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Title: | 探討Contactin 4於大腸直腸癌對細胞能量代謝的影響 Study of Contactin 4 – mediated changes in cellular energy metabolism in colorectal cancer |
Authors: | Po-Lin Chen 陳柏霖 |
Advisor: | 楊雅倩(Ya-Chien Yang) |
Keyword: | 大腸直腸癌,Contactin 4,CREB磷酸化,能量代謝,粒線體, colorectal cancer,Contactin 4,CREB phosphorylation,energy metabolism,mitochondria, |
Publication Year : | 2019 |
Degree: | 碩士 |
Abstract: | 大腸直腸癌在世界癌症死亡率排名位於前三名,顯示需要更多研究了解此疾病。先前本實驗室研究於人類第三號染色體3p26.3 鑑定Contactin 4 (CNTN4) 於大腸直腸癌可能扮演抑癌基因的角色。於HCT116細胞表現CNTN4可抑制細胞增生、固著及非固著依賴性胞落形成的能力;在動物實驗則顯示CNTN4可抑制HCT116細胞於裸鼠皮下腫瘤的生成及血管新生。目前對於CNTN4調控的抑癌機轉尚未明確,先前研究指出CNTN4可能透過結合蛋白PTPRG進行訊息調控路徑,因此本論文先利用實驗室收集51對大腸直腸癌檢體檢測PTPRG,結果顯示在大腸直腸癌PTPRG基因表現與配對正常黏膜組織相比無異;同時以免疫共沉澱法證實PTPRG與CNTN4的交互作用。本論文進一步利用人類磷酸化蛋白激酶晶片檢測單一穩定表現CNTN4之HCT116細胞株,探討可能受PTPRG影響的蛋白磷酸化機制,結果顯示HCT116 細胞表現CNTN4後可大幅降低CREB磷酸化;以西方點墨法亦確認CREB及其上游分子Erk1/2,磷酸化程度明顯和CNTN4表現量呈負相關;當以siRNA抑制CNTN4表現,可恢復CREB及Erk1/2之磷酸化。另外,結合TCGA及GSEA進行生物資訊分析,其結果顯示氧化磷酸化(OXPHOS)、三羧酸循環(TCA cycle)及丙酮酸代謝(Pyruvate metabolism)之能量代謝相關路徑與CNTN4表現呈現負相關。為探討CNTN4是否藉由調控能量代謝抑制HCT116細胞生長,以海馬生物能量測定儀檢測細胞能量代謝狀態,結果顯示CNTN4表現於HCT116細胞明顯弱化粒線體功能運作,後續以西方點墨法及即時定量聚合酶連鎖反應確認HCT116 細胞表現CNTN4可明顯降低粒線體生合成相關的重要轉錄因子PGC-1a表現,而以siRNA抑制CNTN4表現則可恢復PGC-1a表現,同時也觀察到PGC-1a調控下游TFAM表現量亦受CNTN4抑制。利用流式細胞儀及即時定量聚合酶連鎖反應確認HCT116 細胞表現CNTN4後大幅降低粒線體質量及粒線體DNA拷貝數,以siRNA抑制CNTN4表現後,則可恢復之。利用螢光染色觀察粒線體形態變化,當HCT116 細胞表現CNTN4可出現似粒線體分裂聚集現象。綜合以上,我們先確認CNTN4表現能抑制HCT116細胞的CREB及其上游Erk1/2之磷酸化,並且藉由生物資訊分析假設能量代謝可能為CNTN4影響的重要路徑;再以海馬生物能量測定結果支持表現CNTN4可弱化粒線體功能,而其可能是透過抑制CREB磷酸化而減少下游PGC-1a表現,進而影響粒線體生合成及降低粒線體DNA拷貝數之機制。 Colorectal cancer (CRC) is one of the top three leading causes of cancer death in the world. Although the annual incidence due to timely screening has been decreased in these years, its annual mortality keeps the highest among human cancers indicating it still needs more study. Previously, we have identified Contactin 4 (CNTN4) as a novel tumor suppressor gene located at chromosome 3p26.3, which is lost in CRC tumors at a high frequency. Ectopic expression of CNTN4 in HCT116 cells could reduce cell proliferation, anchorage-dependent and anchorage-independent colony formation in vitro. Furthermore, CNTN4 expression could inhibit the tumorigenesis of subcutaneous xenograft and tumor angiogenesis in nude mice. However, the mechanism of CNTN4 is still not clear. It showed CNTN4 might interact with PTPRG to modulate cell signaling pathway. Therefore, we detected and observed the PTPRG gene expression upregulation in 51 pair CRC primary tumor when compaired with normal mucosa. We also confirmed the interaction between CNTN4 and PTPRG by co-immunoprecipitation. In the study, by Human Phospho-Kinase Array, we found ectopic expression of CNTN4 in HCT116 cells could obviously reduce CREB phosphorylation compared with HCT116/Mock and the downregulation of phospho-CREB as well as its upstream regulation, phospho-Erk1/2 was further observed by Western Blot. Additionally, by knockdown of CNTN4 expression with siRNA, the phosphorylation of CREB and ERK1/2 could be restored. On the other hand, we utilized thecolorectal adenocarcinoma dataset of TCGA and GSEA software to perform bioinformatic analysis, and the result showed that CNTN4 expression is nagtively correlated with energy metabolism pathway, including OXPHOS, TCA cycle and pyruvate metabolism. To investigate whether CNTN4 inhibits HCT116 cell proliferation through modulating metabolic activity, we detected energy metabolic status of HCT116 cells expressing CNTN4 by Seahorse analysis. The result showed ectopic expression of CNTN4 decreased mitochondrial function. In addition, we found CNTN4 expression in HCT116 cells reduced PGC-1a expression, an important transcription factor affecting the synthesis of mitochondria, and the phenomen could be restored by CNTN4 knockdown with siRNA. Futhermore, TFAM, a target gene of PGC-1a, were also reduced by CNTN4 expression. Finally, we found ectopic expression of CNTN4 in HCT116 cells reduced mitochondrial mass and mitochondrial DNA. In the cells, the mitochondrial morphology became punctated cluster, which is associated with mitochondrial fission. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71476 |
DOI: | 10.6342/NTU201900417 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 醫學檢驗暨生物技術學系 |
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