Zhang Shuyu, Xue Jing, Zheng Jie, Wang Shuai, Zhou Jundong, Jiao Yang, Geng Yangyang, Wu Jinchang, Hannafon Bethany N, Ding Wei-Qun
School of Radiation Medicine and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Medical College of Soochow University, Suzhou 215123, China; Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
School of Radiation Medicine and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Medical College of Soochow University, Suzhou 215123, China; Department of Radio-Oncology, Nanjing Medical University Affiliated Suzhou Hospital, Suzhou 215001, China.
Free Radic Biol Med. 2015 Aug;85:33-44. doi: 10.1016/j.freeradbiomed.2015.04.012. Epub 2015 Apr 20.
Superoxide dismutase 1 (SOD1) is ubiquitously expressed and the predominant dismutase in the cytoplasm. Whereas transcriptional regulation of the SOD1 gene has been well characterized, posttranscriptional regulation of the gene remains largely unknown in eukaryotes. In this study, a full-length 3'UTR of the SOD1 transcript was cloned and characterized for its ability to regulate SOD1 gene expression in human cancer cells. Inclusion of the SOD1 3'UTR in the pGL3 reporter construct dramatically enhanced the reporter activity by 10- to 220-fold in various cell lines. RT-PCR analysis, however, indicated that the reporter gene mRNA levels were only modestly altered by the SOD1 3'UTR, suggesting that the SOD1 3'UTR enhances the reporter gene activity not simply by stabilizing the mRNA but primarily by promoting translation of the protein. Bioinformatics analysis showed multiple stem and loop structures of the SOD1 3'UTR, and alterations in this secondary structure led to remarkably reduced reporter gene activity. Importantly, introducing the SOD1 3'UTR into cancer cells attenuated endogenous SOD1 expression in a concentration-dependent manner, indicating the involvement of RNA trans-acting factors in this process. Using siRNA and RNA immunoprecipitation techniques, we identified AUF-1, an RNA-binding protein, as a positive regulator of SOD1 expression through its 3'UTR. Consequently, AUF-1 was found to regulate redox balance in our cell model systems. Furthermore, in human ovarian, esophageal, and pancreatic cancer tissues, the expression of SOD1 was significantly correlated with that of AUF-1, further supporting the importance of AUF-1 in regulating SOD1 gene expression.
超氧化物歧化酶1（SOD1）在全身广泛表达，是细胞质中主要的歧化酶。虽然SOD1基因的转录调控已得到充分表征，但该基因在真核生物中的转录后调控仍基本未知。在本研究中，克隆了SOD1转录本的全长3'UTR，并对其在人类癌细胞中调节SOD1基因表达的能力进行了表征。在pGL3报告基因构建体中包含SOD1 3'UTR，可在各种细胞系中将报告基因活性显著提高10至220倍。然而，RT-PCR分析表明，报告基因mRNA水平仅受到SOD1 3'UTR的适度改变，这表明SOD1 3'UTR增强报告基因活性并非仅仅通过稳定mRNA，而是主要通过促进蛋白质翻译。生物信息学分析显示SOD1 3'UTR具有多个茎环结构，这种二级结构的改变导致报告基因活性显著降低。重要的是，将SOD1 3'UTR导入癌细胞以浓度依赖的方式减弱了内源性SOD1表达，表明RNA反式作用因子参与了这一过程。使用siRNA和RNA免疫沉淀技术，我们鉴定了一种RNA结合蛋白AUF-1，它通过其3'UTR作为SOD1表达的正调节因子。因此，在我们的细胞模型系统中发现AUF-1可调节氧化还原平衡。此外，在人类卵巢癌、食管癌和胰腺癌组织中，SOD1的表达与AUF-1的表达显著相关，进一步支持了AUF-1在调节SOD1基因表达中的重要性。
