除後的其餘基因表現量，我們發現TGFBI為PD-L1的下游標的，並和PD-L1一同調控肺癌細胞的生長。更甚者，我們發現PD-L1-TGFBI信號會受TGF-β刺激而影響CITED2-p21CIP對細胞生長的調控。綜觀而言，我們的研究確立了PD-L1會因表觀基因及基因調控而影響正常細胞的分化、腫瘤細胞異質性，及癌細胞進程。

    PD-L1 has become a biomarker and therapeutic target in immunotherapy. However, how PD-L1 is regulated by genetic and epigenetic factors to affect cellular differentiation and cancer cell progression is unclear. In this study, we revealed that PD-L1 expression is under H3K27ac epigenetic regulation during stem cell differentiation and reprogramming. We observed that lung cancer cells display heterogeneous PD-L1 expression, which is associated with H3K27ac modification. We discovered that lung tumors harbored PD-L1 deletion, and silencing PD-L1 in lung cancer cells promoted cellular proliferation, suggesting an inhibitory role of PD-L1 in lung cancer growth. Through gene profiling analysis, we identified TGFBI as a downstream target of PD-L1, knockdown of which promoted lung cancer cell growth. We observed that PDL-TGFBI signaling is induced by TGF-β stimulation to affect CITED2-p21CIP mediated cell growth control. Together, our findings support that PD-L1 is under genetic and epigenetic controls to patriciate in normal cell differentiation, tumor cell heterogeneity, and cancer cell progression.

    摘要 1 abstract 2 致謝 3 目錄 4 Introduction 6 Lung cancer 6 Immunotherapy 6 PD-L1 8 Epigenetic regulation and cell differentiation 9 TGF-β signaling 10 TGFBI 11 CITED2 12 Materials and Methods 13 Bioinformatic analysis 13 Quantitative real-time PCR (qPCR) 14 Cell culture 14 Chromosomal Immunoprecipitation (ChIP) assay 15 Lentiviral transfection 15 Immunoblotting 16 Clonogenic assay 17 Statistical analysis 17 Table 1: Sample names and origin of ENCODE H3K27ac pattern 19 Table 2: qPCR primers 20 Table 3: Primers of PD-L1 PCR 21 Results 22 PD-L1 expression and its H3K27 acetylation in normal tissues 22 PD-L1 expression is under epigenetic control during cell differentiation and reprogramming. 22 Epigenetic regulation of PD-L1 contributes to its heterogeneous expression in lung cancer cells. 23 PD-L1 regulates cell growth in NSCLC 24 TGFBI, a downstream of PD-L1, regulates cell growth in ADC 26 TGF-β upregulates the expression of PD-L1 and TGFBI in ADC cells 26 PD-L1 regulates ADC cell growth through TGF-β-PD-L1-TGFBI-CITED2 axis 27 Discussion 29 Epigenetic regulation of PD-L1 heterogeneity 29 TGF-β and PD-L1 in NSCLC 30 PD-L1 and TGFBI 31 CITED2’s function in PD-L1-TGFBI-mediated cell growth arrest 32 Figures 33 Reference 49

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