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Title: | 探討 Contactin 4 於大腸直腸癌之抑癌分子機轉 Study of contactin 4-mediated molecular mechanism in colorectal cancer |
Authors: | I-Chien Lin 林宜芊 |
Advisor: | 楊雅倩(Ya-Chien Yang) |
Keyword: | 大腸直腸癌,Contactin 4,PTPRG,CREB磷酸化,c-Jun磷酸化,粒線體生合成,細胞凋亡, Colorectal cancer,Contactin 4 (CNTN4),Protein tyrosine phosphatase γ (PTPRG),CREB phosphorylation,c-Jun phosphorylation,apoptosis,mitochondrial biogenesis, |
Publication Year : | 2020 |
Degree: | 碩士 |
Abstract: | 大腸直腸癌在世界癌症死亡率排名位於前三名。本實驗室先前研究提出位於人類第三號染色體3p26.3 之Contactin 4 (CNTN4) 於大腸直腸癌可能扮演抑癌基因的角色。於HCT116細胞株表現CNTN4可抑制細胞增生、固著及非固著依賴性胞落形成的能力;在活體動物實驗中,則顯示CNTN4可抑制HCT116細胞株於裸鼠皮下腫瘤的生成及血管新生;針對抑癌機轉初步推測CNTN4可與Receptor-type protein tyrosine phosphatase gamma (PTPRG)交互作用,進而抑制ERK1/2及其下游CREB之磷酸化,並使PGC-1α表現下降,影響粒線體生合成及降低粒線體DNA拷貝數。本論文為進一步了解CNTN4與PTPRG之相互作用,首先發現表現CNTN4可增加PTPRG蛋白表現。為確認以HCT116/CNTN4穩定表現細胞株所觀察到的抑癌機制,本論文藉由慢病毒轉導CNTN4質體於其他大腸直腸癌細胞株,並建立HCT15/CNTN4及COLO205/CNTN4穩定表現細胞株。藉由分離細胞膜蛋白及細胞質蛋白,發現過表現CNTN4可表現至細胞膜,然而,大部分為CNTN4-V5蛋白表現於細胞質。於HT29、SW620、HCT15及COLO205大腸直腸癌細胞表現CNTN4後皆可抑制細胞增生。於HCT15細胞表現CNTN4可降低CREB及其上游蛋白ERK1/2之磷酸化,下游路徑之PGC-1α表現量亦隨之下降,亦可抑制粒線體DNA拷貝數及粒線體質量;然而,於COLO205細胞表現CNTN4僅可降低CREB之磷酸化和PGC-1α表現量,以及抑制粒線體質量,因於此細胞之上游調控路徑須進一步研究。於臨床檢體檢測CNTN4表現與粒線體DNA含量,發現於大腸直腸癌腫瘤部分CNTN4表現量與粒線體DNA表現量呈現負相關趨勢。因先前利用人類磷酸化蛋白激酶晶片發現HCT116 細胞表現CNTN4後可降低c-Jun磷酸化,目前已知c-Jun受ERK1/2、JNK及p38等MAPK家族蛋白調控,故於HCT116/CNTN4細胞確認c-Jun及其上游分子ERK1/2磷酸化程度下降。已知c-Jun參與細胞凋亡及細胞增生的調控路徑,故進一步確認HCT116/CNTN4細胞的Bcl-2蛋白表現下降,並增加cleaved caspase 3生成,而於HCT116/CNTN4細胞之自發性細胞凋亡增加;當利用H2O2處理HCT116和HCT15細胞,表現CNTN4可促進細胞凋亡,據此推測HCT116細胞表現CNTN4可減少ERK1/2及c-Jun磷酸化,進而抑制Bcl-2表現和增加活化態caspase 3,因而可增加細胞凋亡比例。綜合上述,CNTN4會與PTPRG交互作用影響下游pERK/pCREB/PGC-1α路徑,進而改變粒線體生合成;同時調控pERK/p-c-Jun/Bcl-2路徑,促進細胞凋亡。 Colorectal cancer (CRC) is the third leading cause of cancer death in the world. Our previous study has identified that Contactin 4 (CNTN4) located at human chromosome 3p26.3 plays an important role as a tumor suppressor gene in CRC. Ectopic expression of CNTN4 in HCT116 cells could inhibit cell proliferation, anchorage-dependent and anchorage-independent colony formation in vitro. Furthermore, CNTN4 expression could inhibit the subcutaneous xenograft tumor formation and angiogenesis in nude mice. The mechanism study of CNTN4 indicated that CNTN4 could interact with protein tyrosine phosphatase γ (PTPRG), and then inhibit the phosphorylation of ERK1/2 and its downstream protein, including phospho-CREB and PGC-1α, resulting in the modulation of energy metabolism. CNTN4 would not only promote mitochondrial fission, but also decrease mitochondrial DNA copy number and mitochondrial mass via interaction between PGC-1α and TFAM. In the study, to establish more CRC cell models, lentiviral transduction was conducted to transiently express CNTN4 in HCT15 and SW620 cells. At the same time, I established HCT15/CNTN4, COLO205/CNTN4 and SW620/CNTN4 single stable transfectants. I first showed that overexpressed CNTN4 could present on the membrane, but still mainly in the cytosol. Next, I verified that ectopic expression of CNTN4 in HCT116, HCT15 and COLO205 cells could increase expression of PTPRG protein. CNTN4 expression inhibited cell proliferation in all of transient CNTN4 expression cell lines and single stable clones. Meanwhile, CNTN4 expression decreased mitochondrial DNA copy number and mitochondrial mass via pERK/pCREB/PGC-1α pathway in HCT15 cells, which is consistent with the results observed in HCT116/CNTN4 stable clones. However, CNTN4 expression in COLO205 cells could only downregulate pCREB, PGC-1α, and mitochondrial mass. Therefore, the other molecules involved in the mechanism should be further addressed. In the 50 paired CRC tissues, there was a negative trend between relative CNTN4 expression and mitochondrial DNA copy number. On the other hand, CNTN4 expression in HCT116 cells could reduce c-Jun phosphorylation compared with HCT116/Mock by using Human Phospho-Kinase array. We further confirmed the downregulation of pERK1/2 and phospho-c-Jun in HCT116/CNTN4 transfectants. It is well known that c-Jun can regulate cell proliferation and apoptosis. Therefore, we determined the expression of Bcl-2 and cleaved caspase 3. Ectopic expression of CNTN4 in HCT116 cells indeed downregulated Bcl-2 and increased active caspase 3. As expected, CNTN4 expression could increase spontaneous apoptosis in HCT116 cells and H2O2-induced apoptosis in HCT116 and HCT15 cells, suggesting that CNTN4 might promote apoptosis through pERK/p-c-Jun/Bcl-2 pathway. In conclusion, CNTN4 expression could interact with PTPRG to mediate mitochondrial biogenesis through pERK/pCREB/ PGC-1α pathway and promote apoptosis through pERK/p-c-Jun/Bcl-2 pathway. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59084 |
DOI: | 10.6342/NTU202003449 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 醫學檢驗暨生物技術學系 |
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