Arkadia和UCH37表达载体的构建及其对TGF-β1/Smads信号通路的影响

doi:10.3864/j.issn.0578-1752.2012.09.021

摘要/Abstract

摘要： 【目的】构建泛素启动酶Arkadia和泛素羧基末端水解酶-5（亦称UCH37）的表达载体，并探讨Smad7蛋白的泛素化修饰对马兜铃酸肾病中TGF-β1/Smads信号通路的影响。【方法】通过观察绿色荧光的表达量及real-time PCR反应优化转染条件，转染Arkadia和UCH37 siRNA，real-time PCR检测mRNA表达抑制率。分别构建Arkadia和UCH37的表达载体pGPU6/GFP/Neo，经鉴定后转染细胞，real-time PCR和Western blotting检测Arkadia、UCH37和Smad7的表达变化。【结果】①30 pmol siRNA和1.5 μL LipofectamineTM 2000为最佳转染条件；②Arkadia-1490和UCH37-721的干扰效果最明显；③载体的插入序列完全正确，且pGPU6/GFP/Neo-Arkadia能上调马兜铃酸肾病模型中Smad7的表达，而pGPU6/GFP/Neo-UCH37使Smad7表达下降。【结论】成功构建了表达载体pGPU6/GFP/Neo-Arkadia和pGPU6/GFP/Neo-UCH37，且Arkadia和UCH37可分别放大、抑制TGF-β1/Smads信号通路。

关键词: Arkadia基因, UCH37基因, 转染, 表达载体, TGF-&beta, 1/Smads信号通路

Abstract: 【Objective】The objective of the study is to construct expression vectors of ubiquitination starting enzyme Arkadia and ubiquitin COOH-terminal hydmlase-L5 (also known as UCH37), and to investigate the effect of Smad7 modified by ubiquitination on TGF-β1/Smad7 signal pathway in aristolochic acid nephropathy (AAN). 【Method】The transfection condition was optimized by observing expressions of green fluorescences and undertaking real-time PCR reactions. After transferring the siRNA of Arkadia and UCH37, mRNA inhibition ratios were detected by real-time PCR. Then the expression vectors pGPU6/GFP/Neo of Arkadia and UCH37 were constructed, and transferred into cells after evaluations, and the expression changes of Arkadia, UCH37 and Smad7 were detected by real-time PCR and western blotting. 【Result】 Results showed that 30 pmol siRNA and 1.5 μL LipofectamineTM 2000 were the best transfection conditions. The interferences of Arkadia-1490 and UCH37-721 were most obvious. The inserted sequences of vectors were found to be correct, and pGPU6/GFP/Neo-Arkadia could increase Smad7 expression in AAN, while pGPU6/GFP/Neo-UCH37 was the opposite.【Conclusion】Expression vectors pGPU6/GFP/Neo-Arkadia and pGPU6/GFP/ Neo-UCH37 were constructed successfully, and it was found that Arkadia and UCH37 could separately enlarge, and inhibit TGF-β1/ Smad7 signal pathway.

Key words: Arkadia gene, UCH37 gene, transfection, expression vector, TGF-β1/Smad7 signal pathway

廖芳芳, 苑嗣纯, 张中文, 吴国娟. Arkadia和UCH37表达载体的构建及其对TGF-β1/Smads信号通路的影响[J]. 中国农业科学, 2012, 45(9): 1848-1856.

LIAO Fang-Fang, YUAN Si-Chun, ZHANG Zhong-Wen, WU Guo-Juan. Constructions of Arkadia and UCH37 Expression Vectors and Effects on TGF-β1/Smad7 Signal Pathway[J]. Scientia Agricultura Sinica, 2012, 45(9): 1848-1856.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.09.021

https://www.chinaagrisci.com/CN/Y2012/V45/I9/1848

参考文献

[1]Vanherweghem J L, Depierreux M, Tielemans C. Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs. Lancet, 1993, 341: 387-391.

[2]Depierreux M, van Damme B, Vanden H K. Pathologic aspects of a newly described nephropathy related to the prolonged use of Chinese herbs. American Journal of Kidney Diseases, 1994, 24: 172-180.

[3]Rastaldi M P. Epithelial-mesenchymal transition and its implications for the development of renal tubulointerstitial fibrosis. Journal of Nephrology, 2006, 19(4): 407-412.

[4]Vanhaelen M, Vanhaelen-Fastre R, But P. Identification of aristolochic acid in Chinese herbs. Lancet, 1994, 343:174.

[5]Arlt V M, Alunni-Perret V, Quatrehomme G. Ariotolochic acid(AA)- DNA adduct as maker of AA exposure and risk factor for AA nephropathy-associated cancer. International Journal of Cancer, 2004, 111(6): 977-980.

[6]Cheng J, Grande J P. Smad expression during kidney development. Experimental Biology and Medicine, 2002, 227(11): 943-956.

[7]Nakao A, Afrakhte M, Moren A. Inentification of Smad7, a TGF-β- inducibe antagonist of TGF-β signaling. Nature, 1997, 389(9): 631-635.

[8]Schiffer M, von Gersdorff G, Bitzer M. Smad proteins and transforming growth factor-beta singalling. Kidney International, 2000, 77: 45-52.

[9]Wang Y Y, Zhang Z W, Shen H, Lu Y, Li H R, Ren X M, Wu G J. TGF-beta1/Smad7 signaling stimulates renal tubulointerstitial fibrosis induced by AAⅠ. Journal of Receptor and Signal Transduction Research, 2008, 28(4): 413-428.

[10]邱小波, 王琛, 王琳芳. 泛素介导的蛋白质降解. 北京: 中国协和医科大学出版社, 2008: 23-25.

Qiu X B, Wang C, Wang L F. Ubiquitin-Mediated Proteolysis. Beijing: Press of China Union Medical University, 2008: 23-25. (in Chinese)

[11]Gondi C S, Rao J S. Concepts in vivo siRNA delivery for cancer therapy. Journal of Cellular Physiology, 2009, 220(2): 285-291.

[12]Brummelkamp T R, Bernards R, Agami R. A system for stable expression of short interfering RNAs inmammalian cells. Science, 2002, 296(5567): 550-553.

[13]Weber M, Moller K, Welzeck M. Effects of lipolysaccharide on transfection efficiency in eukaryotic cells. Biotechniques, 1995, 19: 930-940.

[14]Elbashir S M, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22- nucleotide RNAs. Genes and Development, 2001, 15: 188-200.

[15]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△Ct method. Methods, 2001, 25(4): 402-408.

[16]唐永凯, 贾永义. 荧光定量PCR 数据处理方法的探讨. 生物技术, 2008,18(3): 89-91.

Tang Y K, Jia Y Y. The processing method study of real-time PCR data. Biotechnology, 2008, 18(3): 89-91. (in Chinese)

[17]Derrick G, Olivier V. Control of RNA silencing and localization by endolysosomes. Trends in Cell Biology, 2010, 20(8): 491-501.

[18]Berns K, Hijmans E M, Mullenders J. A large-scale RNAi screen in human cells identifies new components of the p53 pathways. Nature, 2004, 428: 431-437.

[19]Bantounas I, Phylactou L A, Uney J B. RNA interference and the use of small interfering RNA to study gene function in mammalian systems. Journal of Molecular Endocrinology, 2004, 33: 545-557.

[20]Jin H L, Hongjian Z, Xiao H. Smad7 inhibits fibrotic effect of TGF-beita on renal Tubular epithelial cells by blocking Smad2 activation. Journal of the American Society of Nephrology, 2002, 13(6): 1464-1472.

[21]Mohammed A, John J. R, Dongho K. Approaches for chemically synthesized siRNA and vector-mediated RNAi. FEBS Letters, 2005, 579(26): 5974-5981.

[22]Elbashir S M, Harborth J, Lendecke W. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411(66): 494-498.

[23]Ramaswamy G, Frank J S. siRNA: a guide for RNA silencing. Chemistry and Biology, 2002, 9(10): 1053-1057.

[24]Hirohiko H. Enhancement of RNAi activity by improved siRNA duplexes. FEBS Letters, 2004, 557(1/3): 193-198.

[25]Liu F Y, Li X Z, Peng Y M. Arkadia regulates TGF-beta signaling during renal tubular epithelial to mesenchymal cell transition. Kidney International, 2008, 73(5): 588-594.

[26]Wicks S J, Haros K, Maillard M. The deubiquitinating enzyme UCH37 interacts with Smads and regulates TGF-beta signalling. Oncogene, 2005, 24(54): 8080-8084.

[27]Wicks S J, Grocott T, Haros K. Reversible ubiquitination regulates the Smad/TGF-beta signalling pathway. Biochemical Society Transactions, 2006, 34(5): 761-763.