Cd對(duì)雄激素受體AR轉(zhuǎn)錄活性的作用機(jī)制研究
發(fā)布時(shí)間：2018-07-17 03:01
【摘要】：重金屬鎘(Cadmium, Cd)是一種重要的工業(yè)和環(huán)境污染因素,在空氣、土壤和水中廣泛存在,被美國毒理管理委員會(huì)(ATSDR)列為第六位危及人體健康的有毒物質(zhì)。Cd具有內(nèi)分泌干擾作用,可以干擾生物體激素的合成、釋放、轉(zhuǎn)運(yùn)、與受體結(jié)合、代謝等途徑,從而影響內(nèi)分泌系統(tǒng)功能,破壞機(jī)體內(nèi)環(huán)境的協(xié)調(diào)和穩(wěn)定。有資料顯示,Cd可誘導(dǎo)睪丸、附睪和精囊腺等組織器官發(fā)生結(jié)構(gòu)和功能上的退行性變化,引起生精障礙,甚至不育。流行病學(xué)數(shù)據(jù)表明,Cd能夠增加個(gè)體罹患前列腺癌(Prostate Cancer, PCa)的風(fēng)險(xiǎn)。前列腺等生殖相關(guān)組織器官都是內(nèi)分泌依賴的器官,其生長(zhǎng)、分化受到雄激素水平的調(diào)節(jié),而雄激素發(fā)揮作用依賴于其受體AR (Androgen receptor)。AR是一個(gè)轉(zhuǎn)錄因子,具有轉(zhuǎn)錄激活活性,可激活下游基因的轉(zhuǎn)錄,同時(shí)AR信號(hào)通路在前列腺的生長(zhǎng)發(fā)育、疾病發(fā)生中發(fā)揮著極其重要的作用。目前關(guān)于Cd對(duì)AR介導(dǎo)的干擾作用不甚清楚,其機(jī)制尚未明確。 本課題以雄激素受體AR為切入點(diǎn)研究鎘的內(nèi)分泌干擾毒性,探討Cd對(duì)AR表達(dá)、轉(zhuǎn)錄活性及下游信號(hào)通路的影響,并對(duì)其影響機(jī)制進(jìn)行研究,以此來尋找Cd為代表的重金屬類內(nèi)分泌干擾物的毒理學(xué)機(jī)制,為其他物質(zhì)的雄性激素內(nèi)分泌干擾毒性篩選提供新的方法,進(jìn)一步為鎘污染所造成疾病的預(yù)防控制提供新的策略。 首先利用CCK8實(shí)驗(yàn)研究了Cd對(duì)LNCaP細(xì)胞的細(xì)胞毒性作用,發(fā)現(xiàn)16μ.M以下濃度無明顯細(xì)胞毒性；流式細(xì)胞技術(shù)檢測(cè)不同濃度的Cd處理對(duì)LNCaP細(xì)胞周期的影響,發(fā)現(xiàn)Cd可以使LNCaP細(xì)胞的S期增加,這表明Cd可能促進(jìn)細(xì)胞的增殖；利用熒光素酶試驗(yàn),將帶有熒光素酶報(bào)告基因的雄激素反應(yīng)元件(Androgen response element, ARE)和AR共轉(zhuǎn)染293T細(xì)胞,經(jīng)不同濃度Cd處理,分析Cd對(duì)AR轉(zhuǎn)錄活性的影響,發(fā)現(xiàn)Cd可增強(qiáng)AR的轉(zhuǎn)錄活性；然后利用qRT-PCR和western檢測(cè)不同濃度Cd處理后AR及其調(diào)控的下游靶基因——前列腺特異性抗原(Prostate specific antigen, PSA) mRNA和蛋白表達(dá)水平變化,發(fā)現(xiàn)AR可增加PSA的表達(dá),但AR本身的表達(dá)量沒有明顯改變,這表明Cd不是通過改變AR的表達(dá)水平影響AR的功能。 類泛素化修飾(SUMO化)是一種重要的翻譯后修飾,AR經(jīng)類泛素化修飾后轉(zhuǎn)錄活性降低而表達(dá)量不變。文獻(xiàn)報(bào)道,去類泛素化酶SENP1可裂解類泛素與AR之間的共價(jià)連接,降低AR的類泛素化水平。因此我們推測(cè)Cd很可能通過影響SENP1調(diào)節(jié)AR的轉(zhuǎn)錄活性。我們檢測(cè)了Cd對(duì)可破壞AR SUMO化的蛋白酶1(SUMO specific protease, SENP1) mRNA和蛋白表達(dá)水平的變化,發(fā)現(xiàn)Cd增加SENP1的表達(dá)；繼而利用基因沉默技術(shù),檢測(cè)LNCaP細(xì)胞中的SENP1沉默后,AR及其下游靶基因PSA的表達(dá),發(fā)現(xiàn)通過SENP1調(diào)控AR的活性；進(jìn)一步檢測(cè)了Cd對(duì)/AR SUMO化水平的影響,發(fā)現(xiàn)Cd處理后AR分子的SUMO化水平降低。綜上所述,Cd通過SENP1降低雄激素受體AR的SUMO化調(diào)節(jié)AR的活性。以上結(jié)果分析了鎘雄激素內(nèi)分泌干擾毒性的一種新機(jī)制,為其他物質(zhì)的內(nèi)分泌毒性篩選提供新的方法,也為尋找環(huán)境Cd污染所致疾病的預(yù)防控制的新策略提供理論支持。
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R114
本文編號(hào)：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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R114
【共引文獻(xiàn)】
相關(guān)期刊論文 前1條
1 李裕;張強(qiáng);王潤元;肖國舉;王勝;;鎘的致癌性與食品中鎘的生物有效性[J];生命科學(xué);2010年02期
相關(guān)會(huì)議論文 前1條
1 李裕;張強(qiáng);王潤元;肖國舉;;食品中鎘的生物有效性與影響因素[A];第27屆中國氣象學(xué)會(huì)年會(huì)干旱半干旱區(qū)地氣相互作用分會(huì)場(chǎng)論文集[C];2010年
，本文編號(hào)：2128717
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