新型雌激素受体调节因子GA5的功能研究
摘要
雌激素受体(ER)是核受体超家族成员之一,它包括ERα和ERβ两种亚型。ER在乳腺癌的发生、发展中发挥重要作用,目前认为ER是乳腺癌内分泌治疗的靶标和预后的指标之一。当前,乳腺癌内分泌的治疗主要是通过降低ER转录活性而实现。因此调节ER转录活性的蛋白因子的发现和相应功能研究对于有效开发治疗雌激素相关疾病的药物具有重要的意义。
雌激素受体对其下游基因的表达调节是1个复杂的、多种因素参与的过程。其中,分布于不同组织中的共调节因子(包括共激活因子和共抑制因子)能够与雌激素受体结合进而影响其转录激活活性。相对于ERα,ERβ发现的比较晚,其共调节因子发现得就更少,为深入研究ERβ在雌激素应答基因转录调节中的作用方式,发现新型的转录共调节因子,我们以ERβ的AF-2区段为诱饵从人乳腺cDNA文库中筛选到了一个功能未知的候选新基因GA5。为了深入研究GA5基因的功能,我们做了以下研究并取得相应的成果。
(1)成功构建了GA5原核表达载体PGEX-KG-GA5,在大肠杆菌中诱导表达的GST-GA5蛋白经纯化后免疫小鼠得到多克隆抗体,该抗体可特异识别GA5蛋白。
(2)用自制多克隆抗体进行Western blot检测分析GA5蛋白在不同乳腺癌细胞内源表达量的差异,得知GA5在不同乳腺癌细胞中的表达量不一样,且有被修饰的可能。
(3)构建了GA5 siRNAs真核表达载体,通过Western blotting实验证明,构建的siRNA能有效抑制GA5基因的表达,为进一步研究GA5在肿瘤中的功能提供了理想的研究工具。
(4)酵母双杂交实验已证明GA5与ER在体内外均存在相互作用,因此,GA5有可能通过与ER相互作用,参与雌激素信号通路的调控。为此,我们构建了ERα、ERβ真核表达载体pIRESpuro2-Flag-ERα和pIRESpuro2-Flag-ERβ,证明Flag-ERα和Flag-ERβ融合蛋白在293T细胞中成功表达;用荧光素酶ERE-luc检测pIRESpuro2-Flag-ERα和pIRESpuro2-Flag-ERβ转录活性,发现在雌激素存在的情况下,pIRESpuro2-Flag-ERα和pIRESpuro2-Flag-ERβ转录活性分别提高了7倍和6倍。
Estrogen receptor(ER)is one of the members of the nuclear receptor superfamily,which contains two subtypes ERαand ERβ.ER plays an important role in the development of breast cancer.Recently ER is thought to act as a target of the therapy for breast cancer and one of indexes to evaluate prognosis. Until now,the endocrine therapy of mammary carcinoma is achived mainly by decreasing ER transcriptional activity.Therefore,it is of great significance to discover and characterize the proteins that regulate ER activity in developing new drugs to treat the diseases related to estrogen.
It is a complex process for ER to regulat its downstream genes' expression, as multiple factors,such as co-regulators distributing in many different tissues, are involved in the process.These co-regulators(including Co-activators and coinhibitors) can affect transcription activation of ER through binding to it. Relatively,the discovery of ERβis much later than ERα,and the number of its co-regulators even much less than that of ERβ.For further exploring the role of ER in transcription regulation of the genes corresponding to estrogen, also for discovering new co-regulators of ERβ,we found a new candidate gene GA5 from a human gland cDNA library using ERβAF-2 sector as a bait.To further understand the function of GA5,a series of experiments were carried out, and some good results were obtained.
(1)prokaryotic expression vector pGEX-KG-GA5 was successfully constructed,and GST-GA5 fusion protein was induced to express in E.coli BL21.Purified GST-GA5 fusion protein was inoculate BLAB/c mouse for three times,and polyclonal anti-GA5 antibody was preparied.Western blotting analysis showed that the antibody was specific to GA5 protein.
(2)GA5 expression in several different breast cancer cell lines was determined using the above polyclonal anti-GA5 antibody.The result showed that GA5 expression in those breast cancer cell lines was different and that GA5 protein may be modified by other molecules.
(3)The expression vectors of GA5 siRNAs were constructed and confirmed by DNA sequencing.Western blotting analysis showed that GA5 siRNA could effectively inhibit the expression of GA5 gene.It is helpful for further study the functions of GA5 in tumor.
(4)The Yeast double-hybrid experiment had proven that GA5 and ER interact each other in vitro and in vivo,therefore,it is expected that GA5 is involved in regulation of the estrogen signaling pathway through the interaction. The eukaryotic expression vector pIRESpuro2-Flag-ERαand pIRESpuro2-Flag-ERβwas successfully constructed,westen blotting assay illustrated that Flag-ERαand Flag-ERβfusion protein were expressed in 293T cells.The ranscription activities of pIRESpuro2-Flag-ERαand pIRESpuro2-Flag-ERβwere determined by luciferase ERE-luc.The results showed that,in the presence of strogen,the transcription activities of pIRESpuro2-Flag-ERαand pIRESpuro2-Flag-ERβincreased by 7%and 6% respectively.
雌激素受体对其下游基因的表达调节是1个复杂的、多种因素参与的过程。其中,分布于不同组织中的共调节因子(包括共激活因子和共抑制因子)能够与雌激素受体结合进而影响其转录激活活性。相对于ERα,ERβ发现的比较晚,其共调节因子发现得就更少,为深入研究ERβ在雌激素应答基因转录调节中的作用方式,发现新型的转录共调节因子,我们以ERβ的AF-2区段为诱饵从人乳腺cDNA文库中筛选到了一个功能未知的候选新基因GA5。为了深入研究GA5基因的功能,我们做了以下研究并取得相应的成果。
(1)成功构建了GA5原核表达载体PGEX-KG-GA5,在大肠杆菌中诱导表达的GST-GA5蛋白经纯化后免疫小鼠得到多克隆抗体,该抗体可特异识别GA5蛋白。
(2)用自制多克隆抗体进行Western blot检测分析GA5蛋白在不同乳腺癌细胞内源表达量的差异,得知GA5在不同乳腺癌细胞中的表达量不一样,且有被修饰的可能。
(3)构建了GA5 siRNAs真核表达载体,通过Western blotting实验证明,构建的siRNA能有效抑制GA5基因的表达,为进一步研究GA5在肿瘤中的功能提供了理想的研究工具。
(4)酵母双杂交实验已证明GA5与ER在体内外均存在相互作用,因此,GA5有可能通过与ER相互作用,参与雌激素信号通路的调控。为此,我们构建了ERα、ERβ真核表达载体pIRESpuro2-Flag-ERα和pIRESpuro2-Flag-ERβ,证明Flag-ERα和Flag-ERβ融合蛋白在293T细胞中成功表达;用荧光素酶ERE-luc检测pIRESpuro2-Flag-ERα和pIRESpuro2-Flag-ERβ转录活性,发现在雌激素存在的情况下,pIRESpuro2-Flag-ERα和pIRESpuro2-Flag-ERβ转录活性分别提高了7倍和6倍。
Estrogen receptor(ER)is one of the members of the nuclear receptor superfamily,which contains two subtypes ERαand ERβ.ER plays an important role in the development of breast cancer.Recently ER is thought to act as a target of the therapy for breast cancer and one of indexes to evaluate prognosis. Until now,the endocrine therapy of mammary carcinoma is achived mainly by decreasing ER transcriptional activity.Therefore,it is of great significance to discover and characterize the proteins that regulate ER activity in developing new drugs to treat the diseases related to estrogen.
It is a complex process for ER to regulat its downstream genes' expression, as multiple factors,such as co-regulators distributing in many different tissues, are involved in the process.These co-regulators(including Co-activators and coinhibitors) can affect transcription activation of ER through binding to it. Relatively,the discovery of ERβis much later than ERα,and the number of its co-regulators even much less than that of ERβ.For further exploring the role of ER in transcription regulation of the genes corresponding to estrogen, also for discovering new co-regulators of ERβ,we found a new candidate gene GA5 from a human gland cDNA library using ERβAF-2 sector as a bait.To further understand the function of GA5,a series of experiments were carried out, and some good results were obtained.
(1)prokaryotic expression vector pGEX-KG-GA5 was successfully constructed,and GST-GA5 fusion protein was induced to express in E.coli BL21.Purified GST-GA5 fusion protein was inoculate BLAB/c mouse for three times,and polyclonal anti-GA5 antibody was preparied.Western blotting analysis showed that the antibody was specific to GA5 protein.
(2)GA5 expression in several different breast cancer cell lines was determined using the above polyclonal anti-GA5 antibody.The result showed that GA5 expression in those breast cancer cell lines was different and that GA5 protein may be modified by other molecules.
(3)The expression vectors of GA5 siRNAs were constructed and confirmed by DNA sequencing.Western blotting analysis showed that GA5 siRNA could effectively inhibit the expression of GA5 gene.It is helpful for further study the functions of GA5 in tumor.
(4)The Yeast double-hybrid experiment had proven that GA5 and ER interact each other in vitro and in vivo,therefore,it is expected that GA5 is involved in regulation of the estrogen signaling pathway through the interaction. The eukaryotic expression vector pIRESpuro2-Flag-ERαand pIRESpuro2-Flag-ERβwas successfully constructed,westen blotting assay illustrated that Flag-ERαand Flag-ERβfusion protein were expressed in 293T cells.The ranscription activities of pIRESpuro2-Flag-ERαand pIRESpuro2-Flag-ERβwere determined by luciferase ERE-luc.The results showed that,in the presence of strogen,the transcription activities of pIRESpuro2-Flag-ERαand pIRESpuro2-Flag-ERβincreased by 7%and 6% respectively.
引文
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