Identification of MicroRNAs controlling human ovarian cell steroidogenesis via a genome‐scale screen

Abstract

The aim of our studies was to identify miRNAs affecting the release of the major ovarian steroid hormones progestagen, androgen and estrogen by human ovarian cells. The effect of transfection of cultured primary ovarian granulosa cells with 80 different gene constructs encoding human pre-miRNAs on release of progesterone, testosterone and estradiol was evaluated by enzyme immunoassay. In addition, effect of two selected antisense constructs blocking corresponding miRNA on progesterone release was tested. Efficiency of transfection (incorporation transfection reagent) and silencing of marker substances (GAPDH mRNA, GAPDH and CREB-1) were validated by fluorescent microscopy, real-time reverse transcription-PCR analysis and immunocytochemical analysis. Thirty-six out of 80 tested miRNA constructs resulted in inhibition of progesterone release in granulosa cells, and 10 miRNAs promoted progesterone release. Transfected of cells with antisense constructs to two selected miRNAs blocking progesterone release induced increase in progesterone output. Fifty-seven miRNAs tested inhibited testosterone release, and only one miRNA enhanced testosterone output. Fifty-one miRNAs suppressed estradiol release, while none of the miRNAs tested stimulated it. This is the first demonstration that miRNAs can control reproductive functions resulting in enhanced or inhibited release of ovarian progestagen, androgen and estrogen. We hypothesize that such miRNA-mediated effects could be potentially used for regulation of reproductive processes, including fertility, and for treatment of reproductive and other steroid-dependent disorders. J. Cell. Physiol. 219: 415–420, 2009.