Unique features of KGN granulosa-like tumour cells in the regulation of steroidogenic and antioxidant genes

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.