摘要： 目的 探究FOXO3和SOHLH1对卵母细胞特异性表达基因Bmp15的调控机制。方法 构建Foxo3-pcDNA3.0表达质粒、Bmp15-pGL3.0荧光质粒，用细胞免疫荧光实验验证这2种表达质粒在HEK293T细胞中的表达情况，双荧光素酶报告基因实验检测FOXO3和转录因子SOHLH1对Bmp15启动子区的转录活性的影响，染色质免疫共沉淀实验验证FOXO3和转录因子SOHLH1在Bmp15启动子区的结合情况。结果 Foxo3-pcDNA3.0表达质粒、Bmp15-pGL3.0荧光质粒构建成功。Foxo3-pcDNA3.0、Sohlh1-pcDNA3.0质粒成功转染HEK293T细胞并表达于其细胞核、质中。SOHLH1对Bmp15启动子（-2 000 bp，+200 bp）区域有正调控作用，该作用可被FOXO3抑制，且呈剂量依赖性。SOHLH1蛋白能与Bmp15启动子（-170 bp，80 bp），（-450 bp，-220 bp），（-745bp，-560 bp），（-1 410 bp，-1 200 bp）区间结合。FOXO3能与Bmp15启动子（120 bp，183 bp），（-84 bp，-75 bp），（-442bp，-395 bp），（-546 bp，-470 bp），（-674 bp，-665 bp）区间结合。结论 FOXO3能抑制转录因子SOHLH1对卵母细胞特异性表达基因Bmp15的转录活性。

FOXO3和转录因子SOHLH1对卵母细胞特异性表达基因Bmp15转录活性的调控

刘源, 杜冰, 张雪, 金美玉, 郑志红

中国医科大学实验动物部, 沈阳 110122

收稿日期:2019-03-27出版日期:2020-07-30发布日期:2020-07-02
通讯作者:郑志红E-mail:zhihongzheng@163.com
作者简介:刘源(1993-),男,硕士研究生.
基金资助:辽宁省科学技术计划（2015408002）


关键词: 卵泡信号交流, SOHLH1, FOXO3, Bmp15
Abstract: Objective To investigate the FOXO3 and SOHLH1 (germ-cell-specific transcription factor) regulated transcriptional activity of Bmp15,an oocyte-specific gene. Methods Foxo3-pcDNA3.0 expression plasmids and Bmp15-pGL3.0 fluorescent plasmids were constructed and transfected into HEK293T human kidney cells. Immunofluorescence assay was performed to detect the expression of SOHLH1 and FOXO3 proteins in HEK293T cells. Dual-luciferase reporter gene assay was used for establishing the Bmp15 promoter activity. The transcriptional activity of FOXO3 and SOHLH1 on the Bmp15 promoter region was detected by polymerase chain reaction. Chromatin immunoprecipitation assay was performed to confirm the binding of FOXO3 and transcription factor SOHLH1 in the Bmp15 promoter (-2 000 to +200 bp). Results The expression of FOXO3 and SOHLH1 proteins was detected in the nucleus and cytoplasm of HEK293T cells after 24 and 48 hours of transfection,respectively. The results showed that SOHLH1 had a positive regulatory effect on the Bmp15 promoter. The binding of SOHLH1 to Bmp15 promoter was detected at the following regions:-170 to +80 bp,-450 to -220 bp,-745 to -560 bp,and -1 410 to -1 200 bp. Further,Foxo3 showed dose-dependent inhibition of SOHLH1-associated positive regulation of Bmp15. Binding of FOXO3 to Bmp15 promoter was detected at the following regions:+120 to +183 bp,-84 to -75 bp,-442 to -395 bp,-546 to -470 bp,and -674 to -665 bp. Conclusion FOXO3 inhibits the transcriptional activity of SOHLH1 on the oocytespecific gene,Bmp15.
Key words: follicular signal exchange, SOHLH1, FOXO3, Bmp15
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