利用 cut&tag 技术探究 LHX9 基因在颗粒细胞的调控机制  

Exploring the Regulatory Mechanism of LHX9 in Granulosa Cell Line by Cut&tag

作　　者：吴婧 徐悦 曹仁 张漫漫 乔洁[2] 宋怀东[1] WU Jing;XU Yue;CAO Ren;ZHANG Man-man;QIAO Jie;SONG Huai-dong(The Core Laboratory in Medical Center of Clinical Research,Department of Molecular Diagnostics,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai,200001,China;Department of Endocrinology,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School ofMedicine,Shanghai,200001,China;School ofLife Science and Technology,Shanghai Tech University,Shanghai,201210,China)

机构地区：[1]上海交通大学医学院附属第九人民医院分子诊断科,上海200001 [2]上海交通大学医学院附属第九人民医院内分泌科,上海200011 [3]上海科技大学生命科学与技术学院,上海201210

出　　处：《现代生物医学进展》2023年第24期4601-4606,共6页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(82270826,81873652)。

摘　　要：目的:在人卵巢颗粒细胞癌细胞株KGN中探索转录因子LHX9下游主要的靶基因及其调控。方法:首先我们采用实时荧光定量PCR观察KGN细胞在卵泡刺激素(FSH)干预前后性腺分化和性激素合成过程中重要基因的表达情况,同时我们利用cut&tag测序技术在两组细胞中识别LHX9下游靶基因,并采用双荧光素酶报告实验对重要靶基因NR5A1的调控进行了验证。结果:实时荧光定量PCR结果显示,通过加FSH处理12小时后,LHX9和NR5A1基因的mRNA水平表达降低,相反的,StAR和CYP19A1基因的mRNA水平表达增高;通过cut&tag测序技术和生物信息学分析,我们发现LHX9下游基因主要分布在内吞作用、细胞衰老、肿瘤相关通路、细胞周期、凋亡、卵母细胞减数分裂、雌激素信号转导等通路上。LHX9可以转录调控性腺分化以及类固醇激素合成的一些重要基因,其中包括NR5A1和SOX9等,荧光素酶报告基因证实了LHX9可以直接结合NR5A1基因的启动子区。结论:本研究利用cut&tag测序技术,发现转录因子LHX9对性腺分化和性激素合成的关键基因有转录调控作用,对深入理解LHX9基因对生殖系统的作用具有重要意义。Objective:To explore the downstream target genes and the regulatory mechanism of transcription factor LHX9 in human ovarian granulocyte cancer cell line KGN.Methods:Firstly,we used real-time PCR to observe the expression of key genes in gonadal differentiation and sex hormone synthesis in KGN cells before and afer FSH intervention.Next,we used cut&tag sequencing to identify the target genes of LHX9 in two groups of cells,and we verified the transcriptional regulation of NR5A1 by LHX9 with dual-lu-ciferase reporter assay.Results:According to real-time PCR result,we found that the expression of LHX9 and NR5A1 genes decreased at mRNA level after 12 hours of FSH treatment,and conversely,the expression of StAR and CYP19A1 gene increased.Through cut&tag sequencing and bioinformatics analysis,we found that LHx9 downstream genes are mainly distributed in endocytosis,cell aging,tumor-related pathways,cell cycle,apoptosis,oocyte meiosis,estrogen signaling and other pathways.LHX9 transcriptional regulates some key genes which regulate gonadal differentiation and steroid hormone synthesis,including NR5A1 and SOX9.And dual-luciferase reporter assay confirmed that LHX9 directly binds to the promoter region of the NR5A1.Conclusions:In this study,we use cut&tag sequencing technology to characterize that LHX9 has transcriptional regulation of key genes during the process of gonadal differentiation and sex hormone synthesis,which is of great significance for understanding of the role of LHx9 gene on the reproductive system.

关 键 词：LHX9 cut&tag NR5A1 性腺分化 

分 类 号：R-33[医药卫生] Q593.4[生物学—生物化学] Q492