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首页> 外文期刊>BMC Medical Genomics >Interplay between estrogen receptor and AKT in Estradiol-induced alternative splicing
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Interplay between estrogen receptor and AKT in Estradiol-induced alternative splicing

机译：雌二醇诱导的选择性剪接中雌激素受体和AKT的相互作用

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Background Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. Methods MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. Results We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF. Conclusion E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.
机译：背景技术选择性剪接对于响应细胞外信号产生复杂的蛋白质组至关重要。包括雌激素受体α（ERα）及其配体在内的核受体促进了选择性剪接。 ERα：雌二醇（E2）介导的选择性剪接的内源性靶标和磷酸化ERα的细胞外激酶对E2诱导剪接的影响尚不清楚。方法MCF-7及其抗雌激素衍生物用于大多数测定。 CD44 mini基因被用来测量E2和AKT对选择性剪接的影响。进行ExonHit阵列分析以鉴定E2和AKT调控的内源性选择性剪接凋亡相关基因。进行定量逆转录聚合酶链反应以验证替代剪接。 ERα结合剪接的基因通过染色质免疫沉淀法验证。进行溴脱氧尿苷掺入-ELISA和膜联蛋白V标记测定以分别测量细胞增殖和凋亡。结果我们确定了结合E2基因的剪接阵列和基因表达阵列，从而确定了E2诱导的选择性剪接的目标，并解构了围绕E2诱导的剪接的一些机制。 E2诱导的可变剪接基因分为至少两个亚组：与E2调控的转录偶联，以及ERα与该基因的结合，而不会影响转录速率。此外，磷酸化ERα和剪接因子的AKT以基因特异性方式影响ERα：E2依赖性剪接。可选择剪接的基因包括FAS / CD95，FGFR2和AXIN-1。 E2增加FGFR2 C1亚型的表达，但在mRNA水平降低C3亚型。 E2诱导的MCF-7细胞中FAS和FGFR2的可变剪接分别与对FAS激活诱导的凋亡的抵抗和对角质形成细胞生长因子（KGF）的响应有关。 MCF-7乳腺癌细胞对抗雌激素他莫昔芬的抗性与ERR依赖的FGFR2过表达有关，而对氟维司群的抗性与ERα依赖的同工型转换有关，这与对KGF的反应改变有关。结论E2可能通过替代剪接部分改变细胞蛋白质组，而其对E2诱导的替代剪接事件中转录起始和畸变的影响不相关，可能影响对抗雌激素的反应。

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