Hsa_circ_0043533 modulates apoptosis and viability of granulosa cells via miR-409-3p/BCL2 and EMT signalling in PCOS: providing a novel perspective of metformin

doi:10.21203/rs.3.rs-2993985/v1

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Hsa_circ_0043533 modulates apoptosis and viability of granulosa cells via miR-409-3p/BCL2 and EMT signalling in PCOS: providing a novel perspective of metformin

https://doi.org/10.21203/rs.3.rs-2993985/v1

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PCOS (polycystic ovary syndrome) is an important cause of infertility in women of child birthing age. It was confirmed granulosa cells were associated closely with the process of anomalous follicle formation and ovulation in PCOS. The apoptosis of granulosa cells in PCOS patients is significantly low but the growth is high, which cause accumulation granulosa cells, leading to polycystic ovaries. Non-coding RNAs was demonstrated involving in PCOS, and we found high hsa_circ_0043533 in PCOS. A series of experiments were carried out to investigate the role of hsa_circ_0043533 in PCOS and the mechanism within. Knockdown of hsa_circ_0043533 promoted apoptosis and restrained migration, proliferation, and viability of KGN cells. The regulation of hsa_circ_0043533 on miR-409-3p/BCL2 axis and key EMT (Epithelial-mesenchymal transition) markers was discovered. And the hsa_circ_0043533/miR-409-3p/BCL2 axis was regulated by metformin. This project revealed novel insights into the molecular mechanisms of granulosa cell proliferation and apoptosis in PCOS, demonstrated the new molecular pathogenesis of PCOS.

PCOS

hsa_circ_0043533

miR-409-3p

apoptosis

EMT

metformin

Polycystic ovary syndrome (PCOS) is the most common reproductive endocrine disorder in women of reproductive age (1). PCOS patients usually have complex clinical manifestations including irregular menstruation, follicular dysplasia, hyperandrogen, insulin resistance, and infertility (2). Despite mounting evidence associated with environmental and genetic factors, the present state of knowledge of PCOS etiology is considered inadequate (3). Previous researches were certificated that the secretion of factors, including gonadal steroids, growth factors, and cytokines, by granulosa cells was fundamental to the survival of the granulosa cells and eventual follicle growth (4, 5), and granulosa cells were associated closely with the process of follicle formation and ovulation (6, 7).

Moreover, granulosa cells underwent apoptosis earlier than oocytes and theca cells in atretic follicles, suggesting that granulosa cells were the initiators of follicular atresia (5, 8). The apoptosis rate of granulosa cells from PCOS patients was significantly lower than that from women with normal ovulation cycles, mechanism could be associated with decreased expression of apoptotic effectors and increased expression of a cell survival factor (9). Therefore, excessive growth together with insufficient apoptosis of granulosa cells were regarded as the causes of PCOS.

CircRNAs are single stranded RNAs with 5' and 3' ends covalently closed RNA (10). MicroRNAs are small non-coding RNAs composed of 21–25 nucleotide residues (11), which pair with protein-coding mRNAs to guide their post transcription (12). CircRNAs could act as the sponges recruiting miRNAs, regulating a series of pathogenic signalling pathways including apoptosis, vascularization, invasion, metastasis and EMT (Epithelial-mesenchymal transition) via the circRNA-miRNA axis (13, 14). Due to the rapid development of high-throughput sequencing technology, more and more circRNA expression abnormalities have been found in PCOS models (15). Research about circRNA on apoptosis or proliferation of granulosa cells in the progression of PCOS are flourishing (16–18). Our team reported hsa_circ_0043533 was significantly higher in GCs of PCOS than that in non-PCOS validated by QPCR (19). However, the potential mechanisms underlying the function and regulation of hsa_circ_0043533 in PCOS are unclear. We proposed the hypothesis that hsa_circ_0043533 regulate some specified miRNA through the sponge adsorption, downstreaming impact the key molecules essential in the granulosa cell function.

Metformin is a first-line hypoglycaemic agent for type 2 diabetes (20). A growing number of reports indicated the role of metformin in addition to controlling blood sugar, such as anti-cancer, regulating metabolism, and treating PCOS (21–23). The utility of metformin on PCOS was treating the metabolism disorder. However, studies have not clarified the role played by metformin on GCs’ cell function in PCOS thoroughly, as well as the mechanism within.

To verify our hypothesis, we designed the project aiming to discover the regulation of hsa_circ_0043533 on the cell behaviour of KGN cells, and the molecular sponge potential of which interacting with miRNA and the downstreaming markers, further investigate the regulation mechanism of metformin.

Clinical samples

The PCOS clinical cohort was established based on the patients in our department (description of the cohort was concluded in supplementary 1). The clinical characteristics of all subjects in both groups are exhibited in Table 1. We gathered the granulosa cells from the cohort and the expression of miRNA was assessed using Q-PCR.

Table 1

Comparison of clinical characteristics and hormone levels.
Variables	PCOS (n = 20)	non-PCOS (n = 20)	p value
Age (years)	31.38 ± 1.50	31.06 ± 1.69	NS
Infertility (years)	3.81 ± 2.22	3.31 ± 1.82	NS
BMI (kg/m²)	25.55 ± 3.37	23.07 ± 3.75	NS
LH(mIU/ml)	12.32 ± 4.05	5.38 ± 2.32	< 0.0001
FSH (mIU/ml)	6.08 ± 0.89	6.86 ± 1.14	NS
LH/FSH	2.03 ± 0.61	0.77 ± 0.28	< 0.0001
PRL (ng/ml)	15.14 ± 6.00	18.61 ± 6.37	NS
P (ng/ml)	0.32 ± 0.12	0.43 ± 0.19	NS
AMH (ng/ml)	12.34 ± 6.12	4.67 ± 3.12	< 0.001
E₂ (pg/ml)	51.56 ± 25.33	44.87 ± 21.26	NS
T (ng/ml)	0.59 ± 0.12	0.28 ± 0.09	< 0.0001
FBG(mmol/L)	5.04 ± 0.41	4.74 ± 0.37	NS
INHB (ng/L)	114.8 ± 46.48	143.7 ± 47.46	NS
Oocytes	16.92 ± 4.94	11.44 ± 3.50	< 0.01
Embryo formation rate¹	0.60 ± 0.23	0.59 ± 0.26	NS
Data are presented as the mean ± SD, p < 0.05 considered statistically significant, NS no significant difference.
Embryo formation rate¹ = number of embryos/ number of oocytes obtained.
BMI body mass index, AMH anti-Müllerian hormone, LH luteinizing hormone, FSH follicle-stimulating hormone, E2 estradiol, T testosterone, P progesterone, PRL prolactin, INHB inhibin B, FBG Fasting blood glucose

RNA extraction and cDNA synthesis

According to the manufacturer's instruction of SPARKeasy Tissue/Cell RNA kit (SparkJade, Shandong, China), total RNA was extracted. MiRNA was reverse transcribed into the first-strand complementary DNA (cDNA) using miRcute Plus miRNA First-Strand cDNA Synthesis Kit (TIANGEN, Beijing, China), and circRNA and mRNA using the SPARKscript II RT Plus Kit (SparkJade, Shandong, China).

Quantitative polymerase chain reaction (Q-PCR)

The expression assays were performed using AceQ® qPCR SYBR® Green Master Mix (Vazyme, Nanjing, China). The primer sequences for Q-PCR were designed and synthesized by Tsingke (Beijing, China) and manifested in Supplementary Table S1. GAPDH is used as loading control for circRNA and mRNA, and U6 for miRNA.

Cells lines and cell culture

The human ovarian granulosa cell line KGN, ovarian epithelial cell line IOSE80, human ovarian granular tumor cell line COV434, and human embryonic kidney cell line HEK 293T were purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China).All the cells were cultured in DMEM (Hyclone Laboratories, Logan, UT, USA) with 10% fetal bovine serum (FBS, Hyclone, South Logan, UT, USA) and 1% gentamicin sulfate. All cells were maintained at a 37°C, humidity environment with 5% CO₂.

Cell transfection

The miR-409-3p mimics, inhibitors or the NCs were purchased from GenePharma (Shanghai, China) and the siRNAs against hsa_circ_0043533 with corresponding scrambled control were designed and synthesized by GenePharma (Shanghai, China), (sequences shown in Supplementary Table S2). Lipofectamine 3000 kit (Invitrogen, USA) was used for transfections complying with the manufacturer's instructions.

Cell survival assay

Cell Counting Kit-8 (CCK-8) assay was applied to evaluate the cell survivability. Cells were seeded into 96-well plates with a concentration of 3*10³cells/well. 10uL CCK-8 solution (Solarbio, Beijing, China) was added to the cells after incubation for 2 hours. The absorbance of each well was detected at the wavelength of 450nm in microplate reader (BIOTEK, Vermont, USA). However, we could only detect the existing cells using CCK-8, and the results were referred to the consequence of combination of growth and apoptosis. We normalized the cell viability from the CCK8 assay by the apoptosis rate from the flow cytometry to investigate the absolute value of the cell proliferation.

Cell-Light EDU DNA Kit (RiboBio, Guangzhou, China) was employed to measure the survival. Cells were cultured in medium containing 1:1000 EDU solution for 2 hours, then fixed by 4% paraformaldehyde (PFA) and stained using Apollo Dye Solution. Hoechst33342 reaction solution was employed to stain nucleic acids according to the manual of the kit. The EDU positive cells were observed and photographed under fluorescence microscopy.

Cell apoptosis assay

Fluorescein isothiocyanate (FITC) combined with Annexin V staining and propidium iodide (PI) was conducted for cell apoptosis assays. Cells were centrifuged at 4℃, 300g for 5 minutes. The cells were harvested and then stained with 5µL Annexin V-FITC and 10µL PI staining solution (Yisheng, Shanghai, China) for 15 minutes without light. The apoptosis rate was then evaluated by flow cytometry.

Calculation of actual cell proliferation

Since cell apoptosis was significantly regulated, the cell survivability we obtained using CCK-8 and EDU staining was the result of the combined action of cell proliferation and apoptosis. So, we calculated the actual proliferation of the cell to measure the viability of the cell using the survivability plus the apoptosis capacity.

Wound-healing cell migration assay

In our study, the migration of KGN cells was assessed using a wound-healing assay. Cells were plated into 6-well plates to 90% confluence and wounds were scratched with a 200µL pipette tip. Photographs were taken with an inverted microscope (IX71, Olympus, Tokyo, Japan). Wound widths were computed to calculate decreasing area.

Trans-well cell migration assay

Cell migration assays were performed using trans-well inserts (Corning, NY, USA). Cells in concentration of 3*10⁴ cells/well were seeded in trans-well inserts. After a 24-hour incubation, the upper surface cells of the inserts were wiped with cotton swabs, and the lower surface cells were fixed with 4% methanol for 10 minutes, then stained with 0.1% crystal violet. Subsequently, the stained cells were photographed using a light microscope and five fields were randomly selected for counting.

Protein extraction and western blotting

Total proteins were lysed with RIPA lysis buffer consisting of proteinase inhibitor (Sigma, USA) for electrophoresis. After electro-transfer, the membranes were blocked in 5% skinned milk powder, and incubated with primary and secondary antibodies. All the antibodies used in this project were showed in Supplementary Table S3. The bands were detected under Tanon 5200 chemiluminescence imaging system (Shanghai, China) and analyzed by ImageJ (National Institutes of Health, Bethesda, MD, USA).

Dual-luciferase reporter assay(DLRA)

The wild-type and mutation plasmids of hsa_circ_0043533 and BCL2 were constructed by Tsingke (Beijing, China). The plasmids were co-transfected into HEK 293T cells. 48 hours later, the luciferase reporter activity was determined by Dual luciferase reporter assay Kit (Vazyme, Nanjing, China).

Statistical analysis

All statistical analyses were exhibited as datasets consisting of at least three independent experiments. SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA) was used in this project. Student-Newman-Keuls test was performed to analyze data between two groups, and One-way ANOVA was performed to analyze data among multiple groups.

Expression of hsa_circ_0043533 in human granulosa cell lines, and inhibition of hsa_circ_0043533 in KGN cell line.

Q-PCR result revealed that the expression level of hsa_circ_0043533 was higher in KGN cells than that in COV434 cells and normal IOSE80 cells (Fig. 1A), therefore the KGN cell line was chosen as the experimental cell model in this project. Q-PCR showed that transfection of si-RNAs targeting hsa_circ_0043533 in KGN cells decreased the hsa_circ_0043533 level (si-circ_0043533 group) successfully, compared to those transfected with blank si-RNAs (si-NC group), and wild type KGN cells (WT group) (Fig. 1B).

Knockdown of hsa_circ_0043533 repressed the survival, proliferation, migration and assisted to apoptosis of KGN cells.

Results of the in vitro cell survival assay using CCK-8 manifested that knockdown of hsa_circ_0043533 significantly inhibited KGN cells’ survivability (Fig. 1C, D). EDU staining indicating the DNA replication activity showed the same result that silencing hsa_circ_0043533 decreased the survival of KGN cells more intuitively through fluorescence staining, together with the quantification of the fluorescence intensity (Fig. 1E). Flow cytometry demonstrated that downregulated hsa_circ_0043533 dramatically increased the apoptosis rate of the KGN cells (Fig. 2A). And we calculate that the actual proliferation of KGN cells was inhibited after knockdown of hsa_circ_0043533 (Fig. 2B).

In vitro wound healing migration assay showed loss of hsa_circ_0043533 shrunk the migrated area of KGN cells, indicating the migration potential of the hsa_circ_0043533 knockdown cells was limited with statistical significance (Fig. 2C). In vitro trans-well migration assay also showed a low level of hsa_circ_0043533 markedly decreased the quantity of the cells with migration ability (Fig. 2D). All the results revealed the hsa_circ_0043533 was largely involved in the regulation on KGN cell behaviours.

Key EMT markers were influenced by hsa_circ_0043533 in KGN cells.

The protein level of several essential EMT markers, E-cadherin, N-cadherin and Vimentin, were examined by Western blotting when the EMT process was triggered by 10 ng/ml TGF-β1 in KGN cells. The treatment of TGF-β1 could reduce E-cadherin but increase N-cadherin and Vimentin expression level significantly in WT KGN cells. The treatment of TGF-β1 could down-regulate E-cadherin level of cells in WT and si-NC group, but not in si-circ_0043533 group; similarly, the treatment of TGF-β1 up-regulated the N-cadherin and Vimentin level of cells in both WT and si-NC group instead of the si-circ_0043533 group (Fig. 3A).

Key apoptotic markers were influenced by hsa_circ_0043533 in KGN cells.

Western blotting results showed the protein level of fundamental pro-apoptotic proteins, BAX, Cleaved CASP3, CASP3, Cleaved CASP9, CASP9, were significantly increased in cells from si-circ_0043533 group compared to those from si-NC group, while the expression of anti-apoptosis proteins, BCL2, in si-circ_0043533 group was obviously decreased. We found in si-circ_0043533 group, the ratio of the pro- and anti-apoptotic molecules BAX/BCL2 was rather high since BAX and BCL2 was increased and decreased, respectively. We also found the ratio of the precursor form (CASP3. 9) and activated form (Cleaved CASP3. 9) was also elevated in si-circ_0043533 group. (Fig. 3B, C and D)

Hsa_circ_0043533 targeted miR-409-3p directly.

We conducted sequence analysis using online circular RNA interactome database (available from https://circinteractome.irp.nia.nih.gov/). The results of the sequence analysis demonstrated that there were six complementary pairing bases between wild type hsa_circ_0043533 and miR-409-3p, suggesting hsa_circ_0043533 and miR-409-3p could binding to each other (Fig. 4A). Co-transfection of miR-409-3p-NC/mimics and luciferase reporter plasmids containing hsa_circ_0043533-WT/MUT into HEK 293T cells revealed that the luciferase activity of cells with miR-409-3p mimics and hsa_circ_0043533-WT plasmid was significantly decreased, confirmed the binding between hsa_circ_0043533 and miR-409-3p (Fig. 4B). Q-PCR was performed using granulosa cell samples from our PCOS cohort. Examine showed a downregulation of miR-409-3p in the granulosa cells from PCOS patients (Fig. 4C).

Inhibition of miR-409-3p overturned the effect of hsa_circ_0043533 knockdown on viability, migration, and apoptosis of KGN cells.

In vitro cell survival assay using CCK-8 revealed KGN cells with low level of both hsa_circ_0043533 and miR-409-3p were accompanied with rescued higher survivability than the cells with low level of hsa_circ_0043533 alone (Fig. 4D). Similarly, EDU staining showed that compared with loss of hsa_circ_0043533 alone, additive inhibition of miR-409-3p rescued KGN cells’ survivability (Fig. 4E). In vitro trans-well and wound healing assay demonstrated that targeting miR-409-3p in si-circ_0043533 cells significantly enhanced migration which was inhibited by si-circ_0043533 alone (Fig. 4F, G). Loss of hsa_circ_0043533 promoted KGN cells’ apoptosis, and down-regulation of miR-409-3p in si-circ_0043533 cells decreased the apoptosis rate significantly (Fig. 4H). All the results indicated the regulation of hsa_circ_0043533 on KGN cell behaviours required the assistance of miR-409-3p.

BCL2 was targeted by miR-409-3p directly in vitro.

We carried out sequence analysis using online Targetscan database (available from http://www.targetscan.org/mamm_31/). Results of the targeting sequence analysis revealed the potential correlation between miR-409-3p and BCL2 gene: multiple bases in the 3'-UTR region of BCL2 gene were complementary to miR-409-3p (Fig. 5A). In order to verify the interaction of miR-409-3p and BCL2, we adopted dual-luciferase reporter assay. We altered the complementary sequence in 3'-UTR region of BCL2 (MUT BCL2), which lost the potential binding ability to miR-409-3p (Fig. 5A). The dual-luciferase reporter assay was carried out. In HEK 293T cells transfected with WT BCL2, co-transfection of miR-409-3p-mimics decreased the luciferase activity compared to miR-409-3p-NC; whereas in those cells transfected with MUT BCL2, neither miR-409-3p-mimics nor miR-409-3p-NC could decrease the luciferase activity (Fig. 5B). The luciferase reporter assay demonstrated the miR-409-3p could bind to wild type 3'-UTR region of the BCL2 gene. Q-PCR and Western blotting results illustrated that in KGN cells, both transcriptional and protein level of BCL2 were inhibited by overexpressed miR-409-3p, which was achieved by the direct combination of miR-409-3p and 3'-UTR region of BCL2 gene (Fig. 5C, D).

Multiplied effect of metformin on the survival, migration, proliferation, and apoptosis of KGN cells.

The EDU staining showed the higher concentration of metformin was accompanied with stronger inhibition on the cell survival (Fig. 6A). CCK-8 assay revealed that in general, metformin inhibited the cell survival, and no matter how long of the treatment, high concentration of metformin was always accompanied with more inhibition on cell viability. Low concentrations (2.5mM) of metformin had no effect on survival; after treating for longer time (48 hours), middle to high concentration (5-40mM) of metformin inhibited survival, and the extremely high concentration (40mM) killed almost all cells after 72 hours (Fig. 6B). To analyse the effect of metformin on survival in a time- and dose-dependent manner, we discovered that both higher concentration and longer treatment duration had more powerful repression on KGN cells’ survival (Fig. 6C). And we concluded the same trends in terms of IC50 (half maximal inhibitory concentration) of metformin (Table 2).

Table 2

The IC50 values of KGN treated with metformin.
Cell line	IC50 (mM)
KGN	24h	48h	72h
KGN	30.93	15.37	6.92

Apoptosis of KGN cells was detected by flow cytometry. After treatment for 24h, the apoptosis rate of the KGN cells seemed to increase without statistical difference, and the apoptosis rate was quiet low in 24h (Fig. 6D). After 48h, metformin promoted the apoptosis of, and higher concentration lead to higher apoptosis rate; in which the apoptosis rate was nearly 100% after treatment of 20mM metformin for 48h (Fig. 6E).

Metformin treatment for 24h did not impact the normalized absolute proliferation of KGN cells (Fig. 7A). Cells treated with 2.5mM metformin showed upregulated proliferation but treated with 10mM metformin showed downregulated proliferation, and treated with other two concentration in between (5 and 10mM) made no differences (Fig. 7B),meanwhile the time- and dose-dependent manner (Fig. 7C, D).

We chose two concentrations, 2.5 and 5mM, for trans-well assay to exclude migration in 24h. There was a significant decrease in KGN cells treated metformin, and the decreasing was proportional to concentration of metformin (Fig. 7E). These data demonstrated that metformin significantly depressed cell growth and migration of KGN cells in a time and dose-dependent manner.

Effect of metformin on the of KGN cells’ behaviour via regulating miR-409-3p.

We treated KGN cells with 10 and 20mM metformin for 24 and 48h to further investigate regulation of metformin on miR-409-3. The expression of miR-409-3p was significantly up-regulated detected by Q-PCR (Fig. 7F), and the up-regulated expression of miR-409-3 was in the manner of both time-dependent and dose-dependent (Fig. 7G, H).

PCOS is becoming increasingly common in women of childbearing age, accompanied by lifelong complications (24). The pathogenesis of PCOS have not been fully elucidated, and it is a daunting challenge to understand the molecular and cellular mechanism of the disease.

It has been wildly confirmed that granulosa cells have an essential regulating role in the growth and maturation of oocytes and follicles. Granulosa cells synthesize and secret steroidal hormones, nutrients, cAMP/cGMP, and paracrine factors to maintain a specialized microenvironment for oocytes development. The imbalance between granulosa cell growth and apoptosis is a key factor in many diseases, such as PCOS (4). In the last decade, circRNA has been recognized as a new and important regulator of gene expression, as their altered expression is closely associated with a variety of diseases. Recently, the identification of aberrantly expressed circRNAs in PCOS patients provides new clues to explore novel mechanism (25, 26). On this basis, we aimed to investigate the role of hsa_circ_0043533 in PCOS, and the mechanism hsa_circ_0043533 within.

High expression of hsa_circ_043533 was disclosed in GCs of PCOS patients in the host lab (19), so we hypothesized hsa_circ_043533 could be regarded as a promoter in the development of PCOS. KGN cells with the most abundant expression of hsa_circ_043533 were chosen to establish the cell model in this project. After knockdown hsa_circ_043533 in KGN cells, a series of in vitro cell survival and EMT related function such as apoptosis, survival, and migration. Low level of hsa_circ_043533 enhanced the apoptosis rate and declined cell survival and migration, as well as the absolute proliferation significantly. As known to all, PCOS is characterized by ovulatory dysfunction (27), some crucial processes in which such as primary follicle pool formation, follicle recruitment and dominant follicle selection were all regulated by the balance of apoptosis and viability of granulosa cell (28). The increased viability and decreased apoptosis of granulosa cells in follicles of PCOS patients lead to rather abundance of granulosa cells, which hinder the follicle maturation, resulting in the accumulation of small antral follicles without dominant follicle, leading to polycystic ovaries (24). We considered that hsa_circ_043533 played a promoting role in these processes. The increased proliferation and decreased apoptosis by high expressed hsa_circ_043533 in PCOS acted together, leading to the development of PCOS.

Based on the results of the cellular function assays, related key molecules involved in apoptosis and EMT procedures were examined. The key proteins involved in the corresponding cell behaviour may be the target of hsa_circ_043533. We found the expression of pro-apoptotic proteins were increased and the expression of anti-apoptotic protein was decreased in the circ_0043533 knockdown group. These key apoptotic proteins involved in the mitochondrial apoptotic pathway, which might be regulated by hsa_circ_043533.

CircRNAs can act as the miRNA sponges (29). Complementary sequences between circRNA and miRNA are the premise of interaction. Continuously, exploring the regulatory mechanism between hsa_circ_043533 and key cell function involved proteins, we found out miR-409-3p with a complementary sequence to hsa_circ_043533, and the direct binding between was also proved. Effect of knockdown hsa_circ_043533 on cell function, including increased apoptosis and decreased survival, migration, were all rescued by targeting miRNA in KGN cells. Knockdown hsa_circ_043533 released the miR-409-3p adsorbed on sponges, thereby affecting cell function, while antagonizing the effect of intracellular miR-409-3p proved that miR-409-3p is just the downstream target of hsa_circ_043533 to regulate KGN cells.

MiRNAs can work as the gene transcription factors, which affect protein synthesis by regulating gene transcription, thereby further regulating cells function (30). Similarly, we discovered complementary sequences existed between miR-409-3p and 3'-UTR region of the BCL2 gene, and we confirmed the direct interaction between miR-409-3p and 3′-UTRs of BCL2. MiR-409-3p inhibitor reversed the effect of hsa_circ_0043533 silencing on KGN cellular behaviour by targeted regulation of BCL2 gene. BCL2 is an important silencer cell apoptosis (31). In brief, the sufficient hsa_circ_0043533 adversely affected PCOS progression via competitively binding to miR-409-3p, inducing the expression of BCL2 and subsequently regulating mitochondrial apoptotic pathway, decreased apoptosis of granulosa cells in PCOS.

Metformin, the first-line oral hypoglycemic agent for type 2 diabetes, has been widely used in the treatment of PCOS patients with insulin resistance (23, 32). Insulin resistance is an important clinical manifestation of PCOS patients, and metformin is often used to regulate metabolic disorders in PCOS patients. We found that the effect of metformin on the function of KGN cells was complex. In general, higher concentration and longer-term treatment of metformin can inhibit cell survival and reduce migration. While the effect of apoptosis is time-dependent: regardless of the concentration of metformin, effect on apoptosis was weak after treatment for 24h, and the apoptosis rate increased significantly after 48h treatment. Normalized the cell survival by apoptosis rate, we also found the impact of metformin on absolute proliferation of KGN cells was biphasic. Treatment for 24h showed no influence on proliferation; after 48h, treating with metformin in low concentrations (2.5mM) promoted proliferation, while treating with metformin in high concentrations (20mM) restrained proliferation. We could find out the bidirectional regulation of metformin on proliferation concluded as promotion in low dose and inhibition in high dose. These biphasic regulation suggested that when giving metformin clinically, physicians should fully consider the dosage of the drug to avoid excessive stimulation of the growth of granulosa cells, and it is necessary to allow the drug to act for a sufficient time, at least over 24h to achieve full efficacy of promoting apoptosis. Previous studies showed treating with metformin in PCOS patients could reduce some abnormally highly expressed miRNAs, like miR-122, miR-223 and miR-29a, to improve follicular development (33). The modulation of metformin on the hsa_circ_043533/miR-409-3p axis was also revealed since the miR-409-3p was upregulated by metformin. One of the impact mechanisms of metformin on PCOS was regulating expression level of miR-409-3p, achieving the remission of high hsa_circ_043533 induced imbalance of verbality and apoptosis of granulosa cells.

Conclusion

Through a series of experiments, we conclude that hsa_circ_0043533 participates in PCOS by regulating EMT related markers such as E-cadherin, N-cadherin and Vimentin; and apoptosis markers such as BAX, BCL2 and Caspase family. The upregulation of hsa_circ_0043533 on BCL2 was achieved through competitive interaction with miR-409-3p. Suppression of hsa_circ_0043533 attenuated PCOS progression via miR-409-3p, down-streaming; and metformin had an intervention effect on this. Both hsa_circ_0043533 and miR-409-3p may serve as diagnostic as well as potential therapeutic biomarkers of PCOS, and miR-409-3p could act as the evaluation criteria for metformin treatment in PCOS. This research provides new insights into the molecular mechanisms of granulosa cell behaviour in PCOS. Future studies focusing on these biomarkers could shed further light on the development of new therapeutic intervention strategies.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the institutional ethics committee of Medical College of Qingdao University. All participants provided written informed consent prior to the commencement of the study.

CONSENT FOR PUBLICATION

The author declares that all work described here has not been published before and that its publication has been approved by all co-authors.

FUNDING INFORMATION

The National Natural Science Foundation of China, Grant/Award Number: 81671416.

DATA AVAILABILITY

Data will be made available on request.

ACKNOWLEDGEMENTS

We are grateful to the College of Basic Medical Sciences, Qingdao University for their kindly assistance, and we would like to acknowledge all the members in our department.

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No competing interests reported.

Supplementary1.docx
Supplementary 1. Description of the clinical cohort.
SupplementaryTableS1.docx
Supplementary Table S1. Sequence of the primers used in this project for Q-PCR.
SupplementaryTableS2.docx
Supplementary Table S2. Sequence of mimics, inhibitor, and NC.
SupplementaryTableS3.docx
Supplementary Table S3. Information of antibodies used in the project.

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Hsa_circ_0043533 modulates apoptosis and viability of granulosa cells via miR-409-3p/BCL2 and EMT signalling in PCOS: providing a novel perspective of metformin

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1. INTRODUCTION

2. METHODS

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4. DISCUSSION

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