The interrelationships between the microRNA miR-34a and the plant flavonoids apigenin and quercetin in the regulation of ovarian cell functions.

In the present study, porcine ovarian granulosa cells were transfected with miR-34a mimics, miR-34 inhibitor, or their respective negative controls (NC mimics and NC inhibitor). As shown in Fig.  1 , FAM-labeled NC mimics and NC inhibitor were predominantly localized in the cell cytoplasm. The transfection efficiency was greater than 75%, as assessed by transfection of fluorescein-labeled (FAM-labeled) NC mimics and NC inhibitor (Fig.  2 ). RT‒qPCR was used to confirm that the miR-34a expression level was increased by several fold in granulosa cells after miR-34a mimic transfection, whereas it was decreased after transfection with the miR-34a inhibitor (Fig.  3 ). Fig. 1 Transfection efficiency of negative control (NC) mimics and NC inhibitor in porcine granulosa cells after 48 h of transfection with these oligonucleotides. Fluorescence microscopy analysis of green fluorescein-labeled (FAM-labeled) negative control (NC) mimics and FAM-labeled NC inhibitor. DAPI was used to stain cellular nuclei. Scale bars: 1 cm = 20 μm. Transfection efficiency of negative control (NC) mimics and NC inhibitor in porcine granulosa cells after 48 h of transfection with these oligonucleotides. Fluorescence microscopy analysis of green fluorescein-labeled (FAM-labeled) negative control (NC) mimics and FAM-labeled NC inhibitor. DAPI was used to stain cellular nuclei. Scale bars: 1 cm = 20 μm. Fig. 2 Transfection efficiency of negative control (NC) mimics and NC inhibitor in porcine granulosa cells after 48 h of transfection with these oligonucleotides. Fluorescence microscopy and subsequent cytometry of proportion of cells containing green fluorescein-labeled (FAM-labeled) negative control (NC) mimics and FAM-labeled NC inhibitor. The values are means ± SEMs. Results are expressed as the mean ± SEM from at least 3 independent experiments. Transfection efficiency of negative control (NC) mimics and NC inhibitor in porcine granulosa cells after 48 h of transfection with these oligonucleotides. Fluorescence microscopy and subsequent cytometry of proportion of cells containing green fluorescein-labeled (FAM-labeled) negative control (NC) mimics and FAM-labeled NC inhibitor. The values are means ± SEMs. Results are expressed as the mean ± SEM from at least 3 independent experiments. Fig. 3 Evaluation of miR-34a expression levels in cells transfected with miR-34a mimics, miR-34a inhibitor or their respective negative controls (NC mimics and NC inhibitor) by reverse transcription-quantitative polymerase chain reaction (RT‒qPCR). * - the effect of miR-34a mimics and of miR-34a inhibitor: a significant ( P  < 0.05) difference between the cells transfected with miR-34a oligonucleotides and the corresponding negative controls. Results are expressed as the mean ± SEM from at least 3 independent experiments. Evaluation of miR-34a expression levels in cells transfected with miR-34a mimics, miR-34a inhibitor or their respective negative controls (NC mimics and NC inhibitor) by reverse transcription-quantitative polymerase chain reaction (RT‒qPCR). * - the effect of miR-34a mimics and of miR-34a inhibitor: a significant ( P  < 0.05) difference between the cells transfected with miR-34a oligonucleotides and the corresponding negative controls. Results are expressed as the mean ± SEM from at least 3 independent experiments. Comparison of cells transfected with miR-34a mimics and NC demonstrated the influence of miR-34a on all measured parameters. Transfection of cultured porcine granulosa cells with miR-34a mimics reduced the accumulation and expression of both the proliferation-related peptide PCNA (Fig.  4 A) and the apoptosis-related protein bax (Fig.  4 B) (see miR-34a mi group without apigenin or quercetin). Furthermore, transfection with miR-34a mimics promoted the secretion of progesterone (Fig.  4 C) and IGF-I (Fig.  4 E) but reduced estradiol secretion (Fig.  4 D). Fig. 4 A Effect of administration of apigenin (0, 1, 10, 100 µg/ml) (left) or quercetin (0, 1, 10, 100 µg/ml) (rights) alone and in combination with miR-34a mimics (miR-34a mi) on cell proliferation (accumulation of PCNA) in cultured porcine ovarian granulosa cells. Results shows (a) significant ( P  < 0.05) differences between cells cultured without (0 µg/ml) and with apigenin or quercetin alone; (b) significant ( P  < 0.05) differences between the cells transfected with miR-34a mi and its NC alone. Results are expressed as the mean ± SEM from at least 3 independent experiments. B Effect of administration of apigenin (0, 1, 10, 100 µg/ml) (left) or quercetin (0, 1, 10, 100 µg/ml) (rights) alone and in combination with miR-34a mimics (miR-34a mi) on cytoplasmic apoptosis (accumulation of bax) in cultured porcine ovarian granulosa cells. Results shows (a) significant ( P  < 0.05) differences between cells cultured without (0 µg/ml) and with apigenin or quercetin alone; (b) significant ( P  < 0.05) differences between the cells transfected with miR-34a mi and its NC alone; (ab) significant ( P  < 0.05) differences between the cells treated with apigenin or quercetin alone and apigenin or quercetin in combination with miR-34a mi. Results are expressed as the mean ± SEM from at least 3 independent experiments. C Effect of administration of apigenin (0, 1, 10, 100 µg/ml) (left) or quercetin (0, 1, 10, 100 µg/ml) (rights) alone and in combination with miR-34a mimics (miR-34a mi) on the release of progesterone by cultured porcine ovarian granulosa cells. Results shows (a) significant ( P  < 0.05) differences between cells cultured without (0 µg/ml) and with apigenin or quercetin alone; (b) significant ( P  < 0.05) differences between the cells transfected with miR-34a mi and its NC alone; (ab) significant ( P  < 0.05) differences between the cells treated with apigenin or quercetin alone and apigenin or quercetin in combination with miR-34a mi. Results are expressed as the mean ± SEM from at least 3 independent experiments. D Effect of administration of apigenin (0, 1, 10, 100 µg/ml) (left) or quercetin (0, 1, 10, 100 µg/ml) (rights) alone and in combination with miR-34a mimics (miR-34a mi) on the release of estradiol by cultured porcine ovarian granulosa cells. Results shows (a) significant ( P  < 0.05) differences between cells cultured without (0 µg/ml) and with apigenin or quercetin alone; (b) significant ( P  < 0.05) differences between the cells transfected with miR-34a mi and its NC alone; (ab) significant ( P  < 0.05) differences between the cells treated with apigenin or quercetin alone and apigenin or quercetin in combination with miR-34a mi. Results are expressed as the mean ± SEM from at least 3 independent experiments. E Effect of administration of apigenin (0, 1, 10, 100 µg/ml) (left) or quercetin (0, 1, 10, 100 µg/ml) (rights) alone and in combination with miR-34a mimics (miR-34a mi) on the release of insulin-like growth factor I (IGF-I) by cultured porcine ovarian granulosa cells. Results shows (a) significant ( P  < 0.05) differences between cells cultured without (0 µg/ml) and with apigenin or quercetin alone; (b) significant ( P  < 0.05) differences between the cells transfected with miR-34a mi and its NC alone; (ab) significant ( P  < 0.05) differences between the cells treated with apigenin or quercetin alone and apigenin or quercetin in combination with miR-34a mi. Results are expressed as the mean ± SEM from at least 3 independent experiments. A Effect of administration of apigenin (0, 1, 10, 100 µg/ml) (left) or quercetin (0, 1, 10, 100 µg/ml) (rights) alone and in combination with miR-34a mimics (miR-34a mi) on cell proliferation (accumulation of PCNA) in cultured porcine ovarian granulosa cells. Results shows (a) significant ( P  < 0.05) differences between cells cultured without (0 µg/ml) and with apigenin or quercetin alone; (b) significant ( P  < 0.05) differences between the cells transfected with miR-34a mi and its NC alone. Results are expressed as the mean ± SEM from at least 3 independent experiments. B Effect of administration of apigenin (0, 1, 10, 100 µg/ml) (left) or quercetin (0, 1, 10, 100 µg/ml) (rights) alone and in combination with miR-34a mimics (miR-34a mi) on cytoplasmic apoptosis (accumulation of bax) in cultured porcine ovarian granulosa cells. Results shows (a) significant ( P  < 0.05) differences between cells cultured without (0 µg/ml) and with apigenin or quercetin alone; (b) significant ( P  < 0.05) differences between the cells transfected with miR-34a mi and its NC alone; (ab) significant ( P  < 0.05) differences between the cells treated with apigenin or quercetin alone and apigenin or quercetin in combination with miR-34a mi. Results are expressed as the mean ± SEM from at least 3 independent experiments. C Effect of administration of apigenin (0, 1, 10, 100 µg/ml) (left) or quercetin (0, 1, 10, 100 µg/ml) (rights) alone and in combination with miR-34a mimics (miR-34a mi) on the release of progesterone by cultured porcine ovarian granulosa cells. Results shows (a) significant ( P  < 0.05) differences between cells cultured without (0 µg/ml) and with apigenin or quercetin alone; (b) significant ( P  < 0.05) differences between the cells transfected with miR-34a mi and its NC alone; (ab) significant ( P  < 0.05) differences between the cells treated with apigenin or quercetin alone and apigenin or quercetin in combination with miR-34a mi. Results are expressed as the mean ± SEM from at least 3 independent experiments. D Effect of administration of apigenin (0, 1, 10, 100 µg/ml) (left) or quercetin (0, 1, 10, 100 µg/ml) (rights) alone and in combination with miR-34a mimics (miR-34a mi) on the release of estradiol by cultured porcine ovarian granulosa cells. Results shows (a) significant ( P  < 0.05) differences between cells cultured without (0 µg/ml) and with apigenin or quercetin alone; (b) significant ( P  < 0.05) differences between the cells transfected with miR-34a mi and its NC alone; (ab) significant ( P  < 0.05) differences between the cells treated with apigenin or quercetin alone and apigenin or quercetin in combination with miR-34a mi. Results are expressed as the mean ± SEM from at least 3 independent experiments. E Effect of administration of apigenin (0, 1, 10, 100 µg/ml) (left) or quercetin (0, 1, 10, 100 µg/ml) (rights) alone and in combination with miR-34a mimics (miR-34a mi) on the release of insulin-like growth factor I (IGF-I) by cultured porcine ovarian granulosa cells. Results shows (a) significant ( P  < 0.05) differences between cells cultured without (0 µg/ml) and with apigenin or quercetin alone; (b) significant ( P  < 0.05) differences between the cells transfected with miR-34a mi and its NC alone; (ab) significant ( P  < 0.05) differences between the cells treated with apigenin or quercetin alone and apigenin or quercetin in combination with miR-34a mi. Results are expressed as the mean ± SEM from at least 3 independent experiments. In nontransfected cells, the addition of apigenin stimulated the accumulation of the proliferation marker PCNA (Fig.  4 A), reduced the accumulation of the apoptosis-related peptide bax (Fig.  4 B) and the release of progesterone (Fig.  4 C) and IGF-I (Fig.  4 E), but promoted estradiol secretion (Fig.  4 D). In contrast, in cells transfected with miR-34a mimics (miR-34a + apigenin), apigenin did not promote but rather reduced PCNA accumulation (Fig.  4 A). Furthermore, in the transfected cells, apigenin did not reduce but rather promoted the accumulation of apoptosis-related bax (Fig.  4 B). On the other hand, transfection with miR-34a mimics did not change the inhibitory character of apigenin effect on the release of progesterone (Fig.  4 C) and IGF-I (Fig.  4 E) and the stimulatory action of apigenin on estradiol (Fig.  4 D) release (see the differences between effects of apigenin given at 0 µg/ml and in other doses). Apigenin promoted the inhibitory effect of miR-34a on cell proliferation (Fig.  4 A) but mitigated the suppressive effect of miR-34a on apoptosis (Fig.  4 B), the stimulatory effect of miR-34a on progesterone (Fig.  4 C) secretion and the inhibitory effect of miR-34a on estradiol (Fig.  4 D) and IGF-I release (Fig.  4 E). Quercetin, when administered alone, inhibited proliferation (PCNA accumulation, Fig.  4 A), apoptosis (bax accumulation, Fig.  4 B), and the secretion of estradiol (Fig.  4 D), IGF-I (Fig.  4 E) and progesterone (Fig.  4 C) by nontransfected cells. In transfected cells, quercetin did not affect proliferation (PCNA accumulation, Fig.  4 A), but it promoted apoptosis (bax accumulation, Fig.  4 B). Furthermore, quercetin reduced progesterone (Fig.  4 C) and IGF-I (Fig.  4 E) secretion, but it did not affect estradiol secretion (Fig.  4 D) by transfected cells. Quercetin did not substantially modify the inhibitory effect of miR-34a on cell proliferation (Fig.  4 A) but mitigated the inhibitory effect of this miRNA on apoptosis (Fig.  4 B) and IGF-I secretion (Fig.  4 E) and the stimulatory action on progesterone release (Fig.  4 C) but not the inhibitory effect of miR-34a on estradiol secretion (Fig.  4 D).

MiR-34a mimics (double-stranded RNAs that mimic mature endogenous miR-34a and enhance miRNA activity, representing a gain-of-function assay) and their respective negative control (NC) labeled with fluorescein (Table  1 ) were purchased from GenePharma Co., Ltd. (Shanghai, China). These oligonucleotides were synthesized and purified by using high-performance liquid chromatography (according to the manufacturer’s data, more than 97% purity was observed by using mass spectrometry). Table 1 The sequences of MiRNA mimics, MiRNA inhibitor and primers for RT-qPCR. Oligonucleotides Sequence miR-34a mimics Sense 5′- UGGCAGUGUCUUAGCUGGUUGU-3′ Antisense 5′-AACCAGCUAAGACACUGCCAUU-3′ miR-NC (negative control) mimics Sense 5′-UUCUCCGAACGUGUCACGUTT-3′ Antisense 5′-ACGUGACACGUUCGGAGAATT-3′ miR-34a inhibitor Sense 5′-ACAACCAGCUAAGACACUGCCA-3′ miR-NC (negative control) inhibitor Sense 5′-CAGUACUUUUGUGUAGUACAA-3′ Primer Sequence miR-34a Forward 5′-ACCTTCGCTGGCAGTGTCTT-3′ Reverse 5′-TATGGTTGTTCACGAGTCCTTGTC-3′ U6 Forward 5′-CTCGCTTCGGCAGCACA-3′ Reverse 5′-AACGCT TCACGAATTTGCGT-3′ The sequences of MiRNA mimics, MiRNA inhibitor and primers for RT-qPCR. Twenty porcine ovaries were collected from Landrace prepubertal gilts (6–8 months of age) at the slaughterhouse of Chovmat F.U. in Rastislavice (Slovakia). The ovaries were individually stored in a thermos in a physiological solution at room temperature and processed within 6 h of slaughter. Ovarian granulosa cells were isolated from porcine ovarian follicles (4.5–6.5 mm diameter) without visible signs of atresia (including weak vascularization, thin follicular walls, and pale follicular fluid) by aspiration with a syringe. After aspiration and cell isolation via centrifugation for 10 min at 1,500 rpm, the granulosa cells were washed with sterile DMEM/F12 1:1 medium (BioWhittakerTM; Lonza, Verviers, Belgium) and resuspended in the same medium supplemented with 10% fetal calf serum (BioWhittakerTM) and 1% antibiotic-antimycotic solution (Sigma‒Aldrich, St. Louis, MO, USA). The cells were counted using a Buerker chamber, and their concentration was adjusted to the required concentration (10 6 cells/ml medium). The cell suspension was seeded in 24-well culture plates (NuncTM, Roskilde, Denmark; 1 ml suspension/well) for enzyme-linked immunosorbent assay (ELISA) or in 16-well chamber slides (Nunc Inc., International, Naperville, IL, USA; 200 µl/well) for immunocytochemistry. The cells were precultured in medium at 37.5 °C in 5% CO 2 until a 75% confluent monolayer was achieved (2–3 days). The experimental cells were transfected with miR-34a mimics and their respective negative control (NC) mimic and inhibitor by using Lipofectamine ® RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. For each transfection, oligonucleotides were transfected at a final concentration of 25 nM. Control groups were composed of nontransfected cells or cells that were transfected with NC mimic or NC mimic and NC inhibitor. After transfection for 24 h, granulosa cells were cultured with and without apigenin (1, 10, or 100 µg/ml; Merck KGaA, Darmstadt, Germany) or quercetin (1, 10, or 100 µg/ml; AppliChem GmbH, Darmstadt, Germany). These doses were comparable with the effective doses of apigenin 7 , 25 , 26 and quercetin 27 , 28 that were used in similar previous in vitro experiments. Immediately before administration to cells, plant flavonoids were first dissolved in 0.1% DMSO and then in culture medium. Control groups were composed of nontransfected or transfected cells with no apigenin or quercetin treatment. After culture, the culture medium and cells were processed for cytochemistry, immunocytochemistry, reverse transcription-quantitative PCR (RT‒qPCR), and ELISA. After 48 h of culture, the cells that were transfected with NC control mimics and NC inhibitor labeled with fluorescein were fixed with 4% paraformaldehyde for 10 min, mounted in VECTASHIELD Antifade Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI), which has a selective stain for cell nuclear DNA (Vector Laboratories Inc., Burlingame, CA, USA), and analyzed by fluorescence microscopy (Leica Microsystems, Wetzlar, Germany). The percentage of transfected cells with incorporated labeled oligonucleotide was calculated. Levels of proliferation (PCNA) and apoptosis (bax) markers were measured via quantitative immunocytochemistry as previously described 7 , 8 , 25 – 28 by using primary mouse monoclonal antibodies against either PCNA or bax (dilution of 1:500 in PBS; Santa Cruz Biotechnology Inc.), a secondary swine antibody against mouse IgG (dilution of 1:1,000; Santa Cruz Biotechnology Inc.) labeled with horseradish peroxidase (Servac, Prague, Czech Republic), or a secondary goat antibody against mouse IgG (Sigma‒Aldrich) labeled with CruzFluor™ 594 (CFL 594, dilution of 1:500). Cells that were labeled with horseradish peroxidase were stained with 3.3’-diaminobenzidine (DAB) substrate (Roche Diagnostics GmbH, Mannheim, Germany). Moreover, cells that were labeled with CFL 594 were mounted in VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories Inc.). DAPI and CFL 594-labeled secondary antibodies were detected by fluorescence microscopy. Cells that were treated without the primary antibody were used as negative controls. In addition, the number of stained cells and the location of intracellular molecules were determined based on the brown coloration of DAB peroxidase or the red fluorescence emitted by the CFL 594 fluor using a light or fluorescence microscope (Leica Microsystems) and IM500 Leica software. The ratio of stained cells to total cells was determined. After treatment with miR-34a mimics, NC mimics or NC inhibitor for 48 h, total RNA was extracted from transfected granulosa cells by using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. The concentration and quality of the total RNA were measured with using a UV spectrophotometer (Bio-Rad Inc.; Hercules, CA, USA). The expression levels of mature miR-152 were quantified by using the Hairpin-it-miRNAs qPCR kit (GenePharma Co., Ltd) on an ABI 7500 Fast Instrument (Thermo Fisher Scientific). The thermocycling conditions for amplification were as follows: an initial denaturation at 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s, annealing and elongation at 60 °C for 10 s, and 60 °C for 60 s. U6 small nuclear RNA (snRNA) was used as an internal control, and relative gene expression was calculated with the 2 –ΔΔCt method 29 . The primers that were used (Table  1 ) were designed and synthesized by GenePharma Co., Ltd. All of the samples were analyzed in triplicate from the same RNA preparation, and the mean values were calculated. The concentrations of progesterone, 17β- estradiol, and IGF-1 in 25 µl aliquots of the incubation medium were determined by using ELISA, according to the manufacturer’s instructions. Progesterone, 17β- estradiol, and IGF-I ELISA kits were purchased from LDN Immunoassays and Services (Nodhorn, Germany. The characteristics of these assays are presented in Table  2 . This ELISA was validated for the culture medium samples by using dilution tests. Table 2 Characteristics of the immunoassays used in experiments. Substance assayed Specificity of assay (cross-reactivity of antiserum) Sensitivity of assay (ng/ml) Coefficient of variation (%) Intra-assay Inter-assay Progesterone ≤ 1.1% with 11- desoxycorticosterone, ≤ 0.35% with pregnenolone, ≤ 0.30% 17α-OH with progesterone, ≤ 0.20% with corticosterone, < 0.10% with estriol, 17β-estradiol, testosterone, cortisone and 11- desoxycortisol, < 0.02% with DHEA-S and cortisol 0.045 5.4 5.59 17β -estradiol ≤ 9.5% with fulvestrant, ≤ 4.2% with estrone, ≤ 3.8% with E2-3-glucuronide, ≤ 3.6% with E2-3-sulphate, ≤ 0.4% with estriol, ˂0.1% with androstenedione, 17-hydroxyprogesterone, corticosterone, pregnenolone, E2-17-glucuronide, progesterone, and testosterone 0.0062 6.4 4.5 IGF-I 100% with IGF-I, ≤ 3.3% with insulin, and 1.02% with IGF-II 9.75 7.39 12.63 Characteristics of the immunoassays used in experiments. ≤ 1.1% with 11- desoxycorticosterone, ≤ 0.35% with pregnenolone, ≤ 0.30% 17α-OH with progesterone, ≤ 0.20% with corticosterone, < 0.10% with estriol, 17β-estradiol, testosterone, cortisone and 11- desoxycortisol, < 0.02% with DHEA-S and cortisol ≤ 9.5% with fulvestrant, ≤ 4.2% with estrone, ≤ 3.8% with E2-3-glucuronide, ≤ 3.6% with E2-3-sulphate, ≤ 0.4% with estriol, ˂0.1% with androstenedione, 17-hydroxyprogesterone, corticosterone, pregnenolone, E2-17-glucuronide, progesterone, and testosterone 100% with IGF-I, ≤ 3.3% with insulin, and 1.02% with IGF-II The data from this study are reported as the means of values that were obtained in three separate experiments that were performed on separate days with different groups of granulosa cells, and each experiment included at least six ovaries. Each experimental group was represented by four culture wells containing ovarian granulosa cells. For the cytochemical and immunocytochemical analyses, the proportion of antigen-containing cells among at least 1,000 cells per well was calculated. For the ELISA, blank control values of media-containing wells were subtracted from corresponding values exclude any nonspecific background (less than 10% of the total values). The rates of hormone secretion were calculated per 10 6 viable cells/day. Significant differences between the groups were determined by using the Shapiro‒Wilk normality and Student’s t tests, as well as one-way ANOVA followed by Tukey’s tests, with SigmaPlot 11.0 (Systat Software, GmbH, Erkrath, Germany). Differences were considered statistically significant at P levels less than 0.05 ( P  < 0.05).

The presence of proliferation- and apoptosis-related molecules and the ability of cells to release hormones, to take up gene sequences and to respond to additives indicated that the cells used in the experiments were viable and suitable for the present study. Furthermore, the most popular index of luteinization is progesterone and estradiol release 24 . The moderate production of progesterone and relatively high estradiol secretion observed in our experiments suggest that the cells in our experiments were not subjected to profound luteinization. The fluorescence microscopy, cytochemistry, and RT‒qPCR results showing transfection efficiency and miR-34a expression indicate the efficiency and reproducibility of miRNA delivery. Our observations demonstrate the ability of miR-34a mimics to increase miR-34a expression and the ability of a miR-34a inhibitor to reduce it. These observations demonstrate the specific action of these miRNA sequences. The ability of miR-34a to suppress ovarian cancer cell proliferation and to promote their apoptosis is well documented 15 , 18 , 19 . There are fewer reports on the ability of this miRNA to suppress proliferation 6 , 8 and either stimulate 9 or inhibit 6 , 8 apoptosis in healthy ovarian cells. The present observations confirm the significance of miR-34a in inhibiting both proliferation and apoptosis in healthy ovarian cells. It is possible that this miRNA can suppress ovarian cell turnover and, therefore, ovarian follicular growth and development 24 . Some currently available data indicate the endocrine mechanisms of such miR-34a action. Its overexpression was associated with changes in inhibin B gene expression 9 and the release of steroid hormones, IGF-I, oxytocin, and prostaglandin E2 8 , 12 . In the present experiments, miR-34a promoted the secretion of progesterone and IGF-I and suppressed estradiol secretion. The key role of these hormones in the regulation of ovarian cell proliferation, apoptosis and folliculogenesis is well known 23 , 24 . Therefore, the present observations suggest that miR-34a might affect ovarian cell proliferation and apoptosis via changes in steroid hormones and IGF-I secretion. The ability of miR-34a to promote the secretion of progesterone (a marker and stimulator of ovarian cell luteinization 24 ), and to suppress the secretion of estradiol (a marker of ovarian follicular growth and development 24 ), indicates that this miRNA can be a physiological promoter of ovarian luteinization and/or atresia, and it affects these processes via changes in steroid hormones. The possibility that the anti-apoptotic effect of miR-34a may be due to the upregulation of IGF-I observed in the present and previous 8 experiments cannot be excluded because IGF-I is considered an anti-apoptotic factor 24 . Understanding the mechanisms and physiological significance of the observed effects of miR-34a requires further in vitro and in vivo studies. Nevertheless, these effects support speculation about the possible practical application of this miRNA. The possibility that miR-34a can be a marker and predictor of animal and human reproductive state and fecundity cannot be excluded. Moreover, the possibility that activators of miR-34a can be useful for inhibiting or synchronizing the ovarian cycle cannot be excluded, while miR-34a inhibitors could be useful for promoting of ovarian follicullogenesis and fecundity. Finally, the ability of miR-34a to promote apoptosis in ovarian cancer cells and to suppress apoptosis in healthy cells indicates the potential utility of this miRNA for the prediction and safe treatment of ovarian tumorigenesis. There is evidence that suggests that the plant flavonoids apigenin and quercetin exert physiological and therapeutic effects on female reproductive processes. There are reports about the ability of apigenin to promote healthy ovarian functions (proliferation, folliculogenesis, and hormone release) and to suppress ovarian cell tumorigenesis 3 , 7 as well as evidence to support both stimulatory and inhibitory effects of quercetin on these processes 3 , 4 . In the present experiments, apigenin stimulated proliferation, and inhibited apoptosis, and inhibited the release of progesterone and IGF-I, but it promoted estradiol secretion by healthy ovarian cells. These observations are consistent with a previous similar report 7 , 25 about the ability of apigenin to promote these ovarian cell functions; however, in some experiments, apigenin failed to affect cell proliferation, apoptosis and progesterone release 26 . Therefore, the available data indicate that apigenin and apigenin-containing plants could be promoters of female reproductive processes. In our experiments, in contrast to apigenin, quercetin inhibited proliferation, apoptosis, and estradiol and IGF-I secretion, but it did not inhibit progesterone secretion by ovarian cells, i.e., quercetin exhibited properties of a suppressor of ovarian cell functions. These observations are consistent with the results of previous experiments 27 , 28 , 30 , 31 , which demonstrated the ability of quercetin to suppress proliferation and IGF-I secretion in cultured ovarian cells. These data suggest the potential harmful effects of quercetin and quercetin-containing plants on healthy ovarian functions. On the other hand, the potential application of quercetin to synchronize of animal reproductive cycles or as a natural contraceptive could not be excluded. The performed experiments are the first to demonstrate the mutual effects of miR-34a and plant isoflavones. First, the present observations show the ability of miR-34a to induce and even to reverse the effect of plant molecules. In nontransfected cells, apigenin stimulated proliferation and inhibited apoptosis, but in cells overexpressing miR-34a, apigenin reduced proliferation and promoted apoptosis. Quercetin inhibited proliferation, apoptosis, and the release of all measured hormones by nontransfected cells. In transfected cells, quercetin did not affect proliferation, did promote apoptosis, and did inhibit progesterone and IGF-I secretion, but it did not inhibit estradiol secretion. Thus, miRNAs can define the response of ovarian cells to food and medicinal plant molecules. The differences in miRNA expression could explain the variability in the response of cells and whole organisms to food and phytotherapeutical agents. Second, the present observations are the first to demonstrate that plant molecules could modify the effect of miR-34a on healthy ovarian cells. In our experiments, both apigenin and quercetin were able to mitigate the main effects of miR-34a. This observation is consistent with a previous report about the ability of apigenin to prevent some effects of another miRNA, namely, miR-152 7 . These observations demonstrate that plant molecules and corresponding plant foods can substantially modify the response of target cells to miRNAs. The variabilities in the response to miRNAs that were observed in some in vitro and in vivo experiments could be explained by the variabilities in nutritional status/inclusion of plant molecules. Taken together, the present experiments are the first to demonstrate mutual interrelationships between miR-34a and plant flavonoids in the regulation of healthy ovarian cell functions. Such interrelationships indicate that the optimal combination of miRNA technology and phytotherapy could increase the efficiency of both therapies. If such interrelationships occur in in vivo and clinical conditions, they could be applicable for the regulation of animal and human reproduction as well as for the prevention and treatment of reproductive disorders.

Most likely, phytotherapy is oldest strategy for affecting human and animal physiological processes and improving health, including reproductive health 1 , 2 . The primary known physiological effects of medicinal and functional food plants are explained by the presence of polyphenols – flavonoids such as apigenin 3 and quercetin 4 . Many plants and plant polyphenols are able to affect female reproductive processes and to prevent and treat female reproductive pathologies, such as ovarian cancer, polycystic ovarian syndrome, ovarian insufficiency, menopause symptoms, endometriosis and others 3 . Recently, novel, so-called RNA interference-dependent mechanisms by which plants affect ovarian functions have been identified. Short noncoding RNAs called microRNAs (miRNAs) regulate the expression of many genes by either inhibiting translation or degrading messenger RNA 5 . miRNAs are important regulators of key ovarian functions, including proliferation 6 – 8 , apoptosis 6 – 10 , oogenesis and embryogenesis 11 , hormone release 7 , 8 , 12 and hormone reception 7 . Moreover, miRNAs can be both markers and regulators of reproductive dysfunction, including ovarian aging 13 , ovarian follicle atresia 9 , ovarian insufficiency 14 and cancer 15 , 16 . Some plants 15 , 17 and the plant flavonol quercetin can affect ovarian 18 and non-ovarian 19 cancer by modulating the regulatory effects of miRNAs on cell proliferation and apoptosis. The role of another plant flavonoid, namely flavone apigenin, on inducing miRNA expression and its effect on healthy ovarian cells has also been reported 7 . In contrast, miRNAs might influence the response of ovarian cancer cells to plant molecules. For example, miR-152 prevents the effect of apigenin on some healthy ovarian cell functions 7 . To our knowledge, the influence of miR-34a on the effects of plant molecules on healthy ovarian cells and vice versa has not yet been demonstrated. Understanding the interrelationships between plant molecules and miRNAs in the regulation of ovarian cell functions could expand the existing knowledge about the regulation of reproductive processes. Moreover, such knowledge can help to develop new approaches, including a combination of phytotherapy and miRNA technology, to regulate animal and human fecundity and to treat reproductive disorders. The aim of the present study was to understand whether miRNAs can influence the response of healthy ovarian cells to the plant flavonoids apigenin and quercetin and whether these plant flavonoids can modify the effects of miRNAs. As a model, we used cultured healthy porcine ovarian granulosa cells that were transfected and not with oligonucleotide sequences encoding miR-34a, whose importance in the regulation of the functions of both cancerous 20 and healthy 6 – 9 , 12 ovarian cells has been previously demonstrated. We compared miR-34a-overexpressing and control cells that were cultured in the presence or absence of flavone apigenin and flavonol quercetin at different doses. After culture, we analyzed markers of proliferation (accumulation of PCNA 21 ), markers of cytoplasmic apoptosis (accumulation of bax 22 ), and the release of steroid (progesterone, estradiol) and peptide (IGF-I) hormones, which are considered markers and regulators of ovarian functions 23 , 24 .