雌激素硫酸转移酶SULT1E1的功能及调控研究

发布时间：2018-08-28 14:04

【摘要】：雌激素作为类固醇激素,主要通过和细胞核(膜)上的雌激素受体结合,在人体的靶器官和组织中发挥重要的调控作用,可影响分泌、新陈代谢以及生殖和发育等正常的生理功能。机体内雌激素的水平由合成和代谢途径来达到平衡。雌激素的合成由芳香酶、17β-羟基类固醇脱氢酶Ⅰ(17β-HSD1)和硫酸酯酶(STS)参与；而雌激素的代谢则主要通过硫酸化途径完成,可将雌激素转化为硫酸化的雌激素。其中,雌激素硫酸转移酶SULT1E1是雌激素硫酸化反应的关键酶。目前,关于雌激素硫酸转移酶SULT1E1的研究多集中在它和激素依赖性肿瘤之间的关系,具体调节机制尚不十分清楚；另外,SULT1E1在雌激素的另一个靶组织——血管内皮中的功能尚未见研究报道。因此,本课题分三个部分,分别研究雌激素硫酸转移酶SULT1E1在激素依赖性肿瘤以及血管内皮细胞中的功能及调控：第一部分：雌激素可通过调节细胞增殖和凋亡影响激素依赖性肿瘤包括乳腺癌和子宫内膜癌的发生发展。硫酸化反应是雌激素代谢的主要途径,雌激素硫酸转移酶SULT1E1是该反应的关键酶,PAPSS(包括PAPSS1和PAPSS2)则提供反应底物硫酸基团。首先,通过组织芯片染色以及临床病理标本分析,我们发现雌激素硫酸转移酶SULT1E1和PAPSS在肿瘤及其癌旁(正常)组织中的表达存在差异性,SULT1E1和PAPSS在肿瘤组织的表达较正常或者癌旁组织高。体内实验发现注射腺病毒SULT1E1可以抑制裸鼠移植瘤的生长。在MCF-7细胞体外过表达SULT1E1和PAPSS1后,细胞增殖实验结果显示,过表达SULT1E1和PAPSS1可以抑制雌激素引起的细胞增殖；流式细胞检测表明：雌激素可以保护H202诱导的MCF-7细胞凋亡,而SULT1E1和PAPSS1可通过硫酸化雌激素来加速细胞的凋亡。为进一步阐明其发生机制,我们检测了增殖和凋亡相关的基因／蛋白的表达水平。结果发现,过表达SULT1E1和PAPSS1可下调癌基因c-myc,细胞周期蛋白cyclin Dl以及抗凋亡分子bcl-2的表达,而上调促凋亡分子bax的水平。说明SULT1E1和PAPSS1通过调节细胞增殖和凋亡相关分子的水平抑制肿瘤细胞增殖,促进细胞凋亡。第二部分：雌激素在心血管系统发挥重要的作用,可保护血管内皮细胞的功能。雌激素可被硫酸转移酶SULT1E1催化生成硫酸化雌激素。但迄今为止,尚未见有关SULT1E1在内皮细胞中的功能的研究报道。我们推测,SULT1E1可能影响内皮细胞的功能。我们检测了SULT1E1在脐静脉内皮细胞(HUVEC)中的表达,结果显示：SULT1E1在HUVEC中表达较丰富且在前几代细胞中持续表达。因此,我们设计合成了针对SULT1E1的siRNA干扰片段。干扰SULT1E1之后可以下调SULT1E1的表达,减弱酶的活性以及降低雌激素硫酸化水平。体内酶活性检测表明：SULT1E1可转化80nM的雌激素,而干扰SULT1E1只能转化40nM的雌激素。通过实时PCR基因筛选,我们发现干扰SULT1E1后可调节炎症因子的释放、胆固醇水平以及细胞迁移,并影响其相关基因和蛋白的表达。而干扰PPARγ或者加入PPARγ抑制剂GW9662可拮抗SULT1E1的调节作用。为,我们进一步探讨了雌激素是否会影响SULT1E1的作用。和不加雌激素相比,加入80nM的雌激素可以逆转SULT1E1对炎症因子、脂质代谢和细胞迁移相关分子的调节作用,而雌激素受体ERp干扰或者雌激素受体ER拮抗剂IC1182780处理后,雌激素的调节作用减弱。为解释这一现象,我们加入雌激素E2和硫酸化雌激素E2S检测它们对上述基因的调节。E2S可以上调PPARγ、IL-4和脂质代谢相关基因的表达,E2作用刚好相反。这说明,在外源性雌激素存在的情况下,对照组只有E2S的调节作用,而干扰SULT1E1组有E2和E2S的双重调节。该结果表明,干扰SULT1E1影响了E2和E2S的比例,进而调节PPARγ影响HUVEC的炎症因子释放、胆固醇代谢和细胞迁移能力。第三部分：基于雌激素硫酸转移酶SULT1E1在雌激素功能中的影响的重要性,针对SULT1E1的调控显得尤为重要。目前,关于SULT1E1的调节方面的研究不多。有文献报道,IGF-1可以增加乳腺癌细胞中雌激素硫酸酯酶STS的活性；另外,PPARα对硫酸转移酶的调节尚存在争议。因此,我们推测IGF-1和PPARα可能调节SULT1E1的表达。我们检测了HUVEC和HUASMC中SULT1E1的表达,结果显示SULT1E1在这两种细胞中均有表达。加入IGF-1后,SULT1E1的表达上调；而加入PPARa激动剂WY14643后,SULT1E1的表达下调。通过体内SULT1E1酶活性检测发现,在HUASMC细胞中,IGF-1增强SULT1E1酶的活性使硫酸化雌激素的生成增加；WY14643减弱SULT1E1酶的活性而减少了硫酸化雌激素的生成。我们构建含SULT1E1启动子区的荧光素酶报告基因载体。通过荧光素酶活性检测发现,IGF-1使SULT1E1的荧光素酶活性增加；而加入PPARα激动剂WY14643后,SULT1E1的荧光素酶活性下降。综上所述,我们得出以下结论：雌激素硫酸转移酶SULT1E1和PAPSS可通过调节细胞增殖和凋亡来减慢裸鼠移植瘤的生长并且抑制雌激素诱导的肿瘤细胞促增殖和抗凋亡作用；另外,SULT1E1可通过调节PPARγ影响脐静脉内皮细胞的炎症因子分泌、胆固醇代谢以及细胞迁移。胰岛素样生长因子IGF-1和PPARα激动剂WY14643可通过转录水平调节SULT1E1的表达，，并可影响SULT1E1酶的活性。该研究为雌激素硫酸转移酶SULT1E1对雌激素功能的影响提供了新的研究思路。
[Abstract]:Estrogen, as a steroid hormone, plays an important regulatory role in the target organs and tissues of the human body by binding to estrogen receptors in the nucleus (membrane), which can affect the secretion, metabolism, reproduction and development of normal physiological functions.
Estrogen synthesis is mediated by aromatase, 17beta-hydroxysteroid dehydrogenase I (17beta-HSD1) and sulfatase (STS). Estrogen metabolism is mainly accomplished by sulfation, which converts estrogen into sulfated estrogen. SULT1E1 is a key enzyme in the sulfation of estrogen. At present, the research on the relationship between SULT1E1 and hormone-dependent tumors is mostly focused on, and the specific regulatory mechanism is still unclear. In addition, the function of SULT1E1 in the vascular endothelium, another target tissue of estrogen, has not been reported. Therefore, this study is divided into three parts, respectively, to study the function and regulation of estrogen sulfate transferase SULT1E1 in hormone-dependent tumors and vascular endothelial cells.
Part I: Estrogen affects the development of hormone-dependent tumors, including breast and endometrial cancers, by regulating cell proliferation and apoptosis. Sulfation is the main pathway of estrogen metabolism. Estrogen sulfate transferase SULT1E1 is the key enzyme in this reaction, and PAPSS (including PAPSS1 and PAPS2) provides the substrate sulfate. Firstly, through tissue chip staining and clinicopathological analysis, we found that the expression of estrogen sulfate transferase SULT1E1 and PAPSS was different in tumor and adjacent (normal) tissues. The expression of SULT1E1 and PAPSS in tumor tissues was higher than that in normal or adjacent tissues. The proliferation of MCF-7 cells overexpressing SULT1E1 and PAPS1 in vitro showed that the overexpression of SULT1E1 and PAPS1 could inhibit the proliferation of MCF-7 cells induced by estrogen. Flow cytometry showed that estrogen could protect the apoptosis of MCF-7 cells induced by H202, while SULT1E1 and PAPS1 could sulfate the female. To further elucidate the mechanism of apoptosis, we examined the expression of genes/proteins associated with proliferation and apoptosis. Overexpression of SULT1E1 and PAPS1 down-regulated the expression of oncogene c-myc, cyclin Dl and anti-apoptotic molecule bcl-2, and up-regulated the expression of apoptotic molecule bax. These results suggest that SULT1E1 and PAPS1 can inhibit the proliferation of tumor cells and promote apoptosis by regulating the levels of cell proliferation and apoptosis-related molecules.
Part II: Estrogen plays an important role in the cardiovascular system and protects the function of vascular endothelial cells. Estrogen can be catalyzed by sulfate transferase SULT1E1 to produce sulfated estrogen. However, so far, there are no reports about the function of SULT1E1 in endothelial cells. We speculate that SULT1E1 may affect endothelial cells. Function. We examined the expression of SULT1E1 in human umbilical vein endothelial cells (HUVEC). The results showed that SULT1E1 was abundantly expressed in HUVEC and was continuously expressed in previous generations of cells. Therefore, we designed and synthesized siRNA interference fragments targeting SULT1E1. Estrogen sulfation level.In vivo enzyme activity assay showed that SULT1E1 could transform 80 nM estrogen, while interfering with SULT1E1 could only transform 40 nM estrogen.Through real-time PCR gene screening, we found that interfering with SULT1E1 could regulate the release of inflammatory factors, cholesterol levels and cell migration, and affect the expression of related genes and proteins. In order to further explore whether estrogen affects the function of SULT1E1, estrogen can reverse the regulation of SULT1E1 on inflammatory factors, lipid metabolism and cell migration-related molecules, while estrogen can reverse the regulation of SULT1E1 on inflammatory factors, lipid metabolism and cell migration-related molecules without estrogen. In order to explain this phenomenon, we added estrogen E2 and sulfated estrogen E2S to detect their regulation of the above genes. E2S could up-regulate the expression of PPAR-gamma, IL-4 and lipid metabolism-related genes, and E2 had the opposite effect. In the presence of exogenous estrogen, only E2S could be regulated in the control group, while E2 and E2S could be regulated in the SULT1E1 group by interfering with SULT1E1.
Part III: Based on the importance of the role of estrogen sulfate transferase SULT1E1 in estrogen function, the regulation of SULT1E1 is particularly important. At present, there are few studies on the regulation of SULT1E1. The expression of SULT1E1 in HUVEC and HUASMC was detected. The expression of SULT1E1 was up-regulated after the addition of IGF-1, and down-regulated after the addition of PPARa agonist WY14643. In vivo SULT1E1 enzyme activity assay showed that in HUASMC cells, IGF-1 enhanced the activity of SULT1E1 enzyme and increased the production of sulfated estrogen; WY14643 decreased the activity of SULT1E1 enzyme and decreased the production of sulfated estrogen. We constructed a luciferase reporter gene vector containing the promoter region of SULT1E1. IGF-1 increased the luciferase activity of SULT1E1, while the luciferase activity of SULT1E1 decreased when PPARa agonist WY14643 was added.
In conclusion, we conclude that estrogen sulfate transferase SULT1E1 and PAPSS can slow down the growth of xenografts in nude mice by regulating cell proliferation and apoptosis and inhibit estrogen-induced proliferation and Anti-apoptosis of tumor cells. In addition, SULT1E1 can affect the inflammatory factors of umbilical vein endothelial cells by regulating PPAR gamma. Insulin-like growth factor-1 and PPAR alpha agonist WY14643 can regulate the expression of SULT1E1 at transcriptional level and affect the activity of SULT1E1 enzyme. This study provides a new research idea for the effect of estrogen sulfate transferase SULT1E1 on estrogen function.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【相似文献】