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.

Involvement of miRNAs in ovarian follicular and luteal development

“…Whether, physiologically, miR-224, miR-383 and miR-378 are each involved in regulation of aromatase during different stages of follicle development has not been determined. In addition, large-scale functional testing in human granulosa cells revealed effects of multiple miRNAs on the production of progesterone, testosterone and oestradiol, as well as in the expression of the markers of proliferation and apoptosis, PCNA and BAX, although no attempt was made to investigate the mechanisms involved in these responses (Sirotkin et al 2009(Sirotkin et al , 2010.…”

Dynamics and role of MicroRNAs during mammalian follicular development

“…A recent study showed that level of miR-378 in the follicular wall was correlated with estradiol, the estradiol: progesterone ratio and CYP19A1 (Donadeu et al, 2016). MiR-34a and miR-320 inhibit estradiol release from human granulosa cells and murine ovaries, respectively (Sirotkin et al, 2009;Yao et al, 2014). However, miR-320 could stimulate testosterone and progesterone in murine ovaries.…”

“…The effect of miRNAs on the release of major ovarian steroid hormones was determined by transfecting human primary granulosa cells with 80 different human pre-miRNAs constructs, and the release of progesterone was inhibited by 36 miRNAs. Whereas, 10 miRNAs promoted progesterone release (Sirotkin et al, 2009). MiR-378 decreases ovarian estradiol production by targeting aromatase.…”

The role of micro-RNAs in the female reproductive tract

“…Unfortunately, these studies neither explored the targets of these miRNAs, which may be regulated to modulate cell apoptosis/ proliferation, nor did they examine what factors may modulate the expression of these miRNAs. Sirotkin et al (2009) used an identical approach to identify miRNAs that regulate ovarian cell steroidogenesis. Thirty-six miRNAs that inhibited progesterone release and ten miRNAs that enhanced progesterone release were identified ( Table 2).…”