Inhibition of microRNA-19b promotes ovarian granulosa cell proliferation by targeting IGF-1 in polycystic ovary syndrome.

The purpose of the present study was to investigate the functional role of microRNA (miR)-19b in polycystic ovary syndrome (PCOS) and try to elucidate its underlying mechanisms. Expression of miR‑19b and insulin‑like growth factor 1 (IGF-1) was examined in ovarian cortexes [(from 18 women with PCOS and 10 who did not have PCOS (non‑PCOS)] and KGN cells. Cell proliferation assays (cell viability and colony formation assay) were performed following overexpression or inhibition of miR‑19b and IGF‑1 or following insulin treatment in KGN cells. Expression levels of the cell cycle-associated protein cyclin D1 and cyclin‑dependent kinase (CDK) 1 were analyzed following overexpression or inhibition of miR-19b and IGF-1. Potential miR‑19b targets were identified by bioinformatics. Luciferase assay, reverse transcription‑quantitative polymerase chain reaction and western blotting were performed to determine whether IGF‑1 was a target of miR‑19b. miR‑19b expression was significantly decreased in the PCOS ovarian cortex and KGN cells and its identified target, IGF‑1, was upregulated. miR‑19b overexpression inhibited cell proliferation at G2/M phrase. Overexpression of IGF‑1 promoted cell viability and colony formation ability in KGN cells. The expression of cyclin D1 and CDK1 was statistically increased by inhibition of miR‑19b and overexpression of IGF‑1. High concentrations of insulin decreased levels of miR‑19b, stimulated KGN cell proliferation, and elevated IGF‑1 levels. Inhibition of miR‑19b promoted ovarian granulosa cell proliferation by targeting IGF‑1 in PCOS. Insulin decreased the expression levels of miR‑19b and stimulated cell proliferation. The present study suggested that overexpression of miR‑19b may be a potential therapeutic approach for PCOS.

Introduction

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder affecting 5–10% of women of childbearing age (1) and is characterized by hyperandrogenemia, chronic anovulation and polycystic ovaries (2). The common clinical manifestations of PCOS are menstrual disorders, subfertility, acne vulgaris, alopecia, seborrhea, obesity, hirsutism and acanthosis (3). It is the most common cause of anovulatory infertility, in addition, women with PCOS have an increased risk of developing insulin resistance, hypertension, type 2 diabetes mellitus, oxidative stress, dyslipidemia, cardiovascular diseases and endometrial cancer (4–6). Although the exact etiology of PCOS remains to be fully understood, granulosa cell survival and proliferation may be responsible for PCOS (7,8). Therefore, an improved understanding of granulosa cell proliferation involved in PCOS may provide novel insight into treatment of PCOS.

MicroRNAs (miRNAs) are a class of highly conserved, small and non-coding RNAs that affect biological functions by regulating mRNA transcription and translation at the post-transcriptional level through imperfect base pairing with the 3′-untranslated region (UTR) of target mRNAs (9). It has been previously demonstrated that miRNAs are involved in several diseases, including metabolic disorders (10) and PCOS (11–14). Several miRNAs, including miR-9 and miR-18b (15), have been identified significantly increased in PCOS granulosa cells, and several miRNAs, including miR-19b and miR-93 (16), have been identified to be significantly decreased in PCOS blastocysts. A recent study demonstrated that miR-93 serves important roles in accelerating cell proliferation in granulosa cells (13). However, little information is available about the functional role of miR-19b in PCOS granulosa cells, and whether miR-19b is involved in granulosa cell proliferation remains unclear.

Therefore, the present study aimed to determine the functional role of miR-19b in PCOS granulosa cells. The expression pattern of miR-19b in PCOS ovary tissues and ovarian granulosa cell-like KGN cells were first investigated. The effects of miR-19b on cell proliferation were then determined by altering the endogenous levels of miR-19b in KGN cells. Subsequently, the direct targets of miR-19b were further predicted in order to elucidate the underlying mechanism of cell proliferation.

Patients and methods

Patients and samples

A total of 18 participants who were diagnosed as PCOS in Guangzhou Women and Children's Medical Center between June 2015 and April 2016 were enrolled in the present study. The ovarian tissues were obtained from all the participants who underwent laparoscopic investigation for infertility. The diagnosis of PCOS was based on the revised Rotterdam European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine criteria (2003) (17). An additional 10 normally menstruating age-matched women who volunteered for the study were recruited between August 2015 and April 2016 in Department of General Gynaecology. These volunteers had no history of diabetes mellitus, glucose disorder, chronic anovulation, hyperandrogenism, endometriosis or other endocrine diseases were recruited as the control group. The ovarian tissues were obtained from these participants when they underwent laparoscopic sterilization or diagnostic laparoscopy for pelvic pain. The present study was approved by the Ethics Committee of Guangzhou Women and Children's Medical Center, and informed written consent was obtained from all women prior to participation. Physical examinations and measurements, including ages, body mass index (BMI), height, waist circumference, hip circumference and the modified Ferriman-Gallwey score (mFG), were completed in the two groups. There were no significant differences between the above indexes.

Cell culture, treatment and transfection

Human ovarian granulosa cell-like KGN cells, normal ovarian surface epithelial IOSE80 cells and 293 cells were all obtained from the American Type Culture Collection. These cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin G and 0.1 mg/ml streptomycin sulfate (Life Technologies; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C with 5% CO2.

Cells were plated in 6-well plates at a density of 2×105/well and were treated with recombinant human (rh) insulin (Invitrogen; Thermo Fisher Scientific, Inc.) At different concentrations (0, 1, 10 or 100 ng/ml). A total of 24 h later, the expression of miR-19b was tested, and then 24 h after measurement of miR-19b, the expression of IGF-1 was determined.

For cell transfection, vectors including miR-19b mimic, miR-19b inhibitor, miR-19b scramble, small interfering RNA (siRNA) against IGF-1 (si-IGF-1), expression vector pcDNA 3.1 (+)-IGF-1, or negative control (si-NC) vector with the scramble IGF-1 sequence were all purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Cell transfection was performed using Lipofectamine 2000 (Life Technologies; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Cell viability

The cell viability was assessed using 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (18). Briefly, cells were placed in the 96-well plates and adjusted to 5×103 cells. The cells were transfected with the vectors (miR-19b mimic, miR-19b inhibitor, miR-19b scramble, si-IGF-1, pcDNA 3.1 (+)-IGF-1 or si-NC) or treated with insulin until cells grew 70–80% confluent. Subsequent to being cultured in DMEM/F12 medium supplemented with 10% FBS for 24 h, the cells were centrifuged at 2,500 × g for 5 min at 4°C, and then the supernatant was removed. The cells were then incubated with 20 µl MTT at 37°C for 4 h and lysed in 150 µl dimethyl sulfoxide (DMSO) at room temperature for 10 min at different time points (24, 48, 72 and 96 h) following transfection. The optical density was measured at 570 nm using an absorption spectrophotometer (Olympus Corporation, Tokyo, Japan). Assays were run in triplicate.

Colony formation assay

Colony formation assay was performed to determine the cell proliferation ability as previously described (19). In brief, following cell transfection or treatment with insulin, the cells were plated into the 60 mm culture dishes at a density of 100 cells/dish. Then the cells were maintained in DMEM/F12 medium supplemented with 10% FBS for 14 days until visible clones appeared. Thereafter, cells were harvested, washed with PBS, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet solution, followed by air drying. Finally, the number of colonies was counted under a fluorescence microscope (IX83; Olympus Corporation).

Cell cycle

The cell cycle was evaluated 48 h following transfection with the vectors (miR-19b mimic, miR-19b inhibitor, or miR-19b scramble). Briefly, following transfection, the cells were trypsinized and suspended with fresh DMEM/F12 medium containing 10% FBS. Subsequently, the cells were pelleted and suspended with cold phosphate-buffered saline and fixed at 4°C for 30 min. Cells were then stained with propidium iodide solution for 30 min and cell cycle and DNA content were assessed using flow cytometry analysis.

Target prediction and luciferase reporter assay

The potential targets of miR-19b were predicted by bioinformatics analysis using TargetScan software version 6.2 (www.targetscan.org). The 3′-UTR sequence of wild-type (WT) or mutated (Mut) IGF-1, containing the putative miR-19b-binding site, was amplified by PCR and cloned into the psiCHECK2 vector (Promega Corporation, Madison, WI, USA). Site-directed mutagenesis was conducted using the QuikChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer's instructions, resulting in Mut 3′-UTR. For the reporter assay, WT or Mut 3′-UTR vectors encoding Renilla luciferase were cotransfected with miR-19b mimic or negative control (miR-control) into 293 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following 48-h transfection, the luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega Corporation). The Renilla luciferase activity was normalized to firefly luciferase activity.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA, including miRNAs, was isolated from cells or tissues using 1 ml TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Complementary DNA (cDNA) was produced from 1 µg RNA according to the manufacturer's protocol. Reagents (20 µl) for the reverse transcription reaction were 5 µM annealed miRNA-specific stem-loop RT primer (1 µl) (Sangon Biotech Co., Ltd., Shanghai, China), 10 mM dNTPs (1 µl) (Life Technologies), MultiScribe reverse transcriptase (1 µl) (Applied Biosystems; Thermo Fisher Scientific, Inc.), RNase inhibitor (1 µl) (Sangon Biotech Co., Ltd.), RNA template (6 µl), nuclease-free water (10 µl), 10X RT buffer and 100 mM Tris-HCl (pH 8). The expression levels of mRNAs were measured by RT-qPCR using SYBR-Green-based quantitative RT-PCR (SYBR-Green PCR Master mix; Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR was run under the following condition: An initial denaturation at 94°C for 5 min, 35 cycles of 94°C for 1 min, annealing at 51°C for 1 min, extension at 72°C for 1 min and final extension at 72°C for 5 min. U6 and GAPDH were used as the internal controls. Primers for targets amplification were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The gene expression was analyzed using the 2−ΔΔCq method (20).

Western blotting

Total protein was extracted from cells or tissues using RIPA buffer and the concentrations were measured by using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's instructions. For western blotting, protein samples (25 µg) was subjected to a 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transferred onto a polyvinylidene fluoride (PVDF) membrane (GE Healthcare Life Sciences, Little Chalfont, UK). Subsequently, the PVDF membrane was blocked in 5% nonfat milk in 0.1% Tris-buffered saline (TBS)-Tween (TBST) for 1 h at room temperature. Thereafter, the membrane was probed with the anti-IGF-1 antibody (ab40789; Abcam, Cambridge, MA, USA), anti-cyclin D1 antibody (#2922; Cell Signaling Technology, Inc., Danvers, MA, USA), or anti-CDK1 (ab18; Abcam) overnight at 4°C. Following this, membranes were incubated with horseradish-peroxidase secondary antibody (Cell Signaling Technology, Inc.) at room temperature for 2 h. Subsequent to being washed 3 times with TBST, the blotted proteins were visualized with enhanced chemiluminescence detection system (EM Millipore, Billerica, MA, USA). GAPDH served as the internal control.

Statistical analysis

The data were expressed as the mean ± standard deviation, and analyzed using SPSS software, version 19.0 (IBM Corp., Armonk, NY, USA). Comparisons between the two groups were calculated using a two-tailed Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Results

miR-19b was decreased in tissues and cells

To explore the functional role of miR-19b in PCOS, we first assessed the expression levels of miR-19b in both tissues and cells by using RT-qPCR analysis. The ovarian tissues were obtained from both women with PCOS and normally menstruating women. No significant differences were observed in ages, BMI, height, waist circumference, hip circumference and mFG between the two groups. For the cell experiments, KGN cells and normal ovarian surface epithelial IOSE80 cells were used. As presented in Fig. 1, the results indicated that the relative expression levels of miR-19b were significantly decreased in PCOS tissues and KGN cells when compared with their corresponding control groups (P<0.01). These data suggested that miR-19b may serve a key role in PCOS.

Figure 1.

Expression of miR-19b in PCOS. (A) Expression of miR-19b in the ovarian cortex of 18 PCOS and 10 non-PCOS patients. (B) Expression of miR-19b in KGN cells. **P<0.01 vs. the control group. miR, microRNA; PCOS, polycystic ovary syndrome.

miR-19b overexpression suppressed cell proliferation

From the above results, it was speculated that miR-19b may be involved in the growth and proliferation of granulosa cells. The expression of miR-19b was altered in KGN cells by transfection with miR-19b mimic or inhibitor. Then the effects of abnormal expression of miR-19b on cell viability and colony formation ability were determined. As indicated in Fig. 2, compared with the control group or miR-19b scramble group, both the cell viability and colony formation ability were significantly downregulated by transfection with the miR-19b mimic, however were markedly upregulated by transfection with miR-19b inhibitor (P<0.05 and P<0.01). These data indicated that miR-19b overexpression suppressed cell proliferation.

Figure 2.

Effects of abnormal expression of miR-19b on cell viability and colony formation ability. (A) Effects of abnormal expression of miR-19b on cell viability. (B) Effects of abnormal expression of miR-19b on colony formation ability and the (C) quantification. *P<0.05, **P<0.01 vs. the control group. miR, microRNA; OD, optical density.

miR-19b overexpression arrested cell cycle at G2/M phase

In addition, the effects of abnormal expression of miR-19b on the cell cycle distribution were investigated using flow cytometry. The results indicated that the cells transfected with miR-19b mimic exhibited a significantly decreased proportion of S phase cells and a significant increase in the proportion of G2/M phase cells (Fig. 3A and B; P<0.05) compared with the control group or miR-19b scramble group. In contrast with the miR-19b mimic, the cells transfected with miR-19b inhibitor exhibited the opposite results. The underlying mechanism was then investigated by analysis of the mRNA and protein expression levels of cell cycle-associated proteins cyclin D1 and CDK1. The results demonstrated that the mRNA and protein expression levels of cyclin D1 and CDK1 were significantly inhibited by suppression of miR-19b, and were markedly increased by overexpression of miR-19b compared with the control group (P<0.05; Fig. 3C and D). These results indicated that miR-19b overexpression arrested the cell cycle at G2/M phase by downregulating the expression of cyclin D1 and CDK1, leading to suppression of cell proliferation.

Figure 3.

Effects of abnormal expression of miR-19b on cell cycle and on cell cycle-associated protein expression. (A) Effects of abnormal expression of miR-19b on the cell cycle and (B) quantification of this. (C) Effects of abnormal expression of miR-19b on mRNA expression of cyclin D1 and CDK1. (D) Effects of abnormal expression of miR-19b on protein expression of cyclin D1 and CDK1. *P<0.05 vs. the control group. miR, microRNA; CDK, cyclin-dependent kinase.

miR-19b directly inhibited IGF-1 expression by targeting its 3′UTR in KGN cells

To determine the underlying mechanism of cell proliferation mediated by miR-19b, its target gene was predicted using TargetScan 6.2 (www.targetscan.org). Of all of the hypothetical targets of miR-19b, IGF-1, an important growth-promoting polypeptide that serves significant roles in cell proliferation, was selected as one of the candidate genes. To determine whether IGF-1 was a target gene of miR-19b, the WT or Mut 3′UTR of the putative miR-19b target sequence was cloned into a luciferase reporter vector. As presented in Fig. 4A, IGF-1 was predicted to be a target of miR-19b. In addition, it was observed that the luciferase activity was significantly decreased by co-transfection of miR-19b with IGF-1 3′UTR WT in KGN cells (P<0.01), however no significant differences were observed by co-transfection of the miR-19b mimic with IGF-1 3′UTR Mut (Fig. 4B). To further ensure the potential role of miR-19b in the regulation of IGF-1, the mRNA and protein expression levels of IGF-1 in KGN cells were evaluated in the presence of miR-19b mimics or the miR-19b inhibitor. As presented in Fig. 4C and E, the results demonstrated that overexpression of miR-19b led to a significant decrease in IGF-1 mRNA and protein levels, whereas inhibition of miR-19b markedly upregulated IGF-1 mRNA and protein levels compared with the negative control groups (P<0.05 or P<0.01). These results suggested that IGF-1 was a direct target of miR-19b and was negatively regulated by miR-19b in KGN cells.

Figure 4.

Target prediction of miR-19b. (A) Potential miR-19b targets. (B) Relative luciferase activity following co-transfection of miR-19b mimic with IGF-1 3′UTR WT or Mut. (C) Relative mRNA expression of IGF-1 following abnormal expression of miR-19b. (D) Relative protein expression of IGF-1 following abnormal expression of miR-19b, and (E) a representative western blotting image for expression of IGF-1. *P<0.05, **P<0.01 vs. the control group. miR, microRNA; IGF, insulin-like growth factor; UTR, untranslated region; WT, wild-type; Mut, mutant.

IGF-1 was increased in tissues and cells and IGF-1 overexpression promoted cell proliferation

It had been confirmed that IGF-1 was a direct target of miR-19b; therefore, the mRNA and protein expression levels of IGF-1 were further analyzed in tissues and cells. It was identified that the mRNA and protein expression levels of IGF-1 were statistically increased in PCOS tissues and KGN cells compared with the control group (P<0.01; Fig. 5A-D). To further investigate the functional contributions of IGF-1 to granulosa cell growth in vitro, the expression of IGF-1 was overexpressed or silenced. Subsequently, the effects of abnormal expression of IGF-1 on cell proliferation were examined by analyzing cell viability and colony formation ability. As demonstrated in Fig. 5E-G, the results demonstrated that overexpression of IGF-1 markedly promoted cell viability and colony formation ability when compared with the control group (P<0.05), whereas the reverse results were obtained by silencing the expression of IGF-1 (P<0.05 or P<0.01). In addition, the expression levels of cyclin D1 and CDK1 were measured. It was observed that the mRNA and protein expression levels of cyclin D1 and CDK1 were significantly elevated by overexpression of IGF-1 (P<0.05), whereas the levels of cyclin D1 and CDK1 were significantly decreased by silencing the expression of IGF-1 (P<0.05) (Fig. 5H-I). The results suggested that IGF-1 overexpression promoted cell proliferation by upregulating the expression of cyclin D1 and CDK1.

Figure 5.

Expression of IGF-1 in PCOS and effects of abnormal expression of IGF-1 on cell proliferation. (A) mRNA expression of IGF-1 in the ovaries cortex of 18 PCOS and 10 non-PCOS patients. (B) mRNA expression of IGF-1 in KGN cells. (C) Protein expression of IGF-1 in the ovaries cortex of 18 PCOS and 10 non-PCOS patients. (D) Protein expression of IGF-1 in KGN cells. (E) Effects of abnormal expression of IGF-1 on cell viability. (F and G) Effects of abnormal expression of IGF-1 on colony formation ability. (H and I) Effects of abnormal expression of IGF-1 on cell cycle-associated protein expression. *P<0.05, **P<0.01 vs. the control group. IGF, insulin-like growth factor; PCOS, polycystic ovary syndrome; OD, optical density; si, small interfering; NC, negative control; CDK, cyclin-dependent kinase.

Insulin decreased miR-19b expression and stimulated cell proliferation

Hyperinsulinemia is one of the most common biochemical abnormalities identified in PCOS. Thus, it was hypothesized that high concentrations of insulin may result in different expression levels of miR-19b. Different concentrations of insulin (0, 1, 10 or 100 ng/ml) were administered to KGN cells and the expression of miR-19b and IGF-1 was examined by RT-qPCR. The results indicated that the expression of miR-19b was significantly decreased by administration of insulin, with a dose-dependent effect (P<0.05 or P<0.01; Fig. 6A). However, the expression of IGF-1 exhibited the opposite results. The expression of IGF-1 was statistically increased by administration of insulin, also with a dose-dependent effect (P<0.05 or P<0.01; Fig. 6B). Thereafter, the concentration of 100 ng/ml insulin was selected for cell proliferation. As demonstrated in Fig. 6C-E, it was observed that insulin (100 ng/ml) significantly stimulated the cell viability and colony formation ability (P<0.05).

Figure 6.

Effects of insulin treatment on expression of miR-19b and IGF-1 and on cell proliferation. Effects of insulin treatment on expression of (A) miR-19b and (B) IGF-1. (C) Effects of insulin treatment on cell viability. (D and E) Effects of insulin treatment on colony formation ability. *P<0.05, **P<0.01 vs. the control group. miR, microRNA; IGF, insulin-like growth factor; OD, optical density.

Discussion

The present study focused upon the functional role of miR-19b in cell proliferation of human ovarian granulosa cell-like KGN cells, in addition to its possible regulatory mechanism. The results demonstrated that the expression of miR-19b was significantly decreased in PCOS ovary tissues and KGN cells. Overexpression of miR-19b statistically decreased the cell viability and colony-formation ability, and arrested cell cycle at G2/M phase. In addition, it was identified that IGF-1 was a direct target gene of miR-19b and was negatively regulated by miR-19b. Overexpression of IGF-1 promoted cell proliferation. In addition, it was observed that high concentrations of insulin decreased levels of miR-19b, stimulated cell proliferation and elevated IGF-1 levels.

Numerous miRNAs have been identified to be involved in multiple biological processes, including cell survival and cell proliferation. Among mRNAs, the functional role of miR-19b has been investigated in various diseases. miR-19b, an important functional representative of miR-19-72 cluster family, has been demonstrated to regulate cellular proliferation, differentiation, cell migration or invasion, apoptosis and metabolism (21). However, the biological functions of miR-19b are complex, due to the fact that it has been identified as an oncogene, however additionally exerts a protective role in the context of different diseases. For example, Livak et al (20) observed that serum levels of miR-19b were significantly higher in patients with non-small cell lung cancer compared with those in controls, and patients with low serum levels of miR-19b achieved a higher overall response rate and longer survival time. Lv et al (21) has suggested that miR-19b promotes tumor growth and metastasis by targeting tumor suppressor TP53 (or p53). Baldin et al (22) identified that inhibition of miR-19b decreased the proliferation and migration of cardiac fibroblasts. In contrast, Hu et al (23) identified that miR-19b was downregulated in both rodent and human cardiac tissues following ischemic injury, and increases of miR-19b may be of therapeutic interest to improve cardiomyocyte cell survival.

Notably, a previous study reported that miR-19b was significantly decreased in blastocysts and was associated with human infertility (16). Therefore, the present study aimed to elucidate the effects of miR-19b on cell proliferation of KGN cells, in addition to the underlying mechanisms. In the present study, the expression levels of miR-19b were identified in both PCOS tissues and KGN cells. The results indicated that compared with the normal tissues and cells, the expression levels of miR-19b were significantly decreased. The results highlighted an important role for miR-19b in the pathogenesis of PCOS. Subsequently, the effects of miR-19b on KGN cell proliferation were evaluated. Following alteration of the endogenous expression of miR-19b, the cell viability and colony-formation ability were assessed. The results demonstrated that miR-19b could be a granulosa cell proliferation inhibitor. The possible mechanism regarding cell proliferation of miR-19b was further investigated. It was observed that miR-19b overexpression arrested cell cycle at G2/M phase. The expression levels of cell cycle-associated protein cyclin D1 and CDK1 were measured. Cyclin D1, a key cell cycle regulator, is essential for G1 phase progression (22), leading to uncontrolled cell growth and malignancy. CDK1, encoded by cell division cycle gene 2, belongs to the serine/threonine protein kinase family. It is an important cell cycle regulator, regulating the progression from G2 to M phase during cell cycle (23). miR-19b reduced expression of cyclin D1 and CDK1, thus significantly affecting cell proliferation and cell cycle progression at G2/M phase, which results in G2/M arrest.

It has been previously documented that miRNAs exert their regulatory functions by targeting genes. IGF-1 is a critical growth-promoting polypeptide, which serves significant roles in cell proliferation, survival and differentiation of numerous cell types (24,25). The functional role of IGF-1 in PCOS has been widely investigated (26–28). The synthesis of androgen synthesis has been reported to be stimulated by both IGF-1 and insulin acting on thecal-interstitial cells in vitro (29–31). The increased insulin and IGF-1 along with elevated luteinizing hormone (LH) are responsible for the hyperandrogenemia observed in PCOS (32–34). Having noted the functional role of IGF-1 in cell proliferation, it was speculated that miR-19b overexpression-mediated inhibition of cell proliferation may act via the regulation of IGF-1. To confirm this hypothesis, bioinformatic predictions were used. The results identified that IGF-1 was a target of miR-19b. Furthermore, the luciferase reporter assay indicated that miR-19b directly targeted the 3′UTR of IGF-1. Administration of miR-19b mimic decreased the mRNA and protein levels of IGF-1 in KGN cells, whereas induction of miR-19b inhibitor reversed the results. In addition, an elevated level of IGF-1 was observed in PCOS tissues and KGN cells. Knockdown of IGF-1 produced the opposite effects on cell proliferation as an inhibition of miR-19b. In addition, the data indicated that high concentration of insulin could decrease the expression of miR-19b, elevate the level of IGF-1, and stimulate the cell proliferation of KGN cells. It has been previously reported that insulin is involved in the modulation of ovarian function, and promotes ovarian granulosa cells (35). A study by Jiang et al (13) reported that miR-93 overexpression promoted KGN cell proliferation, and also that the levels of miR-93 were increased by high concentrations of insulin. The results were in line with those of the present study, that high concentrations of insulin could alter the expression of miRNAs and promote cell proliferation.

In conclusion, the results suggest that miR-19b is decreased in PCOS granulosa cells and miR-19b could be a granulosa cell proliferation inhibitor. miR-19b-mediated cell proliferation may act via directly targeting IGF-1.