DNAJB11促进卵巢颗粒细胞中FOXL2诱导的雌激素合成

发布时间：2017-12-28 03:31

本文关键词：DNAJB11促进卵巢颗粒细胞中FOXL2诱导的雌激素合成　出处：《医学研究生学报》2017年10期 　论文类型：期刊论文

【摘要】：目的转录因子FOXL2是颗粒细胞雌激素合成与颗粒细胞功能维持的关键调控分子,但目前尚不清楚FOXL2蛋白的表达及功能调控机制。文章旨在鉴定调控FOXL2蛋白功能的新分子。方法通过分离小鼠卵巢组织进行免疫组化染色检测DNAJB11的表达定位情况,含有全长DNAJB11基因c DNA序列(Ad-DNAJB11-Flag和Ad-Flag-DNAJB11)的腺病毒载体按Ad Max系统(Microbix)方法进行病毒颗粒的包装。按NE-PERTMNuclear and Cytoplasmic抽提试剂盒所述方法进行细胞核质蛋白的分离。KGN细胞感染不同浓度腺病毒(Ad-DNAJB11和Ad-Lac Z),测量波长450 nm的吸光度值,进行细胞增殖检测。采用Access Immunoassay System 2化学发光自动检测系统(Beckman Coulter)检测细胞培养上清中雌二醇的浓度。采用PCR方法扩增含有人FOXL2和人DNAJB 11基因的全长c DNA序列,并分别亚克隆至p CS2-6XMT载体(Myc-FOXL2)和p FLAG-CMV2载体(Flag-DNAJB11)中。采用Lipofectamine 2000转染试剂将Myc-FOXL2和Flag-DNAJB11表达质粒瞬时转染至细胞HEK-293细胞中。收集HEK-293细胞或FSH处理的KGN细胞的裂解液,进行FOXL2和DNAJB11的免疫共沉淀实验。通过细胞免疫荧光染色和荧光素酶报告基因等实验检测内质网Hsp40/Dna J家族成员DNAJB11调控颗粒细胞的雌激素水平。结果 Western blot表明DNAJB11蛋白在人卵癌颗粒细胞KGN、小鼠原代卵巢颗粒细胞m GC以及小鼠卵巢中高表达,免疫组化染色结果亦证实DNAJB11蛋白表达于小鼠卵巢的卵母细胞质中,并在窦前卵泡、排卵前卵泡的颗粒细胞中持续表达。免疫荧光染色和Western blot分析亦表明外源性激素FSH可以诱导KGN细胞中内源性DNAJB11蛋白从内质网转移到细胞核中。腺病毒介导的DNAJB11-Flag过表达定位于细胞内质网中,但当Flag标签阻断DNAJB11信号肽功能时,外源FlagDNAJB11过表达则定位于颗粒细胞的细胞核,内质网中DNAJB11-Flag蛋白高表达以浓度依赖的方式减少KGN细胞中雌二醇的合成,与Ad-Lac Z(MOI=50)比较,Ad-DNAJB11-Flag(MOI=50)减少,KGN细胞中雌二醇的合成[(10 749.0±801.7)pg/m L vs(7 903.0±409.5)pg/m L,P0.01],而细胞核中高表达Flag-DNAJB11后则刺激KGN细胞中雌二醇的生成,与Ad-Lac Z(MOI=50)比较,Ad-DNAJB11-Flag(MOI=50)刺激雌二醇的生成[(10 749.0±801.7)pg/m L vs(14 217.0±1218.0)pg/m L,P0.01]。免疫共沉淀实验表明当Myc-tagged FOXL2与Flag-tagged DNAJB11分别共转染HEK293细胞,FOXL2与DNAJB11可以相互免疫共沉淀对方,荧光素酶报告基因实验进一步表明细胞核中表达的DNAJB11显著增强FOXL2介导的Cyp19A1启动子活性和Cyp19A1蛋白表达。结论 DNAJB11是转录因子FOXL2新的结合分子并调控FOXL2蛋白的稳定性与转录活性。
[Abstract]:Objective transcription factor FOXL2 is a key regulator of estrogen synthesis and granulosa cell function maintenance in granulosa cells, but the expression and functional regulation mechanism of FOXL2 protein is not yet clear. The aim of this article is to identify new molecules that regulate the function of FOXL2 protein. Methods the expression and location of DNAJB11 were detected by immunohistochemical staining of mouse ovarian tissue. The adenovirus vector containing full-length DNAJB11 gene C DNA sequence (Ad-DNAJB11-Flag and Ad-Flag-DNAJB11) was packaged according to Ad Max system (Microbix) method. The cell nuclear protein was separated according to the method described by NE-PERTMNuclear and Cytoplasmic extraction kit. KGN cells infected different concentrations of adenovirus (Ad-DNAJB11 and Ad-Lac Z), measured the absorbance value of 450 nm at wavelength, and detected the cell proliferation. The Access Immunoassay System 2 chemiluminescence automatic detection system (Beckman Coulter) was used to detect the concentration of estradiol in cell culture supernatant. The full-length C DNA sequence containing human FOXL2 and human DNAJB 11 gene was amplified by PCR and subcloned into P CS2-6XMT carrier (Myc-FOXL2) and P FLAG-CMV2 carrier (Flag-DNAJB11), respectively. Myc-FOXL2 and Flag-DNAJB11 expression plasmids were transiently transfected into cell HEK-293 cells by Lipofectamine 2000 transfection reagent. The lysate of KGN cells treated with HEK-293 or FSH was collected and the immunoprecipitation experiment of FOXL2 and DNAJB11 was carried out. The level of estrogen in granulosa cells regulated by Hsp40/Dna J family member DNAJB11 was detected by immunofluorescence staining and luciferase reporter assay. The results of Western blot showed that the high expression of DNAJB11 protein in primary human cancer cells, KGN egg granules of mouse granulosa cells of M and GC in mouse ovary, immunohistochemical staining also confirmed the expression of DNAJB11 protein in the mouse ovary in oocytes, granulosa cells and follicular, expressed in preantral preovulatory follicles in. Immunofluorescence staining and Western blot analysis also showed that exogenous FSH could induce endogenous DNAJB11 proteins in KGN cells to transfer from the endoplasmic reticulum to the nucleus. Adenovirus mediated overexpression of DNAJB11-Flag located in the endoplasmic reticulum, but when the Flag label blocking DNAJB11 signal peptide function, overexpression of FlagDNAJB11 is localized to the granule cell nucleus, endoplasmic reticulum in the high expression of DNAJB11-Flag protein in a concentration dependent manner to reduce the synthesis of estradiol in KGN cells, Ad-Lac and Z (MOI=50), Ad-DNAJB11-Flag (MOI=50) reduced synthesis of estradiol in KGN cells (10749 + 801.7) pg/m L vs (7903 + 409.5) pg/m, L, P0.01], and the nucleus in the high expression of Flag-DNAJB11 after the stimulation of production of estradiol and KGN cells, and Ad-Lac Z (MOI=50), Ad-DNAJB11-Flag (MOI=50 estradiol stimulates) [(10749 + 801.7) pg/m L vs (14217 + 1218) pg/m L, P0.01]. The experimental results show that when Myc-tagged FOXL2 and Flag-tagged DNAJB11 were co transfected into HEK293 cells by immunoprecipitation, FOXL2 and DNAJB11 can mutually co immunoprecipitation each other, luciferase reporter assays indicated that the nuclear expression of DNAJB11 significantly enhanced FOXL2 mediated by Cyp19A1 promoter activity and Cyp19A1 protein expression. Conclusion DNAJB11 is a new binding molecule of the transcription factor FOXL2 and regulates the stability and transcriptional activity of FOXL2 protein.
【作者单位】：南京农业大学动物医学院;南京大学附属鼓楼医院生殖中心;
【基金】：国家自然科学基金(81370683,81571402) 江苏省“十三五科教强卫工程”医学重点人才项目(ZDRCA2016070)
【分类号】：R339.22
【正文快照】： DNAJB11 promotes the synthesis of FOXL2-induced estradiol in ovarian granulosa cellsMAO Yan1,YAN Qiang2,ZHANG Chun-xue2,ZHEN Xin2,CAO Rui-bing1,YAN Gui-jun2(1.College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,Jiangsu,China;2.R

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