miR-3188 Regulates proliferation and apoptosis of granulosa cells by targeting KCNA5 in the polycystic ovary syndrome

1Department of Endocrinology, Hwa Mei Hospital, University of Chinese Academy of Sciences, Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences, Ningbo City, Zhejiang Province, 315010, China; 2Department of Gynecology, Hwa Mei Hospital, University of Chinese Academy of Sciences, Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences, Ningbo City, Zhejiang Province, 315010, China


INTRODUCTION
The polycystic ovary syndrome (PCOS) is a common heterogeneous endocrine disease, which could result in anovulation and infertility in women of childbearing age (Stanosz et al., 2018;Youssef, 2019). Ovarian biopsy in infertile PCOS patients showed increased accumulation of early follicles than in healthy women, suggesting that abnormal follicle development in the early stages may be the root cause of PCOS (Franks et al., 2008). Further study revealed that the growth rate of granulosa cells, which is critical for follicle maturation, was higher in PCOS patients than in healthy controls (Webber et al., 2007). Abnormal granulosa cell proliferation and differentiation are related to dysregulated folliculogenesis, thereby leading to the pathogenesis of PCOS . Therefore, regulation of follicular granulosa cell proliferation or apoptosis may be the direction of treatment for PCOS.
MiRNAs have been reported to be involved in granulosa cell function (Tu et al., 2019), and the pathogenesis of PCOS (Sorensen et al., 2014). Reduction in miR-16 levels in PCOS could inhibit proliferation and promote apoptosis of ovarian granulosa cells . MiR-222 has been reported to promote proliferation and inhibit apoptosis of ovarian granulosa cells, thus promoting the progression of PCOS (Huang et al., 2019). MiR-3188 levels, with the ability to inhibit cell proliferation in non-small cell lung cancer  and head and neck cancer , were found to be increased in PCOS ovarian granulosa cells . Therefore, we hypothesized that miR-3188 might promote proliferation and inhibit apoptosis of ovarian granulosa cells during PCOS progression.
Potassium voltage-gated channel subfamily A member 5 (KCNA5) has been found to be associated with cell proliferation and apoptosis in various cell types (Ryland et al., 2015;Qu et al., 2018). Recently, KCNA5 has been shown to participate in miR-3940-5p-mediated granulosa cell proliferation in PCOS (Gao et al., 2020). However, whether or not miR-3188 could target KCNA5 to regulate granulosa cell proliferation in PCOS remains elusive.
In this study, the expression level of miR-3188 in granulosa cells of PCOS patients was examined. The role and mechanism of miR-3188-mediated granulosa cell proliferation were then evaluated. The findings from this study suggest that miR-3188 may be a promising therapeutic target for PCOS.

Patient samples
This study was approved by the Ethics Committee of the Hwa Mei Hospital, University of Chinese Academy of Sciences. A total of 28 PCOS patients and 36 healthy controls with written informed consents were recruited from the Hwa Mei Hospital, University of Chinese Academy of Sciences. Peripheral blood samples were collected from the patients and controls into tubes (Pre- AnalytiX, Hombrechtikon, Switzerland). Samples were stored at -80°C for subsequent analyses.

Cell viability
KGN cells (2×10 3 cells/well) were seeded and cultured for 24 hours. Cells were incubated with 20 μL of 5 mg/mL MTT solution (KeyGEN, Nanjing, China) for 4 hours before the measurement of absorbance at 490 nm using an Epoch microplate Reader (BioTek, Winooski, VT, USA).

Flow cytometry
KGN cells (1×10 6 cells) were trypsin digested and harvested, and then stained with 5 µL of 100 µg/mL propidium iodide after incubation with 1 U/ml of ribonuclease (Abcam, Cambridge, MA, USA). Cell cycle distribution was analyzed by the FACS flow cytometer (Attune, Life Technologies, Darmstadt, Germany). For cell apoptosis analysis, cells were incubated with fluorescein isothiocyanate-conjugated annexin V after staining with propidium iodide. Cell apoptosis distribution was also analyzed by the FACS flow cytometer.

Statistical analysis
Data are reported as mean ± standard deviation. Statistical tests were conducted using SPSS 21.0 software (IBM-SPSS, Chicago, IL, USA). Student's t test was used to compare differences between two groups. p<0.05 was considered as statistically significant.

Enhanced miR-3188 level in PCOS increases granulosa cell viability
To determine the expression of miR-3188 in PCOS, blood samples were collected from PCOS patients and healthy controls. qRT-PCR analysis demonstrated a significant increase in miR-3188 level in PCOS patients when compared to the controls (p<0.01) (Fig. 1A). In order to establish the functional roles of miR-3188 in PCOS in vitro, KGN was transfected with miR-3188 mimic or inhibitor (Fig. 1B). The results of the MTT assay showed that miR-3188 overexpression increased cell viability of KGN, while miR-3188 knockdown led to opposite effects (Fig. 1C), suggesting the pro-proliferative role of miR-3188 in PCOS granulosa cells.

Effect of miR-3188 on KGN cell cycle
Cell cycles of KGN transfected with miR-3188 mimic or inhibitor were evaluated to assess the role of miR-3188 in PCOS progression. Results demonstrated that miR-3188 promoted cell cycle of KGN, with a decreased cell number in the G1 phase and increased cell number in the G2 and S phases ( Fig. 2A). In contrast, in KGN cells transfected with miR-3188 inhibitor, the KGN cell number in the G1 phase was increased, whereas there was a lower number of cells in the G2 and S phase ( Fig. 2A). This indicates that miR-3188 knockdown leads to cell cycle arrest in KGN. The levels of cell cycle proteins were also assessed by western blotting, which revealed that miR-3188 overexpression led to increased levels of cyclinD1 and reduced p21 to promote cell cycle progression (Fig. 2B). On the contrary, cyclin D1 level was reduced and that of p21 was increased in KGN transfected with miR-3188 inhibitor, consistent with cell cycle arrest (Fig. 2B). These results confirmed the pro-proliferative role of miR-3188 in PCOS granulosa cells.

Effect of miR-3188 on KGN cell apoptosis
Next, the effect of miR-3188 on cell apoptosis was investigated in KGN, and the result indicated that miR-3188 suppressed cell apoptosis of KGN (Fig.  3A). Moreover, there was an increase in the number of apoptotic cells in KGN transfected with miR-3188 inhibitor (Fig. 3A), demonstrating the anti-apoptotic miR-3188/KCNA5 in polycystic ovary syndrome role of miR-3188 in granulosa cells. The levels of apoptotic proteins were also evaluated by western blotting. Results revealed that miR-3188 overexpression enhanced protein expression of Bcl-2, but reduced the levels of Bax and cleaved caspase-3 (Fig. 3B). In contrast, Bax and cleaved caspase-3 levels were enhanced, and Bcl-2 level was reduced in KGN transfected with miR-3188 inhibitor (Fig. 3B). These results confirmed the anti-apoptotic role of miR-3188 in PCOS granulosa cells.

miR-3188 negatively regulates KCNA5 in PCOS
In order to uncover the underlying mechanisms of miR-3188 in the pathogenesis of PCOS, prediction of potential target genes of miR-3188 was performed using TargetScan (http://www.targetscan.org/vert_72/), followed by subsequent validation by luciferase reporter assays. The predicted binding sites between miR-3188 and KCNA5, as well as mutated KCNA5 binding site, which disrupted the binding ability of KCNA5 with miR-3188 are shown in Fig. 4A. Results from the lucif-  erase activity assays showed that transfection with miR-3188 inhibitor increased luciferase activities of KCNA5 wt compared to NC inhibitor (Fig. 4B), while KCNA5 mut demonstrated no response to miR-3188 inhibitor or NC inhibitor (Fig. 4B). Furthermore, the protein expression of KCNA5 was increased in KGN transfected with miR-3188 inhibitor, while it was decreased by miR-3188 mimic (Fig. 4C), confirming that KCNA5 was regulated  by miR-3188 in granulosa cells. A significant decrease in KCNA5 was found in PCOS patients when compared to controls (p<0.01) (Fig. 4D), and the expression of KCNA5 and miR-3188 revealed a negative correlation in PCOS patients (Fig. 4D). These results demonstrated that miR-3188 negatively regulates KCNA5 in PCOS.

Over-expression of KCNA5 counteracts the promotive effects of miR-3188 on PCOS progression
To explore the role of miR-3188/KCNA5 axis in PCOS granulosa cells, KGN was cotransfected with pcDNA-KCNA5 and miR-3188 mimic. KCNA5 overexpression attenuated miR-3188 mimic-mediated cell viability increase (Fig. 5A). Of note, miR-3188 mimic transfection failed to decrease the protein expression of KCNA5 in KCNA5-overexpressed cells (Fig. 5B). In addition, KCNA5 overexpression also lessened the miR-3188 overexpression-induced increase in cyclinD1 and Bcl-2 levels, and decrease in p21, Bax and cleaved caspase levels in KGN (Fig. 5B), further suggesting that miR-3188 could promote cell viability of granulosa cells in PCOS through down-regulation of KCNA5.

DISCUSSION
PCOS, characterized by such inconvenient symptoms as irregular menstrual bleeding and infertility, has been regarded as a "nuisance disease'' devoid of effective therapeutic strategies (Conway et al., 2014;Peng et al., 2020). Inhibition of granulosa cell survival provides a novel insight into PCOS treatment (Zhong et al., 2018;Cox et al., 2020). Considering the important regulatory role of miRNAs in granulosa cell functions, miRNAs have been regarded as critical mediators in physiological and pathological conditions of PCOS (Tu et al., 2019). To date, miR-3188 has been shown as either suppressor or promoter in various tumors to regulate cell proliferation and apoptosis. The study presented here was performed to uncover the role of miR-3188 in granulosa cell proliferation and apoptosis in PCOS.
Biochemical markers, such as the luteinizing hormone or follicle-stimulating hormone levels, circulating androgens and hyperinsulinemia, are diagnostic criteria for PCOS (Barthelmess & Naz 2014;EBERSOLE & BON-NY 2020). MiRNAs are also considered as diagnostic or prognostic biomarkers for PCOS . Here, in line with previous reports , miR-3188 was found to be increased in the peripheral blood samples of PCOS patients when compared to the healthy controls. However, to further validate the potential of miR-3188 as a diagnostic or prognostic marker for PCOS, the correlation between clinical features of PCOS patients and miR-3188 levels should be investigated in a future study.
Aberrant profiling of miRNAs that participated in steroidogenesis or the cell apoptosis process was detected in follicular fluid or granulosa cells in PCOS patients (Sorensen et al., 2014;Lionett et al., 2020). MiR-93, which has the ability to promote granulosa cell proliferation, could be a therapeutic target for PCOS (Jiang et al., 2015). Here, based on MTT, flow cytometry and western blotting analyses, our findings demonstrated that miR-3188 could increase granulosa cell viability, promote cell cycle progression through increasing cyclin D1 and decreasing p21 levels, and suppress cell apoptosis via promoting expression of Bcl-2 and lowering Bax and cleaved caspase-3 levels. Collectively, our results demonstrated that miR-3188 is a novel key miRNA that promotes granulosa cell proliferation in PCOS. This is in contrast to previous finding, which proposed that miR-3188 suppresses cell proliferation and cell cycle progression in non-small cell lung cancer cells through targeting mammalian target of rapamycin . This prompted the identification of cell type-specific downstream target of miR-3188 in PCOS granulosa cells.
Cation channels have been implicated in the apoptosis of granulosa cells, indicating their role in the etiology of PCOS (Köse & Nazıroğlu 2015). Antagonists of potassium channel regulate granulosa cell proliferation and apoptosis (Manikkam et al., 2002;Traut et al., 2009). Previous study has shown that KCNA in granulosa cells binds to an antagonist, 4-aminopyridine, and decreases progesterone production through the steroidogenic pathway (Li et al., 2003). Here, potassium channel KCNA5 level was found to be reduced in granulosa cells of PCOS, and was validated to be the downstream target gene of miR-3188. Gao et al (Gao et al., 2020) has reported that KCNA5 is involved in miR-3940-5p-promoted granulosa cell proliferation in PCOS. Our functional assays revealed that overexpression of KCNA5 counteracted the promotive role of miR-3188 in granulosa cell viability and cell cycle progression, as well as the suppressive role of miR-3188 in granulosa cell apoptosis. MiRNAs could target steroid receptors or steroid synthesis enzymes involved in PCOS (Sorensen et al., 2014), and antagonists of KCNA could mediate progesterone accumulation in the granulosa cells (Li et al., 2003). The role of miR-3188/KCNA in the steroidogenic pathway of granulosa cells should be investigated in a future study. Additionally, signaling pathways, including transforming growth factor-β or follicle-stimulating hormones' pathway, which are known to be associated with miRNAs-medi-ated granulosa cell homeostasis (Tu et al., 2019), as well as pathways involved in miR-3188/KCNA-modulated granulosa cell proliferation in PCOS should also be investigated.

CONCLUSION
In conclusion, as shown in Fig. 6, our results indicated that increased miR-3188 levels in PCOS patients could promote cell viability and cell cycle progression, and suppress cell apoptosis through down-regulation of KCNA5. These observations provide novel insights into the potential development of miR-3188 as a therapeutic approach for the treatment of PCOS.