转染RNF6基因对肝癌细胞IRS-2表达的影响

生物技术通报 ›› 2014, Vol. 0 ›› Issue (6): 225-228.

• 研究报告 • 上一篇

转染RNF6基因对肝癌细胞IRS-2表达的影响

巩健, 宋健

（淄博职业学院制药与生物工程系，淄博 255314）

收稿日期:2013-12-25 出版日期:2014-06-25 发布日期:2014-06-25
作者简介:巩健，女，副教授，研究方向：生物技术应用；E-mail：ghfh2008@126.com

Effect of Gene Ring Finger Protein 6 on the Expression of Insulin Receptor Substrate-2 in Hepatoma Cells

Gong Jian, Song Jian

（Department of Pharmaceutical and Biological Engineering，Zibo Vocational Institute，Zibo 255314）

Received:2013-12-25 Published:2014-06-25 Online:2014-06-25

摘要/Abstract

摘要： 构建环指蛋白6（RNF6）真核表达载体，并探讨其对胰岛素受体底物1（IRS-2）表达的影响。以人cDNA为模板，PCR扩增RNF6 全长编码基因，并将其克隆至载体pcDNA3.1-CHA中，将重组质粒转染肝癌细胞株HepG2，利用Real time-PCR、Western blot检测细胞内IRS-2 mRNA水平及蛋白表达情况。携带RNF6目的基因的质粒转染HepG2细胞48 h后IRS-2的mRNA表达降低，为对照组的37%，显著低于对照组，差异有统计学意义（P﹤0.01）。 RNF6引起IRS-2的表达下调，这一过程可能由于泛素化导致胰岛素信号转导通路障碍。

关键词: 胰岛素受体底物-2, 泛素化, 环指蛋白6, 胰岛素

Abstract: It was to construct an eukaryotic expression vector of ring finger protein 6（RNF6）gene and investigate the effect of RNF6 on the expression of insulin receptor substrate-2（IRS-2）. The coding sequence of hRNF6 gene was amplified by PCR with human cDNA as template. The pcDNA3.1-CHA-RNF6 was constructed and transfected into hepatocarcinoma cells（HepG2）by routine molecular biology technology. The total RNA was extracted from HepG2 cells 72 hours post-transfection, the expression levels of IRS-2 was detected by real-time quantitative PCR. Western blotting was applied to detect the protein levels of IRS-2. Result showed that the mRNA level of IRS-2 gene in transfected HepG2 was 37% of the control. The expression level of IRS-2 was lower than the control group significantly （P﹤0.01）. The expression of IRS-2 was down-regulated in HepG2 significantly, and the disorder in insulin signal transduction pathway, which may result from enhanced ubiquitylation level of IRS-2.

Key words: Insulin receptor substrate-2, Ubiquitin, RNF6, Insulin

巩健, 宋健. 转染RNF6基因对肝癌细胞IRS-2表达的影响[J]. 生物技术通报, 2014, 0(6): 225-228.

Gong Jian, Song Jian. Effect of Gene Ring Finger Protein 6 on the Expression of Insulin Receptor Substrate-2 in Hepatoma Cells[J]. Biotechnology Bulletin, 2014, 0(6): 225-228.

参考文献

[1] Yen SL, Chiu TY, Lin YC, et al. Obesity and hepatitis B infection are associated with increased risk of metabolic syndrome in university freshmen[J] . Int J Obes（Lond）, 2008, 32（3）：474.
[2] Pagliassotti MJ, Kang J, Thresher JS, et al. Elevated basal PI 3-kinase activity and reduced insulin signalling in sucrose-induced hepatic insulin resistance[J] . Am J Physiol Endocrinol Metab, 2002, 282（1）：170-176.
[3] Myer MG Jr, Sun XJ, White MF. The IRS-1 signaling system[J] . Trends Biochem Sci, 1994, 19（7）：289-293.
[4] Wiedmann M, Tamaki S, Silberman R, et al. Constitutive over-expression of the insulin receptor substrate-1 causes functional up-regulation of Fas receptor[J] . J Hepatol, 2003, 38（6）：803-810.
[5] Kim SJ, DeStefano MA, Oh WJ, et al. mTOR complex 2 regulates proper turnover of insulin receptor substrate-1 via the ubiquitin ligase subunit Fbw8[J] . Molecular Cell, 2012, 48：875-887.
[6] Banker GA, Cowan WM. Rat hippocampal neurons in dispersed cell culture[J] . Brain Res, 1977, 126：397-425.
[7] Banker GA, Cowan WM. Further observations on hippocampal neurons in dispersed cell culture[J] . J Comp Neurol, 1979, 187：469-493.
[8] Zhu L, Tong G, Chen J, et al. Cloning and identification of a novel RNF6 transcriptional splice variant Spg2 in human development[J] . Science in China（Life Sciences）, 2008, 51（4）：302-307.
[9] Xu K, Shimelis H, Linn DE, et al. Regulation of androgen receptor transcriptional activity and specificity by RNF6-induced ubiquitination[J] . Cancer Cell, 15（4）：270-282.
[10] Rome S, Meugnier E, Vidal H. The ubiquitin-proteasome pathway is a new partner for the control of insulin signaling[J] . Curr Opin Clin Nutr Metab Care, 2004, 7（3）：249- 254.
[11] Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolytic pathway：destruction for the sake of construction[J] . Physiol Rev, 2002, 82（2）：373- 428.
[12] Kornitzer D, Ciechanover A. Modes of regulation of ubiquitin mediated protein degradation[J] . J Cell Physiol, 2000, 182（1）：1-11.
[13] Groll M, Huber R. Substrate access and processing by the 20S proteasome core particle[J] . Int J Biochem Cell Biol, 2003, 35（5）：606- 616.
[14] Rhodes CJ. Type 2 diabetes a matter of beta cell life and death[J] . Science, 2005, 307（5）：380-384.
[15] Pederson TM, Kramer DL, Rondinone CM. Serine/threonine phosphorylation of IRS-1triggers its degradation：possible regulation by tyrosine phosphorylation[J] . Diabetes, 2001, 50（1）：24-31.

转染RNF6基因对肝癌细胞IRS-2表达的影响

Effect of Gene Ring Finger Protein 6 on the Expression of Insulin Receptor Substrate-2 in Hepatoma Cells

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