探討 Contactin 4對大腸直腸癌之促血管新生因子 uPA 的調控及表現分泌型 Contactin 4 蛋白

Study of Contactin 4-modulated pro-angiogenic factor uPA expression in colorectal cancer and expression of secretory Contactin 4 protein

蕭聿昕(Yu-Xin Xiao)

指導教授 ： 楊雅倩

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201702204

摘要

大腸直腸癌於全世界及台灣均是癌症死亡原因的前三名，且在台灣大腸直腸癌已攀升為每年發生率最高之癌症。本實驗室先前的研究，於人類第三號染色體 3p25.3-p26.3 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、固著依賴性及非固著依賴性胞落形成的能力，同時，在活體動物實驗則發現表現 CNTN4 可以抑制裸鼠皮下腫瘤的生成。目前 CNTN4 的功能尚未清楚，本論文分為三個部分探討：(一) 以血管新生為主題，探討 CNTN4 能否透過抑制血管新生，以減緩腫瘤之生成，結果顯示，於異種移植腫瘤之小鼠模式發現 CNTN4 可減少腫瘤的血管密度，而於體外細胞模型則發現 CNTN4 可抑制促血管新生因子 uPA 之表現。(二) 以 siRNA 策略降低 CNTN4 表現後，進行體外細胞增生能力分析，以驗證 CNTN4 的抑癌角色，結果顯示，抑制 CNTN4 表現可以恢復 HCT116 細胞之增生能力。(三) 為了進一步了解 CNTN4 於細胞外之功能，故建構分泌型 CNTN4 表現質體，以轉染方式送入大腸直腸癌細胞株 HCT116，使其表現分泌型 CNTN4，並進行體外細胞增生能力分析，結果顯示，含有分泌型 CNTN4 之條件培養液可抑制細胞增生之能力。綜合以上結果，CNTN4 可抑制 uPA 之表現，並降低腫瘤的血管密度，且細胞膜之 CNTN4 與分泌型 CNTN4 皆可抑制大腸直腸癌細胞株 HCT116 之增生能力，對 HCT116 細胞具有生長抑制的功能。

關鍵字

大腸直腸癌 ； Contactin 4 ； 血管新生 ； uPA ； 細胞增生 ； 分泌型 CNTN4

並列摘要

Colorectal cancer (CRC) is one of the top three leading causes of cancer death in the world and Taiwan. Nowadays, CRC is the cancer with the highest annual incidence in Taiwan. In our previous study, we have identified Contactin 4 (CNTN4) as a novel tumor suppressor gene located at chromosome 3p25.3-p26.3 that 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 in nude mice. The function of CNTN4 is still not clear currently. In the study, there are three specific aims. First, we focused on tumor angiogenesis. We investigated whether CNTN4 could inhibit angiogenesis to suppress tumorigenesis. The results showed that CNTN4 could decrease the microvessel density in the xenograft tumor mouse model and inhibit the expression of uPA, which is one of the pro-angiogenic factors. In the second part, by using siRNA strategy, we knockdowned CNTN4 expression of HCT116 cells and performed in vitro cell proliferation assay. The results showed that inhibition of CNTN4 expression could restore cell proliferation and thus confirmed the tumor suppressor function of CNTN4. In the third part, to explore the function of CNTN4 cleaved and shed from cell surface, we constructed a secretory CNTN4-expressing plasmid, pSecTag2 Hygro B/sCNTN4, and then transiently transfected it into CRC cells, HCT116. By in vitro cell proliferation assay, we demonstrated that conditioned medium containing secretory CNTN4 could inhibit cell proliferation. Taken together, CNTN4 could inhibit the expression of uPA and decrease the microvessel density of xenograft tumors. Either membrane-anchored CNTN4 or secretory CNTN4 could inhibit cell proliferation of HCT116. Accordingly, CNTN4 displays growth inhibition function on CRC cells.

並列關鍵字

Colorectal cancer ； Contactin 4 ； Angiogenesis ； uPA ； Cell proliferation ； Secretory CNTN4

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