Molecular assays to profile 10 estrogen receptor beta isoform mRNA copy numbers in ovary, breast, uterus, and bone tissues
Molecular assays to profile 10 estrogen receptor beta isoform mRNA copy numbers in ovary, breast, uterus, and bone tissues
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DOI：
10.1385/endo:22:2:101
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发表时间：
2003-11-01
期刊：
影响因子：
3.7
通讯作者：
Poola, I
中科院分区：
文献类型：
--
作者：
Poola, I
雌激素通过两种在遗传上不同但结构上相关的高亲和力核受体--雌激素受体α和β(ERα和ERβ)来调节多种生殖和非生殖组织中的各种生物学过程。两个具有冗余功能的内质网存在的生理意义尚不清楚。ERβ的一些独特性质及其不同的表达模式被认为是雌激素在不同组织中不同功能作用和选择性雌激素受体调节剂(SERM)相反作用的部分基础。为了了解两个ER的相对表达水平如何与雌激素和SERM似乎不同的作用相关联，需要能够精确测量每种异构体水平的定量方法。以前，量化八种ERA亚型的方法已经被描述[Poola 1.(2003)anal]。生物化学。314,217-226]。本文介绍了基于实时聚合酶链式反应的分子检测方法，它可以区分和定量低至100个拷贝的10个ERbeta亚型的mRNAs，包括ERbeta1、ERbeta2、ERbeta4、ERbeta5和ERbeta外显子2Delta、外显子3Delta、外显子4Delta、外显子5Delta、外显子6Delta和外显子5-6Delta。使用特定的引物对和5‘FAM(羧基荧光素)和3’Tamara(6-羧基四乙基罗丹明)标记的探针，并与用其各自的逆转录cRNA的已知拷贝数构建的标准曲线进行比较，对每个异构体mRNA进行定量。设计的分析方法被应用于四种雌激素敏感组织--卵巢、乳房、子宫和骨骼--的10种ERbeta亚型的分析。根据异构体和组织的不同，在这些组织中检测每种异构体的灵敏度从皮克到纳克的逆转录总RNA不等。研究结果还表明，每个组织都有10个不同亚型的mRNAs。有趣的是，ERα阴性的乳腺癌细胞株和肿瘤表达大量的ERbeta亚型，这表明雌激素在这些组织中存在促有丝分裂刺激。骨组织表达多种异构体，但不存在野生型。除了实验的发展，首次有证据表明ERbeta4和ERbeta5是全长受体，而不是以前的报道，它们是外显子7-8的短受体。预计这里描述的方法将大大有助于描述各种ERbeta亚型的功能作用，并与ERpha亚型分析相结合，将极大地促进设计个性化的组织特异性治疗方法来治疗雌激素相关的病理。
Estrogens regulate various biological processes in a diverse range of reproductive and nonreproductive tissues through two genetically distinct but structurally related high affinity nuclear receptors, the estrogen receptor alpha and beta (ERalpha and ERbeta). The physiological significance of the presence of two ERs that have redundant functions is not known. Several unique properties of ERbeta together with its distinct expression patterns are considered to be, in part, the basis for diverse functional actions of estrogens and opposing actions of selective estrogen receptor modulators (SERMs) in different tissues. To understand how relative expression levels of two ERs correlate to seemingly dissimilar actions of estrogens and SERMs, quantitative methods are required that can precisely measure the levels of every isoform. Previously, methods to quantify eight ERa isoforms have been described [Poola 1. (2003) Anal. Biochem. 314,217-226]. In this article, real-time PCR-based molecular assays are described that can distinguish and quantify as low as 100 copies of 10 ERbeta isoform mRNAs, the ERbeta1, ERbeta2, ERbeta4, ERbeta5, and ERbeta exon 2Delta, exon 3Delta, exon 4Delta, exon 5Delta, exon 6Delta, and exons 5-6Delta. Each isoform mRNA is quantified using a specific primer pair and a 5'FAM (carboxy-fluorescein)- and 3'TAMARA (6-carboxy tetraethyl-rhodamine)-labeled probe and in comparison with a standard curve constructed with known copy numbers of its respective reverse transcribed cRNA. The devised assays were applied to profile 10 ERbeta isoforms in four estrogen-sensitive tissues-ovary, breast, uterus, and bone. The sensitivity of detection of each isoform in these tissues varied from picograms to nanograms of reverse-transcribed total RNA depending on the isoform and the tissue. The results presented also show that each tissue has a distinct profile of 10 isoform mRNAs. Interestingly, ERalpha-negative breast cancer cell lines and tumors expressed significant amounts of ERbeta isoforms suggesting that mitogenic stimulation by estrogen exists in these tissues. Bone tissues expressed several isoforms, although wild type was not present. In addition to the assay development, evidence is presented to demonstrate for the first time that ERbeta4 and ERbeta5 are full length receptors, contrary to previous reports that they are short receptors of exon 7-8. It is expected that the methods described here will significantly contribute to delineating the functional roles of various ERbeta isoforms and in conjunction with ERalpha isoform profiling, will highly facilitate designing of individualized tissue specific therapies to treat estrogen-related pathologies.