Involvement of MicroRNA Mir15a in Control of Human Ovarian Granulosa Cell Proliferation, Apoptosis, Steroidogenesis, and Response to FSH
Alexander V. Sirotkin, Gabriela Kisova, Pauline Brenaut, Dmitriy Ovcharenko, Roland Grossmann and Milos Mlyncek
Affiliation: Animal Production Research Centre Nitra, Hlohovecka 2, 951 41 Luzianky, Slovakia.
Our study aimed to examine the role of micro RNA Mir15a in control of basic ovarian cell functions: proliferation,
apoptosis, and secretory activity. In the first series of experiments, primary human ovarian granulosa cells were
transfected with antisense construct blocking Mir15a (anti-Mir15a) and cultured without hormonal treatments. Accumulation
of markers of proliferation (MAPK/ERK1,2 and PCNA) and apoptosis (caspase 3 and bax), and release of steroid
hormones (progesterone, testosterone, and estradiol) were evaluated by immunocytochemical analysis and by enzyme
immunoassay. In the second series of experiments, granulosa cells were transfected with gene construct encoding Mir15a
precursor (pre-Mir15a) and cultured with and without follicle-stimulating hormone (FSH; 0, 1, 10, and 100 ng/ml). Expression
of markers of proliferation (MAPK/ERK1,2) apoptosis (caspase 3), and steroidogenesis (release of progesterone,
testosterone, and estradiol) were evaluated. Transfection of cells with anti-Mir15a resulted in a significant increase in accumulation
of both proliferation and apoptosis markers, a reduction in progesterone and testosterone release, and an increase
in estradiol release. Transfection of cells with pre-Mir15a had an opposite effect: it reduced accumulation of proliferation-
and apoptosis-related proteins MAPK/ERK1,2 and caspase 3, and promoted release of progesterone and testosterone,
but not estradiol. Moreover, pre-Mir15a reversed the effect of FSH on caspase 3, progesterone, and testosterone, but
not on MAPK/ERK1,2 and estradiol. Our observations demonstrate involvement of Mir15a in control of multiple ovarian
functions: proliferation, apoptosis, release of progesterone, androgen, and estrogen, and response to gonadotropin. Moreover,
this is the first demonstration that miRNAs can affect response of cells to hormonal regulators. We propose that
Mir15 could potentially be used for control of different reproductive processes.
Keywords: Apoptosis, bax, caspase, estradiol, FSH, granulosa, MAP kinase, miRNA, ovary, PCNA, progesterone, proliferation,
steroid hormones, testosterone.
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