In the present study, we found that CSF1 is a target gene of hsa-miR-423-5p. Moreover, we demonstrated that hsa-miRNA-423-5p could influence cycle proliferation of KGN cells, and this effect may be achieved by affecting the expression of CDKN1A. Through measuring the E2 concentration of cell culture medium, we show that the secretion of E2 in KGN cells affected by hsa-miR-423-5p. These results suggest that the altered expression of hsa-miR-423-5p and its target gene CSF1 in ovarian granulosa cells maybe the reason of high ovarian response to exogenous gonadotropins.
Previous studies have shown that CSF1 is a target gene for a variety of miRNAs. For example, miR-1207-5p targeting CSF1 inhibits the implantation and metastasis of lung cancer [39]; miR-148b targets CSF1 and other genes that inhibit the development of breast cancer [40]; miR-142-3p targets CSF1 to induce the conversion of monocytes to macrophages [41]; and miR-214 targets CSF1 to regulate the proliferation, invasion, and migration of gastric cancer cells [42]. To date, there have been no reports of miR-423-5p targeting CSF1, as we have shown in this study. We confirmed that CSF1 is a target gene of hsa-miR-423-5p for the first time.
Cytokines are small proteins that bind to receptors on the surfaces of cell membranes. They are involved in promoting cell growth and in regulating immune responses, as well as having a role in inflammatory reactions. Studies have shown that cytokines play important roles in follicular growth and embryonic development. Ovarian granulosa cells secrete a variety of cytokines to promote follicular growth, ovulation, hormone synthesis, and secretion [43-45]. Cytokine expression levels are also closely related to the maintenance of normal follicular development and to steroid hormone secretion in the ovary [46]. Colony stimulating factor 1 is an important cytokine that mainly acts on mononuclear macrophage cell lines, where it is involved in regulating their proliferation, differentiation, and function [47]. Previous studies have shown that CSF1 is also key to the regulation of female reproductive function. In this study, we showed that CSF1 was a target gene of miR-423-5p and that CSF1 most likely regulated ovarian reactivity to control ovarian stimulation by affecting the cell cycle and hormone secretion.
Colony stimulating factor 1 secreted by ovarian granulosa cells through autocrine or paracrine routes. Once released, it binds to cell surface receptors and affects intracellular metabolism, oocyte meiosis, and follicular growth and maturation. As early as 1995, Nishimura et al. [48] first reported that CSF1 affected follicular development and ovulation in rats. A significant increase in the ovulation rate was observed in female rats after CSF1 treatment. Later Araki et al. [49] found that the number of ovulations, antral follicles, and mature follicles were lower in osteopetrotic (op/op) mutant mice when compared with normal litters. The op/op mice lack the coding region for the CSF1 gene and are completely devoid of CSF1. Moreover, the number of granulosa cells and the proliferative capacity of antral follicles were also reduced in the op/op mice. When the researchers injected supplementary CSF1 into the op/op mice, the numbers of antral follicles, mature follicles, and granulosa cells around the follicle increased. Therefore, they concluded that CSF1 promoted ovarian granulosa cell proliferation and that it participated in regulating follicular production and ovulation. Cohen et al. [50] also observed a decrease in the ovulation rate of op/op mice. In 1997, researchers showed that CSF1 and its mRNA were expressed in human follicular fluid, and confirmed for the first time that CSF1 was involved in regulating human follicular development [51]. Soon after, Nishimura et al. [52] observed that the concentration of CSF1 in the serum increased gradually with the duration of COS, peaking between the egg retrieval day and 2 days later. Moreover, the concentration of CSF1 in the follicular fluid was significantly higher than that in the serum on the day of egg retrieval, and the concentration of CSF1 in the follicular fluid containing the ovum was higher than in that of the empty follicle. This study suggested that CSF1 may be involved in regulating egg maturation and ovulation. In other research, it was reported that the concentration of CSF1 in the follicular fluid was significantly higher than that in the serum. Furthermore, the expressions of CSF1 and its receptor were detected in isolated and cultured human luteinized granulosa cells, confirming that CSF1 is a key factor in follicular development [53].
The concentration of CSF1 in serum or follicular fluid is associated with ovarian response. Salmassi et al. [54] observed that there were difference in serum CSF1 levels depending on ovarian response during COS. They found that the higher a ovarian response with greater egg production was associated with a higher serum CSF1 concentration. Lei Huo [55] obtained similar results in patients undergoing IVF treatment, showing that the CSF1 concentration in the follicular fluid on the day human chorionic gonadotropin was given had a positive correlation with the number of eggs obtained. Thus, it is believed that CSF1 can affect ovarian reactivity and is related to egg maturation.
In our previous study [2], bioinformatics analysis of miRNAs with altered expression in ovarian hyperresponders revealed that the target genes of differential miRNAs were enriched in pathways such as cell-cycle regulation. In this study, we found that increased hsa-miR-423-5p can cause a large number of KGN cells to arrest in the G0/G1 phase, with the number of cells in S phase increased when hsa-miR-423-5p expression was inhibited. Although the percentage of S phase cells was no statistical difference between miR-423-5p overexpression group and the negative control, it showed a downward tendency in the miR-423-5p overexpression group compared to the control. This result indicates that the expression level of hsa-miR-423-5p in KGN cells does affect the cell cycle, which is consistent with our previous study and confirms that hsa-miR-423-5p is involved in the proliferation of KGN cells.
To further clarify the mechanism through which hsa-miR-423-5p regulated the cell cycle, we examined the expression of CDKN1A protein. Cyclin-dependent kinase inhibitor 1A is a negative regulator of the cell cycle that inhibits the activity of Cyclin-dependent kinase (CDK) and blocks cells in the G1 phase, thereby inhibiting cell proliferation [56]. When the hsa-miR-423-5p expression was increased, CDKN1A protein expression was upregulated. Conversely, when hsa-miR-423-5p expression was decreased, CDKN1A protein expression was downregulated. Thus, it appears that hsa-miR-423-5p may affect CDK expression and induce cell arrest in the G0/G1 phase, thereby affecting cell proliferation. In patients with high ovarian response to COS, the decreased expression of hsa-miR-423-5p may lead to decreased inhibition of CSF1 and increased number of S phase cells. In turn, this could result in an abnormal high level of CSF1 in granulosa cells, causing the excessive proliferation of granulosa cells and the development of multiple follicles. This may explain cases of high ovarian response to COS. However, compared with the control group, the percentage of G0/G1 phase cells in miR-423-5p inhibitor group was not significantly reduced. According to our analysis, the possible reason is that the sample size is too small to obtain statistically significant differences in both the S and G0/G1 phases. More samples are required to fully profile the cell proliferation differences between the cells with different miR-423-5p expression in the future.
It is known that high levels of E2 are usually associated with high ovarian response in patients [57]. In this study, the E2 concentration was measured in the culture medium of KGN cells: concentrations decreased when hsa-miR-423-5p expression increased and concentrations increased when intracellular hsa-miR-423-5p expression decreased. Study have shown that CSF1 can help FSH by promoting E2 secretion from ovarian granulosa cells and upregulating FSH receptor expression [58]. Therefore, we hypothesized that hsa-miR-423-5p overexpression inhibited CSF1 expression and led to a decrease in E2 secretion by KGN cells. This effect on hormone secretion was attenuated after hsa-miR-423-5p was inhibited. Downregulation of hsa-miR-423-5p in patients with high ovarian response to COS may lead to an increase in the CSF1 concentration in granulosa cells, thereby stimulating excessive secretion of E2 by cells.
Based on these findings, we can state that hsa-miR-423-5p may regulate the proliferation of ovarian granulosa cells and the secretion of E2 within a proper range. It appears to do so by negatively regulating the expression of its target gene, CSF1, in ovarian granulosa cells. This is characterized by a normal ovarian response. In patients with a high ovarian response, however, hsa-miR-423-5p expression appears to be downregulated, such that the inhibition of CSF1 is weakened. In turn, this may lead to excessive CSF1 secretion, excessive granulosa cell proliferation, abnormal sensitivity to exogenous gonadotropins, simultaneous development of a large number of follicles being promoted, and abnormal E2 elevations. Together, these result in a high ovarian response.
To the best of our knowledge, this is the first study to have linked the expression of granulosa cell miRNAs to the secretion of CSF1. We found an upstream regulatory factor that causes changes in CSF1 concentrations in granulosa cells, thereby adding to current knowledge of the factors associated with ovarian reactivity. However, in this study, we only conducted a preliminary exploration of the mechanism through which miR-423-5p and its target gene, CSF1, regulate ovarian response. Further research is therefore needed to identify the signal pathway through which the miR-423-5p targeting of CSF1 regulates ovarian response. In addition, we will try to find suitable targeted inhibitors affecting miR-423-5p/CSF1 pathway in vitro and in vivo to avoid high ovarian response as much as possible. If the research is successful, it may help prevent ovarian hyperstimulation syndrome (OHSS).