Cd对雄激素受体AR转录活性的作用机制研究
发布时间：2018-07-17 03:01
【摘要】：重金属镉(Cadmium, Cd)是一种重要的工业和环境污染因素,在空气、土壤和水中广泛存在,被美国毒理管理委员会(ATSDR)列为第六位危及人体健康的有毒物质。Cd具有内分泌干扰作用,可以干扰生物体激素的合成、释放、转运、与受体结合、代谢等途径,从而影响内分泌系统功能,破坏机体内环境的协调和稳定。有资料显示,Cd可诱导睾丸、附睾和精囊腺等组织器官发生结构和功能上的退行性变化,引起生精障碍,甚至不育。流行病学数据表明,Cd能够增加个体罹患前列腺癌(Prostate Cancer, PCa)的风险。前列腺等生殖相关组织器官都是内分泌依赖的器官,其生长、分化受到雄激素水平的调节,而雄激素发挥作用依赖于其受体AR (Androgen receptor)。AR是一个转录因子,具有转录激活活性,可激活下游基因的转录,同时AR信号通路在前列腺的生长发育、疾病发生中发挥着极其重要的作用。目前关于Cd对AR介导的干扰作用不甚清楚,其机制尚未明确。 本课题以雄激素受体AR为切入点研究镉的内分泌干扰毒性,探讨Cd对AR表达、转录活性及下游信号通路的影响,并对其影响机制进行研究,以此来寻找Cd为代表的重金属类内分泌干扰物的毒理学机制,为其他物质的雄性激素内分泌干扰毒性筛选提供新的方法,进一步为镉污染所造成疾病的预防控制提供新的策略。 首先利用CCK8实验研究了Cd对LNCaP细胞的细胞毒性作用,发现16μ.M以下浓度无明显细胞毒性；流式细胞技术检测不同浓度的Cd处理对LNCaP细胞周期的影响,发现Cd可以使LNCaP细胞的S期增加,这表明Cd可能促进细胞的增殖；利用荧光素酶试验,将带有荧光素酶报告基因的雄激素反应元件(Androgen response element, ARE)和AR共转染293T细胞,经不同浓度Cd处理,分析Cd对AR转录活性的影响,发现Cd可增强AR的转录活性；然后利用qRT-PCR和western检测不同浓度Cd处理后AR及其调控的下游靶基因——前列腺特异性抗原(Prostate specific antigen, PSA) mRNA和蛋白表达水平变化,发现AR可增加PSA的表达,但AR本身的表达量没有明显改变,这表明Cd不是通过改变AR的表达水平影响AR的功能。 类泛素化修饰(SUMO化)是一种重要的翻译后修饰,AR经类泛素化修饰后转录活性降低而表达量不变。文献报道,去类泛素化酶SENP1可裂解类泛素与AR之间的共价连接,降低AR的类泛素化水平。因此我们推测Cd很可能通过影响SENP1调节AR的转录活性。我们检测了Cd对可破坏AR SUMO化的蛋白酶1(SUMO specific protease, SENP1) mRNA和蛋白表达水平的变化,发现Cd增加SENP1的表达；继而利用基因沉默技术,检测LNCaP细胞中的SENP1沉默后,AR及其下游靶基因PSA的表达,发现通过SENP1调控AR的活性；进一步检测了Cd对/AR SUMO化水平的影响,发现Cd处理后AR分子的SUMO化水平降低。综上所述,Cd通过SENP1降低雄激素受体AR的SUMO化调节AR的活性。以上结果分析了镉雄激素内分泌干扰毒性的一种新机制,为其他物质的内分泌毒性筛选提供新的方法,也为寻找环境Cd污染所致疾病的预防控制的新策略提供理论支持。
[Abstract]:Cadmium (Cd) is an important industrial and environmental pollution factor, which exists widely in air, soil and water. The American toxicology Management Committee (ATSDR) has been listed as sixth toxic substances that endanger human health,.Cd has endocrine disrupting effects, which can interfere with the synthesis, release, transport, and metabolism of the hormones. It has been shown that Cd can induce degenerative changes in the structure and function of the tissues and organs such as testis, epididymis and seminal vesicles, causing spermatogenesis and even infertility. Epidemiological data show that Cd can increase the prostate cancer of the individual (Prostate Cancer, PCa) risk. Prostate and other reproductive organs and organs are endocrine dependent organs, and their growth and differentiation are regulated by androgen levels, while androgens play a role depending on their receptor AR (Androgen receptor).AR as a transcription factor, which has a transcriptional activation activity that activates the transcription of the downstream genes, while AR signals are used. The pathway plays an important role in the growth and development of the prostate and the occurrence of the disease. At present, the interference of Cd on AR is not clear, and its mechanism is not clear.
In this study, the endocrine disrupting toxicity of cadmium was studied with androgen receptor AR as a breakthrough point. The effects of Cd on AR expression, transcriptional activity and downstream signal pathway were investigated, and the mechanism of its influence was studied in order to find the toxicological mechanism of the endocrine disruptors, represented by Cd, and to interfere with the male hormone endocrine disrupting drugs of other substances. Sex screening provides new ways to further provide new strategies for disease prevention and control caused by cadmium pollution.
First, the cytotoxic effect of Cd on LNCaP cells was studied by CCK8 experiment. It was found that there was no obvious cytotoxicity of the concentration below 16.M. Flow cytometry was used to detect the effect of Cd on the cycle of LNCaP cells at different concentrations. It was found that Cd could increase the S phase of LNCaP cells, which indicates that Cd may promote cell proliferation; luciferase test is used. The Androgen response element (ARE) and AR were co transfected to 293T cells, and the effect of Cd on AR transcriptional activity was analyzed by Cd treatment at different concentrations. It was found that Cd enhanced the AR transcriptional activity. The gene, Prostate specific antigen (PSA) mRNA and protein expression level changes, found that AR can increase the expression of PSA, but the expression of AR itself is not significantly changed, which indicates that Cd does not affect AR by changing the expression level of AR.
Ubiquitination modification (SUMO) is an important post-translational modification, and the expression of AR after ubiquitin modification is reduced and the amount of expression is unchanged. It is reported that de ubiquitinase SENP1 can cleave the covalent connection between AR and reduce the ubiquitination level of AR. Therefore, we speculate that Cd is likely to regulate the transcription of AR by affecting SENP1. We detected the changes in the expression level of protease 1 (SUMO specific protease, SENP1) mRNA and protein that could destroy AR SUMO, and found that Cd increased the expression of SENP1. Then, the expression of SENP1 silencing in LNCaP cells was detected by gene silencing, and the expression of the target gene and its downstream target gene were detected. The effect of Cd on the level of /AR SUMO was further detected. It was found that the SUMO level of AR molecules decreased after Cd treatment. To sum up, Cd regulates AR activity by SUMO conversion of androgen receptor AR through SENP1, and the above results are a new mechanism for the endocrine disrupting toxicity of cadmium androgen, which provides a new method for screening the endocrine toxicity of other substances. The method also provides theoretical support for finding new strategies for prevention and control of diseases caused by environmental pollution caused by Cd.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114
本文编号：2128717
[Abstract]:Cadmium (Cd) is an important industrial and environmental pollution factor, which exists widely in air, soil and water. The American toxicology Management Committee (ATSDR) has been listed as sixth toxic substances that endanger human health,.Cd has endocrine disrupting effects, which can interfere with the synthesis, release, transport, and metabolism of the hormones. It has been shown that Cd can induce degenerative changes in the structure and function of the tissues and organs such as testis, epididymis and seminal vesicles, causing spermatogenesis and even infertility. Epidemiological data show that Cd can increase the prostate cancer of the individual (Prostate Cancer, PCa) risk. Prostate and other reproductive organs and organs are endocrine dependent organs, and their growth and differentiation are regulated by androgen levels, while androgens play a role depending on their receptor AR (Androgen receptor).AR as a transcription factor, which has a transcriptional activation activity that activates the transcription of the downstream genes, while AR signals are used. The pathway plays an important role in the growth and development of the prostate and the occurrence of the disease. At present, the interference of Cd on AR is not clear, and its mechanism is not clear.
In this study, the endocrine disrupting toxicity of cadmium was studied with androgen receptor AR as a breakthrough point. The effects of Cd on AR expression, transcriptional activity and downstream signal pathway were investigated, and the mechanism of its influence was studied in order to find the toxicological mechanism of the endocrine disruptors, represented by Cd, and to interfere with the male hormone endocrine disrupting drugs of other substances. Sex screening provides new ways to further provide new strategies for disease prevention and control caused by cadmium pollution.
First, the cytotoxic effect of Cd on LNCaP cells was studied by CCK8 experiment. It was found that there was no obvious cytotoxicity of the concentration below 16.M. Flow cytometry was used to detect the effect of Cd on the cycle of LNCaP cells at different concentrations. It was found that Cd could increase the S phase of LNCaP cells, which indicates that Cd may promote cell proliferation; luciferase test is used. The Androgen response element (ARE) and AR were co transfected to 293T cells, and the effect of Cd on AR transcriptional activity was analyzed by Cd treatment at different concentrations. It was found that Cd enhanced the AR transcriptional activity. The gene, Prostate specific antigen (PSA) mRNA and protein expression level changes, found that AR can increase the expression of PSA, but the expression of AR itself is not significantly changed, which indicates that Cd does not affect AR by changing the expression level of AR.
Ubiquitination modification (SUMO) is an important post-translational modification, and the expression of AR after ubiquitin modification is reduced and the amount of expression is unchanged. It is reported that de ubiquitinase SENP1 can cleave the covalent connection between AR and reduce the ubiquitination level of AR. Therefore, we speculate that Cd is likely to regulate the transcription of AR by affecting SENP1. We detected the changes in the expression level of protease 1 (SUMO specific protease, SENP1) mRNA and protein that could destroy AR SUMO, and found that Cd increased the expression of SENP1. Then, the expression of SENP1 silencing in LNCaP cells was detected by gene silencing, and the expression of the target gene and its downstream target gene were detected. The effect of Cd on the level of /AR SUMO was further detected. It was found that the SUMO level of AR molecules decreased after Cd treatment. To sum up, Cd regulates AR activity by SUMO conversion of androgen receptor AR through SENP1, and the above results are a new mechanism for the endocrine disrupting toxicity of cadmium androgen, which provides a new method for screening the endocrine toxicity of other substances. The method also provides theoretical support for finding new strategies for prevention and control of diseases caused by environmental pollution caused by Cd.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114
【共引文献】
相关期刊论文 前1条
1 李裕;张强;王润元;肖国举;王胜;;镉的致癌性与食品中镉的生物有效性[J];生命科学;2010年02期
相关会议论文 前1条
1 李裕;张强;王润元;肖国举;;食品中镉的生物有效性与影响因素[A];第27届中国气象学会年会干旱半干旱区地气相互作用分会场论文集[C];2010年
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