Abnormal GCs autophagy is associated with the pathogenesis of PCOS. In our previous study, we have found that the expression of ATG7 is up-regulated in ovarian GCs of PCOS patients. However, the precise mechanism of ATG7 participating in the pathogenesis of PCOS by regulating autophagy has not been clarified. In this study, we found the microRNA associated with ATG7 through bioinformatics software and dual-luciferase reporter assay. MiR-17-5P could negatively regulate the expression of ATG7. The detection of clinical GCs samples showed that compared with normal women, the expression of miR-17-5P in ovarian GCs of PCOS patients decreased, while the expression of ATG7 mRNA and protein increased. This proved that miR-17-5P regulates autophagy of GCs by targeting ATG7 and played an important role in PCOS.
Autophagy, as an evolutionarily conserved and highly regulated catabolic process, can affect follicular growth and is closely related to the clinical symptoms of PCOS, such as insulin resistance, hyperandrogenemia, obesity, chronic low-grade inflammation, and so on[10–12]. At present, a large number of studies have confirmed that autophagy is involved in different stages of follicular development. ATG7 influenced the survival of germ cells and primordial cells. The number of germ cells and primordial follicles in the ovaries of ATG7 deficient mice was small, and many follicles changed in structure or lose function . The loss of ATG7 or the dose-dependent reduction of autophagy-specific gene becn1 would not only lead to the significant reduction of germ cells in perinatal female mice but also lead to the excessive loss of primordial follicles in newborn mice. Sun et al. found that overexpression of miR-378-3P induced autophagy and increased the number of primordial follicles. After the miR-378-3P knockdown, autophagy was inhibited and the number of primordial follicles decreased. During the development from primordial follicles to mature follicles, the vast majority of follicles died due to follicular atresia, and only a small part (less than 1%) of follicles could mature and ovulate. Autophagy played an important role in follicular atresia and the selection of dominant follicles. By immunofluorescence staining of rat ovarian autophagy markers (MAP1LC3, LC3), it was found that LC3 showed a high expression level in GCs at all developmental stages. Cell death in the form of autophagy might mainly participate in follicular atresia by affecting GCs . Nicole et al.  found that after stimulating human ovarian GCs with oxidized low-density lipoprotein (oxLDL), autophagy was over-activated and mortality increased. Oxidative stress caused GCs death by initiating programmed cell death in the form of autophagy, which was a common cause of follicular atresia.
Autophagy is involved in the regulation of endometrial function and periodic remodeling of human endometrium. Basal level autophagy is essential for maintaining endometrial homeostasis and mediating endometrial specific functions, including menstrual cycle, embryo implantation, and decidualization. The imbalance of autophagy regulation was also associated with the disorder of the endometrial hyperplasia-secretion cycle, which might directly lead to the decrease in endometrial receptivity. The down-regulation of autophagy gene expression in the endometrium of PCOS patients might lead to a decrease in endometrial receptivity .
Autophagy is associated with a variety of endocrine and metabolic disorders in PCOS patients. First of all, autophagy is associated with elevated androgen levels. The mRNA abundance of autophagy-specific gene becn1, ATG5, and ATG7 in ovarian GCs of PCOS patients was significantly increased. The mRNA abundance of becn1 was positively correlated with the level of serum basal total testosterone. Dihydrotestosterone (DHT) increased the ratio of LC3-II/LC3-I in GCs in a dose-dependent manner. Li et al.  found that androgen inhibited GCs autophagy and proliferation by activating the PI3K / Akt signaling pathway. Secondly, autophagy is linked to insulin resistance. Autophagy has been shown to play an important role in regulating the normal function of pancreatic β-cells and insulin target tissues. Rapamycin, an autophagy inducer, could activate the inhibited autophagy, enhance insulin sensitivity and improve insulin resistance in T2DM rats . High mobility group box-1 (HMGB1) led to insulin resistance in GCs of PCOS patients by inducing abnormal autophagy. While uncontrolled autophagy led to the further release of HMGB1 and reduced insulin sensitivity. Thirdly, autophagy is associated with chronic low-grade inflammation. The expression of inflammatory transcriptome in ovarian GCs was up-regulated in patients with PCOS. The imbalance of inflammation would break the normal pattern of follicular development, impair the quality of follicles, and lead to anovulatory infertility. Autophagy proteins affected almost all cell types involved in the pathogenesis of inflammation, such as macrophages, T cells, lymphocytes, and dendritic cells. Nuclear factor kappa-B (NF-κB) was an important factor regulating the cellular inflammatory response. Early studies have shown that autophagy induces NF-κB activation and enhances the association between NF-κB and Becn1, thereby regulating the inflammatory pathway . Last but not least, autophagy is associated with obesity. Adipose tissue has important endocrine functions. Autophagy is a key regulator of white and brown adipose tissue production. Dysregulated autophagy would damage fat accumulation in vitro and in vivo . Luo et al.  found that Chemerin and its receptor CMKLR1, which were closely related to obesity and metabolic syndrome, were overexpressed in PCOS rats. Chemerin promoted autophagy by inhibiting PI3K / Akt / mTOR and MAPK signaling pathways. In addition, ATG7 played an important role in normal adipogenesis. Inhibiting autophagy by destroying ATG7 had anti-obesity and insulin sensitization effects . These studies suggested that a variety of autophagy genes played a role in the pathogenesis of PCOS. Autophagy excess and autophagy deficiency would affect the normal physiological activities of the body. Maintaining a coordinated and balanced autophagy state was of great significance for the prevention and treatment of PCOS.
As an important autophagy-related protein, ATG7 is involved in two important ubiquitin-binding systems in the autophagy process. One is involved in the ubiquitin-like reaction between LC3-Ⅰ and phosphatidylethanolamine (PE), which is transformed into fat-soluble LC3-Ⅱ and adsorbed on the autophagosome membrane. The other is involved in the ubiquitin-like reaction between ATG12 and ATG5, forming the ATG12-ATG5 linker and binding with ATG16, which is involved in the formation of preautophagosome structure . It can be seen that ATG7 is an essential molecule in the process of autophagy and plays a major role in the process of autophagy. ATG7 can be used as a marker to reflect the level of autophagy. And autophagy can be regulated by interfering with the expression of ATG7. Previous studies have found that the expression of ATG7 is up-regulated in ovarian GCs of PCOS patients. The purpose of this study was to explore the specific mechanism of ATG7 participating in the pathogenesis of PCOS by regulating autophagy.
In this study, we predicted the microRNA associated with ATG7 by bioinformatics software to further explore the pathogenesis of PCOS. Among the 51 associated microRNAs, miR-17-5P associated with autophagy was selected. MiR-17-5P belongs to the miR-17-92 family and is located on human chromosome 13. It can regulate a variety of autophagy-related genes such as ATG7 and ATMN1, and play a regulatory role in different stages of autophagy. At present, research on miR-17-5P regulating autophagy is mainly concentrated in the field of tumor. A study by Zhang et al.  found that miR-17-5P down-regulated the expression of DEAD-Box Helicase 5(DDX5) and inhibited autophagy. In the liver cancer transplantation model, miR-17-5P inhibitor could promote autophagy flux and inhibited tumor growth. Hou et al. found that miR-17-5P targeted beclin-1 to inhibit autophagy. Increasing the expression of miR-17-5P could reduce autophagy activity and play an important role in the radiosensitivity of glioma cells. Targeting miR-17-5P / beclin-1 autophagy pathway might be an effective way to improve the efficacy of glioma radiotherapy. Knockout of miR-17-5P up-regulated the expression of PTEN and inhibited the proliferation and autophagy of thyroid cancer cells. Finally, it could reduce the malignancy of thyroid cancer and inhibit the inactivation of Akt / mTOR pathway, which provided a new way for the treatment of thyroid cancer .
Dual-luciferase reporter assay confirmed that miR-17-5P was the associated microRNA of ATG7, and miR-17-5P could negatively regulate the expression of ATG7. There was still a lack of research on miR-17-5P / ATG7 mediated autophagy in human ovarian tissue. By detecting the expression levels of miR-17-5P and ATG7 in human ovarian GCs, we found that compared with normal women, the expression of miR-17-5P was lower and the expression of ATG7 was higher in ovarian GCs of PCOS patients. This suggested that miR-17-5P might target ATG7 mediated GCs autophagy and played a role in the pathogenesis of PCOS.
The disorder of GCs autophagy could affect the connection and communication between GCs themselves and between GCs and oocytes. Therefore, maintaining the coordinated balance of GCs autophagy could indirectly affect the growth, development, and maturation of oocytes. In previous studies, it was found that autophagy balance was the key link to promote follicular development, improve endocrine and metabolic disorders and improve endometrial receptivity. Our study found that the autophagy pathway miR-17-5P / ATG7 might be involved in the pathogenesis of PCOS. This discovery provided a new idea for the treatment of PCOS by developing miR-17-5P analogs or short peptides that specifically inhibited the activity of ATG7.
The same miRNA can regulate the expression of multiple genes at the same time. Slight changes in miRNA pattern may have a significant impact on cell function. Constantly enriches the theoretical research of miRNA pathway, which is helpful to explore more therapeutic methods. Therefore, compared with the traditional single-target therapies, miRNA-based therapy may be a promising treatment.
In this study, we found a new possible pathogenesis of PCOS. In the future, this mechanism may be used to guide the exploration of reliable and efficient treatment methods for infertile patients with PCOS. However, this study has some limitations. Only in vitro experiments were carried out, and in vivo animal experiments are needed to further confirm these findings.