Silencing circ_0058063 enhances proliferation and inhibits apoptosis in PCOS ovarian granulosa cells

Background Polycystic ovary syndrome (PCOS) is the most common endocrine disease in reproductive-aged women. This study was designed to explore the role of circ_0058063 in PCOS. Methods We recruited nine PCOS patients and nine no-PCOS patients. The concentrations of follicle stimulating hormone (FSH), testosterone (T), luteinizing hormone (LH), progesterone (P4) and estradiol (E2) were measured by radioimmunoassay. The level of aromatase was detected using an ELISA kit. The proliferation and apoptosis of ovarian granulosa cells were assessed using CCK-8 assay and ow cytometry, respectively. Gene and protein expression were evaluated through RT-qPCR and Western blotting assay.


Background
Polycystic ovary syndrome (PCOS) is the most common endocrine disease in reproductive-aged women (1,2), affecting approximately 10% of women of childbearing age (3). The most prominent characteristics of PCOS are abnormal follicular development and hyperandrogenism (4,5). Women with PCOS have short-term and long-term complications of multiple systems throughout the body, including reproduction (hairy, amenorrhea, anovulation, infertility and pregnancy complications), metabolism (type 2 diabetes, cardiovascular disease, and abnormal lipid metabolism) and mental (negative, anxiety), which have brought a heavy burden on individuals and the whole society (4). Presently, PCOS cannot be completely cured. Therefore, the deep exploration of the PCOS development mechanism is particularly signi cant for the treatment of the disease. Circular RNAs (circRNAs) constitute a kind of non-coding RNAs, which is named for its closed loop structure, lack of 5'cap ends, and 3'poly A tails (6, 7). Recently, several studies suggest that circRNAs are widely involved in the PCOS progression (8, 9). Research evidence shows that the expression of hsa_circ_0043533 and hsa_circ_0043532 in the PCOS group increased evidently than that in the non-PCOS group, while the non-PCOS group had a remarkably lower hsa_circ_0097636 expression as compared to the PCOS group (8). Research evidence shows that circ_0058063 functions as an oncogene in bladder cancer (10). However, the involvement of circ_0058063 in PCOS is still not well discovered. Our aim is to explore the function of circ_0058063 in patients with PCOS, which may shed new light on the development of PCOS treatment.

Study Subjects
We recruited nine PCOS patients and nine patients with no-PCOS under the age of 35 who underwent in vitro fertilization and embryo transfer (IVF-ET) in the Huai'an Second People's Hospital between January 2018 and April 2019. We measured the subjects' weight, height, and then calculated body mass index (BMI). Selection criteria for the PCOS group (refer to Rotterdam criteria (11)):1) rare ovulation and/or anovulation; 2) clinical manifestations of hyperandrogenism and/or biochemical changes; 3) polycystic ovary was found by ultrasound examination. Those who met 2 of the above 3 items were enrolled in the PCOS group. The no-PCOS group was subjects with normal ovaries function, no PCOS, no history of ovarian adverse reactions, no endometriosis, and receiving IVF-ET due to male factors. All patients voluntarily participated in this research project and signed informed consent. This study was approved by the Ethics Committee of the Huai'an Second People's Hospital.
2.2 Collection of serum 5 mL of fasting venous blood was collected on the morning of the third day of the menstrual cycle. Blood samples were centrifuged at 1500 r/min for 5 min to draw the upper layer of serum, aliquoted into a 0.5 mL EP tube, and stored in the refrigerator at -80 °C for testing.

Acquisition of follicular uid
All subjects induced ovulation according to the GnRH-a/FSH/ HMG/HCG protocol. The dosage and time were adjusted based on the individual situation. 36 hours after HCG injection, the follicles of both ovaries were sucked through the posterior vaginal foramen under the guidance of ultrasound. The rst tube of clear follicular uid without blood staining was collected and centrifuged at 400x g for 10 min at room temperature. The supernatant was stored at -80 °C until detecting.

Collection, separation and puri cation of ovarian granulosa cells
We collected the discarded follicle puncture uid after the separation of the ovarian crown complex, centrifuged it at 2000 rpm for 10 min, and then discarded the supernatant. The cells were resuspended in PBS and digested using 0.25% trypsin solution at 37℃ for 5 ~ 10min. After termination of digestion, the cell suspension was centrifuged at 2000 rpm for 10min and resuspended in PBS. Next, we added an equal volume of human lymphocyte separation solution (Tianjin Med Paci c Technology, China) to cells.
After centrifugation at 2000 rpm for 20 min, the intermediate cell layer was drawn. The cells were washed with DMEM (Gibco, USA) containing 1% Pen-Strep (Invitrogen, USA) and 10% FBS, centrifuged at 1000 rpm for 10 min, and resuspended with DMEM. Trypan blue staining proved that the cell survival rate was greater than 90%. The cell density was adjusted to 2 × 10 5 / mL and fostered in a 37 °C, 5% CO 2 incubator. After the cells adhered to the growth, they were rinsed once or twice every 24h and then replaced with fresh culture medium to further purify the mixed blood cells and other impurities.

Hormone levels detection
The concentrations of follicle stimulating hormone (FSH), testosterone (T), luteinizing hormone (LH), progesterone (P4) and estradiol (E2) were measured by radioimmunoassay. The concentration of aromatase was detected using an ELISA kit. The kits were provided by Xiamen Huijia Biological Technology (Xiamen, China).

RT-qPCR
After 24-hour transfection, the culture medium was replaced, and cells were continued to foster for 48 hours. The total RNA was extracted by Trizol (Invitrogen, USA), and the RNA quality was detected and quanti ed by ultraviolet spectrophotometry. The cDNA was synthesized using PrimeScript™ RT Master Mix (Takara, China), and qPCR was performed utilizing TB Green® Premix Ex Taq™ (Takara, China) according to the standard protocol. U6 and β-actin were used as internal reference controls. The relative expression of circ_0058063 and Aromatase mRNA was calculated using the 2 −ΔΔCt method.

Western blotting
The total protein of the cell was extracted using RIPA buffer (Biyuntian, China). The protein concentration was assessed via a BCA kit (Biyuntian, China). Next, 50μl protein samples were taken for SDSpolyacrylamide gel electrophoresis to separate the target protein, and the membrane was transferred by the wet transfer method. We sealed the membrane with 5% skimmed milk powder for 2 hours, washed the membrane 3 times with PBST solution, and added primary antibodies (1:1000, CST, USA), including Cyclin A, Cyclin D1, Bcl-2, Bax, and GAPDH. Primary antibodies were incubated at 4 ℃ overnight. Then a secondary antibody (1: 3000) was added and incubated at 37 °C for 2 hours. The ECL kit obtained from Amersham Biosciences (Piscataway, NJ) was applied to visualize the immunocomplexes.

Colony formation assay
Cells were seeded at colony forming density (200 cells/ dish) in a 60-mm tissue-culture dish carrying 5 mL medium at 37 °C under 5% CO 2 . After 14 days, the colony formation rate was observed in each group.
Cells were xed with 4% paraformaldehyde for 15 min after discarding the medium. Next, cells were stained with 0.1% crystal violet for 20 min. In each well, ve randomly elds were selected to calculate the number of colonies. Colony formation rate = (number of colonies/number of inoculated cells) × 100% (12).

Cell-Counting Kit-8 Assay
The cell viability was detected using CCK-8 (Biyuntian, China) based on the instructions. In short, after transfection for 48 hours, 10 μl CCK8 reagent was added to ovarian granulosa cells in each well. The cells were incubated at 37 °C for 4 hours. The microplate reader (Bio-Rad, USA) was used to detect the optical density (OD) at a wavelength of 450 nm.

Flow cytometry
Cell apoptosis was measured utilizing the Annexin V-FITC Apoptosis Detection Kit (Abcam, USA) as the manufacturer's protocol. In short, we added pre-chilled 1xbinding buffer to resuspend the cells, and then added 10µl PI and 5µl Annexin V-FIT. Cells were stained for 15 ~ 25min in the darkroom. Flow cytometry (BD, USA) was used to detect the apoptosis rate.

The establishment of PCOS mouse model
Fifty 25-day immature female BALB/c mice were purchased from Wuhan University Center for Animal Experiment. Mice were randomly divided into PCOS group (n=40) and control group (n=10). Mice in the PCOS group were injected with 6 mg DHEA per 100 g body weight (purchased from Hangzhou Aladdin Information Technology Co., Ltd., China) every day for 20 consecutive days to establish a PCOS mouse model. The control group was injected subcutaneously with 0.4 mL of normal saline daily for 20 days.
The fasting blood glucose and fasting insulin levels were measured 3 weeks after the injection. HOMA-IR = FI (mU/L) × FG (mmol/L) / 22.5 (13). Insulin resistance is de ned as HOMA-IR> 2.6 (14). Thirty PCOS mice with HOMA-IR> 2.6 were randomly divided into si-NC group, si-circ_0058063-1 group and si-circ_0058063-2 group, with 10 mice in each group. Then 5 × 10 8 PFU/mL lentivirus containing si-circ_0058063 (GenePharma, China) or negative control vector was injected to the mice ovary. Mice were detected IR and then sacri ced 2 weeks after infection. Mice ovaries were collected for the further experiment. All animal experiments are in compliance with the ethical requirements of laboratory animals and laboratory animal management regulations of the Huai'an Second People's Hospital.

Immunohistochemistry assay
The Bax protein expression of mice ovaries was examined via immunohistochemistry. The ovarian tissue was taken for sectioning, depara nization, and hydration. After washing 3 times with PBS for 5 min each time, 3% H 2 O 2 were added in the ovarian tissue to block endogenous peroxidase for 10 min at room temperature. Then Bax antibody (Wuhan Boster Biological Technology., Ltd, China) were added in the tissue after washing with PBS (3 times, 5 min each) and incubated overnight at 4 °C. Next, peroxidaselabeled goat anti-rabbit immunoglobulin (Ig) G secondary antibody (Wuhan Boster Biological Technology., Ltd, China) were added. Finally, DAB staining was conducted, and the results were observed using a microscope.
2.14 Statistical analysis SPSS 21.0 software was utilized for statistical analysis. The data were expressed as mean ± standard deviation. The differences between multiple groups were compared with one-way ANOVA followed by LSD multiple comparisons. P<0.05 was considered statistically signi cant.

The general characteristics and hormone levels of patients
Compared with the no-PCOS group, patients' BMI increases signi cantly in the PCOS group. Additionally, the PCOS group has signi cantly higher serum LH and T levels than the no-PCOS group. No statistically signi cant difference was seen in age and FSH levels between two groups in Table 1.

The expression of circ_0058063 is elevated in PCOS ovarian granulosa cells and follicular uid
As presented in Figure 1A and 1B, qRT-PCR results indicate that the circ_0058063 level in ovarian granulosa cells and follicular uid is remarkably higher in the PCOS group than the no-PCOS group. Additionally, the si-circ_0058063 group has a lower relative circ_0058063 expression, compared with the si-NC group in Figure 1C. These outcomes reveal that the expression of circ_0058063 is elevated in PCOS ovarian granulosa cells and follicular uid.

Silencing circ_0058063 increases the levels of Aromatase mRNA, E2 and P4 in PCOS ovarian granulosa cells
We use qRT-PCR to detect the Aromatase mRNA expression in ovarian granulosa cells. Additionally, ELISA assay is applied to assess P4 and E2 levels in ovarian granulosa cells. As can be seen from Figure 2A-C, the levels of Aromatase mRNA, E2, and P4 are signi cantly increased in PCOS ovarian granulosa cells after silencing circ_0058063, compared with the si-NC group.

Silencing circ_0058063 enhances cell proliferation in PCOS ovarian granulosa cells
Cell proliferation is measured by colony formation assay and CCK-8 assay. As demonstrated in Figure 3A-C, the si-circ_0058063 group has remarkably higher ovarian granulosa cell proliferation than the si-NC group. In addition, western blot analysis is utilized to evaluate the Cyclin A and Cyclin D1 proliferation protein levels in each group. As displayed in Figure 3D and 3E, the protein levels of Cyclin A and Cyclin D1 in the si-circ_0058063 group increase signi cantly compared with those in the si-NC group. These results suggest that silencing circ_0058063 enhances cell proliferation in PCOS ovarian granulosa cells.

Silencing circ_0058063 inhibits apoptosis of PCOS ovarian granulosa cells
As presented in Figure 4A and 4B, the results of ow cytometry suggest that cell apoptosis is signi cantly decreased after silencing circ_0058063 in PCOS ovarian granulosa cells, compared with the si-NC group.
In Figure 4C and 4D, western blot analysis indicates that silencing circ_0058063 signi cantly increases the expression of Bcl-2 protein and reduces the expression of Bax protein in the si-circ_0058063 group than in the si-NC group. These data show that silencing circ_0058063 inhibits apoptosis of PCOS ovarian granulosa cells.

Knocking circ_0058063 improves ovary injury of PCOS mice
As presented in Figure 5A and 5B, the results of immunohistochemistry assay revealed that the rate of relative Bax positive cells is signi cantly decreased in the si-circ_0058063-1 group and si-circ_0058063-2 group, compared with the si-NC group. These data indicate that silencing circ_0058063 reduces ovary injury of PCOS mice.

Discussion
PCOS is the main cause of anovulatory infertility, which affects 80% of patients with anovulatory infertility (15,16). At present, the mechanism of ovarian injury and abnormal follicular development in PCOS patients is not fully understood. PCOS is often closely related to repeated spontaneous abortions in infertile patients (17). Therefore, we must pay attention to this group of women of childbearing age, who may suffer adverse consequences due to complications of PCOS.
Our study nds that the expression of circ_0058063 in ovarian granulosa cells and follicular uid in the PCOS group is signi cantly up-regulated. Besides, silencing circ_0058063 increases the levels of Aromatase mRNA, E2, and P4 in PCOS ovarian granulosa cells. In addition, silencing circ_0058063 can promote the proliferation of ovarian granulosa cells in PCOS patients. Furthermore, the results of ow cytometry reveal that after silencing circ_0058063, the survival rate of PCOS ovarian granulocytes is signi cantly increased, and the apoptosis rate is signi cantly reduced, indicating that silencing circ_0058063 may play an inhibiting role in ovarian granulosa cell apoptosis and have a promoting effect on cell survival in PCOS.
Circ_0058063 is only been studied in esophageal squamous-cell carcinomas and bladder cancer (10,18,19). In the progression of bladder cancer, circRNA_0058063 serves as a ceRNA via sponging miR-486-3p/FOXP4 and miR-145-5p/CDK6 axis (10,19).Circ_0058063 stimulates glucose-uptake and increases the expression of GLUT1 in esophageal squamous-cell carcinomas (18). To the best of our knowledge, the present study is the rst to investigate the function of circ_0058063 in PCOS.

Conclusions
In conclusion, silencing circ_0058063 enhances proliferation and inhibits apoptosis in PCOS ovarian granulosa cells. Such ndings may offer vital insights into a therapeutic target for PCOS.

Declarations Ethics approval and consent to participate
This study was approved by the Ethics Committee of the Huai'an Second People's Hospital. All patients voluntarily participated in this research project and signed informed consent. Moreover, all animal experiments are in compliance with the ethical requirements of laboratory animals and laboratory animal management regulations of the Huai'an Second People's Hospital.

Consent for publication
Not applicable.

Availability of data and material
Data available on request from the authors.

Competing interests
The authors con rm that there are no con icts of interest.   Figure 1 The