Inhibition of Melanoma Cell Growth and Pulmonary
Metastasis in an ex-vivo Animal Model Using Adenovirus
Armed Short Hairpin RNA Targeting Transforming Growth
Factor-Beta1 Encoding mRNA
Kuo-Feng Tai1 Sih-Wei Tseng2 Mao-Chin Hung3
Fan-Chieh Meng3 Wei-Chieh Sun3 Chien-Hsing Wang4
1 General Education Center, Tzu Chi College of Technology
2 Department of Medical Research Mackay Memorial Hospital
3 Department of Radiological Technology, Tzu Chi College of Technology
4 Department of Sugery, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien,
Taiwan
Abstract
Objectives: In this study, we used an adenovirus-based shRNA expression system and
successfully constructed Ad/TGF-β1-RNAi which mediated the RNAi for TGF-β1 gene
silencing. We examined the effects of TGF-β1 protein knockdown by RNA interference on the
growth and metastasis of melanoma in C57BL/6 mice induced by the B16F0 cell line.
Materials and Methods: The TGF-β1 hairpin oligonucleotide was cloned into
adenoviral vector. The resulting recombinant adenoviruses infected murine melanoma cell line,
B16F0, and designated as B16F0/TGF-β1-RNAi cells. The blank adenoviral vector also
infected B16F0 cells and designed as B16F0/vector-control cells served as a control. Three
million wild type B16F0 cells, B16F0/vector-control cells and B16F0/TGF-β1-RNAi cells
were injected subcutaneously into the right flanks of adult female syngeneic mice C57BL/6
respectively. C57BL/6 mice were evaluated for pulmonary metastasis following tail vein
injection of two million B16F0 cells, B16F0/vector-control cells and B16F0/TGF-β1-RNAi
cells.
Results: TGF-β1 expression was reduced in B16F0/TGF-β1-RNAi cells compared with
B16F0 cells and B16F0/vector-control cells. The tumor sizes were 756.09 ± 65.35 mm3,
798.48 ± 78.77 mm3 and 203.55 ± 24.56 mm3 at the fourteenth day in the mice receiving
B16F0 cells, B16F0/vector-control cells and B16F0/TGF-β1-RNAi cells respectively. TGF-β1
knockdown in B16F0 cells enhanced the infiltration of CD4+ and CD8+ T cells in the tumor
regions. The pulmonary metastasis also reduced significantly on 14 day and 21day in mice
injected with B16F0/TGFβ1-RNAi tumors.
Conclusions: Our results showed that Ad/TGF-β1-RNAi could induce silencing of the
TGF-β1 gene effectively. Silencing of TGF-β1 expression in B16F0 cells by RNA interference
can inhibit the growth and metastasis of melanoma cells in syngeneic C57BL/6 mice. RNA
interference targeting TGF-β1 by adenoviral vector might be a promising vector for cancer
therapy.
Keywords：Adenoviral vector, RNA interference, TGF-β1znmelanoma.
以腺病毒為載體針對轉型生長因子β1 進行
核糖核酸干擾抑制作用能縮減黑色素瘤細胞
在體內的生長並抑制腫瘤細胞轉移
戴國峰1 曾斯偉2 洪茂欽3 孟凡傑3 孫暐傑3 王健興4
1 佛教慈濟技術學院通識教育中心 2 馬偕紀念醫院醫學研究部
3 佛教慈濟技術學院放射技術系 4 佛教慈濟綜合醫院暨慈濟大學 外科
摘要
目的：核糖核酸干擾抑制技術被應用在抑制特定基因的表現上，已是一個相當重要
的技術，本研究之目的是要以腺病毒載體攜帶轉型生長因子β1 寡核苷酸，藉此達成抑
制小黑鼠黑色素瘤轉型生長因子β1 的表現，並觀察利用這樣的方法在抑制轉型生長因
子β1 的表現後，黑色素瘤在小黑鼠皮下的生長與轉移情形。
材料與方法：我們將設計好的轉型生長因子β1 寡核苷酸構築到一個腺病毒載體，
將此重組腺病毒稱為Ad/TGF-β1-RNAi，之後感染小黑鼠黑色素瘤細胞—B16F0 細胞
株，感染後的細胞命名為B16F0/TGF-β1-RNAi 細胞，另外也將不帶任何轉殖基因的空
白腺病毒載體感染小黑鼠黑色素瘤細胞並命名為B16F0/vector-control 細胞，作為實驗中
的載體控制組細胞，分別將上述3 × 106 個野生型腫瘤細胞（B16F0）、載體控制組細胞
（B16F0/vector-control）或是抑制TGF-β1 表現的腫瘤細胞（B16F0/TGF-β1-RNAi）植入
六至八週大具正常免疫力的小黑鼠（C57BL/6）皮下，比較這些腫瘤細胞在小黑鼠皮下
的生長情形。上述細胞也以尾靜脈注射方式注射到C57BL/6 老鼠體內，來評估這些腫瘤
細胞轉移到肺臟的能力。
結果：與野生型腫瘤細胞（B16F0）或是載體控制組細胞（B16F0/vector-control）比
較，B16F0/TGF-β1-RNAi 細胞內轉型生長因子β1 的表現明顯減少，這三株細胞在體外
培養時生長速率差不多，在植入小鼠皮下後第十四天，植入野生型腫瘤細胞（B16F0）
的小黑鼠腫瘤細胞其大小為756.09 ± 65.35 mm3 ， 植入載體控制組細胞
（ B16F0/vector-control）的小黑鼠腫瘤細胞其大小為798.48 ± 78.77 mm3，植入
B16F0/TGF-β1-RNAi 細胞的小黑鼠腫瘤細胞其大小則只有203.55 ± 24.56 mm3，明顯降
低許多，以one-way ANOVA 統計分析 p<0.01，在B16F0/TGF-β1-RNAi 腫瘤內發現有明
顯的CD4+及CD8+T 細胞浸潤。尾靜脈注射B16F0/TGF-β1-RNAi 腫瘤細胞的老鼠，在注
射後第14 及第21 天都觀察到轉移至肺部的腫瘤細胞明顯減少。
結論：我們所設計帶著轉型生長因子β1 寡核苷酸的重組腺病毒在感染黑色素瘤細
胞（B16F0 細胞株）後，明顯抑制B16F0 細胞株轉型生長因子β1 的表現，並抑制腫瘤
細胞在小黑鼠皮下的生長與肺臟的轉移，這個發現有助於未來臨床上腫瘤治療的應用。
關鍵字：腺病毒載體，核糖核酸干擾抑制，轉型生長因子β1，黑色素瘤