Application of siRNA Technology to Silence Copper Chaperone for SOD1 Gene in Human Umbilical Vein Endothelial Cells_West China Medical Journal

Authors：

1. Department of Transplantation Surgery; 2. Regeneration Medical Research Center; West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding?author：

Keywords：

copper chaperone for SOD1; 基因沉默; 小干擾RNA

DOI：

10.7507/1002-0179.20130227

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目的　采用RNA干擾技術沉默CCS（copper chaperone for SOD1）基因，構建相關小干擾RNA（siRNA），探索出針對CCS的高效siRNA序列。方法　合成用于人臍靜脈內皮細胞（HUVEC）細胞中沉默CCS基因的siRNA。應用脂質體轉染的方法在HUVEC細胞中對CCS基因進行RNA沉默。蛋白免疫印跡Western blotting檢測沉默前后CCS蛋白表達變化的情況，甲基四唑藍法MTT檢測轉染前后細胞活力。最后用單因素方差分析對數據進行統計學分析，以確定有效的siRNA序列。結果　轉染前后細胞形態無肉眼可見變化，轉染后細胞活力分別為98.5%和98.8%。CCS蛋白沉默率分別為63.7%和61.4%。結論　采用siCCS-2和siCCS-3序列轉染條件對HUVEC細胞活力損傷小，CCS沉默效率高，實驗條件穩定，重復性好。為我們繼續研究沉默CCS后抑制血管內皮細胞的生長增殖、血管形成提供了穩定的實驗基礎。

Citation： LIU Jun,CHEN Kefei.. Application of siRNA Technology to Silence Copper Chaperone for SOD1 Gene in Human Umbilical Vein Endothelial Cells. West China Medical Journal, 2013, 28(5): 732-736. doi: 10.7507/1002-0179.20130227 Copy

1.	Yoshiaki F, Andrew ST, Thomas V. Oxygen-induced maturation of SOD1: a key role for disulfide formation by the copper chaperone CCS[J]. EMBO J, 2004, 6 (23): 2872-2881.
2.	祁艾紅,吳健民.銅代謝及其相關基因的研究[J]. 國外遺傳學雜志，2006, 29(5): 356-359.
3.	王剛，張凱倫. 人臍靜脈內皮細胞的研究和應用[J]. 醫學綜述，2011, 17（7）：972-975.
4.	Monika D, Derek MD. Breaking down the barriers: siRNA delivery and endosome escape[J]. J Cell Sci, 2010, 123(8): 1183-1189.
5.	Nina MB, Andrew S, Torres PE, et al. Oxygen and the copper chaperone CCS regulate posttranslational activation of Cu, Zn superoxide dismutase[J]. PNAS, 2004, 101(15): 5518-5523.
6.	Dominik P, Caroline AB, Silvia R, et al. Mitochondrial Ccs1 contains a structural disulfide bond crucial for the import of this unconventional substrate by the disulfide relay system[J]. Mol Biol Cell, 2011, 22(10): 3758-3767.
7.	Graham FB, Stefanie G, Xuwen L, et al. Regulation of the Copper Chaperone CCS by XIAP-Mediated Ubiquitination[J]. Mol Cell Biol, 2010, 30(8):1923-1936.
8.	Barbara A, Kevin JW, Katie BF, et al. BACE1 Cytoplasmic Domain Interacts with the Copper Chaperone for Superoxide Dismutase-1 and Binds Copper[J]. J Biol Chem, 2005, 280(4): 17930-17937.
9.	Heejeong K, Hwa-YS, Sarah MB, et al. Mechanisms for copper acquisition, distribution and regulation[J]. Am J Physiol Gastrointest Liver Physiol, 2009, 296(2): G356-G364.
10.	Youchun J, Corey R, Chang X, et al. Dietary copper supplementation reverses hypertrophic cardiomyopathy induced by chronic pressure overload in mice[J]. J Exper Med, 2007, 204(3): 657-666.
11.	Wenke F, Fei Y, Wanli X, et al. Copper regulation of hypoxia-inducible factor-1 activity[J]. Mol Pharmaco, 2009, 75(1): 174-182.

1. 　Yoshiaki F, Andrew ST, Thomas V. Oxygen-induced maturation of SOD1: a key role for disulfide formation by the copper chaperone CCS[J]. EMBO J, 2004, 6 (23): 2872-2881.
2. 　祁艾紅,吳健民.銅代謝及其相關基因的研究[J]. 國外遺傳學雜志，2006, 29(5): 356-359.
3. 　王剛，張凱倫. 人臍靜脈內皮細胞的研究和應用[J]. 醫學綜述，2011, 17（7）：972-975.
4. 　Monika D, Derek MD. Breaking down the barriers: siRNA delivery and endosome escape[J]. J Cell Sci, 2010, 123(8): 1183-1189.
5. 　Nina MB, Andrew S, Torres PE, et al. Oxygen and the copper chaperone CCS regulate posttranslational activation of Cu, Zn superoxide dismutase[J]. PNAS, 2004, 101(15): 5518-5523.
6. 　Dominik P, Caroline AB, Silvia R, et al. Mitochondrial Ccs1 contains a structural disulfide bond crucial for the import of this unconventional substrate by the disulfide relay system[J]. Mol Biol Cell, 2011, 22(10): 3758-3767.
7. 　Graham FB, Stefanie G, Xuwen L, et al. Regulation of the Copper Chaperone CCS by XIAP-Mediated Ubiquitination[J]. Mol Cell Biol, 2010, 30(8):1923-1936.
8. 　Barbara A, Kevin JW, Katie BF, et al. BACE1 Cytoplasmic Domain Interacts with the Copper Chaperone for Superoxide Dismutase-1 and Binds Copper[J]. J Biol Chem, 2005, 280(4): 17930-17937.
9. 　Heejeong K, Hwa-YS, Sarah MB, et al. Mechanisms for copper acquisition, distribution and regulation[J]. Am J Physiol Gastrointest Liver Physiol, 2009, 296(2): G356-G364.
10. 　Youchun J, Corey R, Chang X, et al. Dietary copper supplementation reverses hypertrophic cardiomyopathy induced by chronic pressure overload in mice[J]. J Exper Med, 2007, 204(3): 657-666.
11. 　Wenke F, Fei Y, Wanli X, et al. Copper regulation of hypoxia-inducible factor-1 activity[J]. Mol Pharmaco, 2009, 75(1): 174-182.

West China Medical Journal

Application of siRNA Technology to Silence Copper Chaperone for SOD1 Gene in Human Umbilical Vein Endothelial Cells

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