臺灣博碩士論文加值系統

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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