PDF(Pubmed)
Abstract：
The ovarian KGN granulosa-like tumour cell line is commonly used as a model for human granulosa cells, especially since it produces steroid hormones. To explore this further, we identified genes that were differentially expressed by KGN cells compared to primary human granulosa cells using three public RNA sequence datasets. Of significance, we identified that the expression of the antioxidant gene TXNRD1 (thioredoxin reductase 1) was extremely high in KGN cells. This is ominous since cytochrome P450 enzymes leak electrons and produce reactive oxygen species during the biosynthesis of steroid hormones. Gene Ontology (GO) analysis identified steroid biosynthetic and cholesterol metabolic processes were more active in primary granulosa cells, whilst in KGN cells, DNA processing, chromosome segregation and kinetochore pathways were more prominent. Expression of cytochrome P450 cholesterol side-chain cleavage (CYP11A1) and cytochrome P450 aromatase (CYP19A1), which are important for the biosynthesis of the steroid hormones progesterone and oestrogen, plus their electron transport chain members (FDXR, FDX1, POR) were measured in cultured KGN cells. KGN cells were treated with 1 mM dibutyryl cAMP (dbcAMP) or 10 μM forskolin, with or without siRNA knockdown of TXNRD1. We also examined expression of antioxidant genes, H2O2 production by Amplex Red assay and DNA damage by γH2Ax staining. Significant increases in CYP11A1 and CYP19A1 were observed by either dbcAMP or forskolin treatments. However, no significant changes in H2O2 levels or DNA damage were found. Knockdown of expression of TXNRD1 by siRNA blocked the stimulation of expression of CYP11A1 and CYP19A1 by dbcAMP. Thus, with TXNRD1 playing such a pivotal role in steroidogenesis in the KGN cells and it being so highly overexpressed, we conclude that KGN cells might not be the most appropriate model of primary granulosa cells for studying the interplay between ovarian steroidogenesis, reactive oxygen species and antioxidants.
摘要：
卵巢KGN颗粒样肿瘤细胞系通常用作人类颗粒细胞的模型，尤其是它能产生类固醇激素.为了进一步探索这一点，我们使用三个公共RNA序列数据集鉴定了KGN细胞与原代人颗粒细胞相比差异表达的基因.意义重大,我们发现抗氧化剂基因TXNRD1（硫氧还蛋白还原酶1）在KGN细胞中的表达非常高。这是不祥的，因为细胞色素P450酶在类固醇激素的生物合成过程中泄漏电子并产生活性氧。基因本体论（GO）分析确定类固醇生物合成和胆固醇代谢过程在原代颗粒细胞中更活跃，而在KGN单元格中，DNA处理,染色体分离和动粒途径更为突出。细胞色素P450胆固醇侧链裂解（CYP11A1）和细胞色素P450芳香化酶（CYP19A1）的表达，这对类固醇激素孕激素和雌激素的生物合成很重要，加上他们的电子传输链成员(FDXR，在培养的KGN细胞中测量FDX1，POR）。KGN细胞用1mM二丁酰基cAMP(dbcAMP)或10μM毛喉素处理,有或没有TXNRD1的siRNA敲低。我们还检查了抗氧化基因的表达，通过AmplexRed测定产生H2O2，并通过γH2Ax染色对DNA进行损伤。通过dbcAMP或毛喉素处理观察到CYP11A1和CYP19A1的显着增加。然而,没有发现H2O2水平或DNA损伤的显着变化。通过siRNA抑制TXNRD1的表达阻断了dbcAMP对CYP11A1和CYP19A1表达的刺激。因此，TXNRD1在KGN细胞的类固醇生成中起着如此关键的作用，并且它是如此的高表达，我们得出的结论是，KGN细胞可能不是研究卵巢类固醇生成之间相互作用的最适合的原代颗粒细胞模型，活性氧和抗氧化剂。
