MiR-23a was downregulated in serum of PCOS patients
Peripheral blood was collected from 50 local PCOS patients for the detection of miR-23a level with 50 healthy women's peripheral blood as the control. Clinical information on age, BMI and sex hormone levels of PCOS patients and normal control samples are all listed in Table 1. As shown in Fig. 1A, the serum miR-23a level in PCOS patients was significantly lower than that in the control group (P < 0.001). then, we detected the level of miR-27a and miR-24-2 using qPCR. As shown in Fig. 1B, miR-27a and miR-24-2 also downregulated in peripheral blood of PCOS patients compared with healthy sample.
The correlation between the expression of miR-23a and clinical index of PCOS patients
We further analyzed the correlation between the expression of miR-23a and clinical index. As shown in Table 1, the BMI of PCOS patients was significantly higher than that of healthy controls (P < 0.001). The correlation analysis showed that there was a positive correlation between serum miR-23a level and BMI in PCOS patients (Fig. 1B, P = 0.0199, r = 0.3285), but no correlation was found in healthy control group (Fig. 1C, P = 0.8632, r = 0.02499). As shown in Table 3, the serum LH concentration in PCOS patients was 9.35 ±1.77 mIU/mL, which was significantly higher than that in healthy women (7.67 ± 1.80 mIU/mL) (P < 0.001). Furthermore, there was a negative correlation between serum miR-23a level and LH concentration in PCOS patients (Fig. 1D, P = 0.0088, r = 0.3665), but no correlation was found in healthy control group (Fig. 1E, P = 0.3210, r = 0.1432). The serum miR-23a level was also negative correlated with GLU (Fig. 1G, P = 0.0215, r = 0.3245), INS (Fig. 1I, P = 0.0013, r = 0.4414) and T (Fig. 1K, P = 0.0110, r = 0.3569) concentration in PCOS patients, but not in healthy control group (GLU: Fig. 1H, P = 0.9361, r = 0.0116; INS: Fig. 1J, P = 0.0678, r = 0.2604; and T, Fig. 1L, P = 0.7091, r = 0.0541).
MiR-23a inhibits the proliferation of human ovarian granulosa cells
In this study, the expression of miR-23a in three human granulosa cell lines was detected by qPCR. As shown in Fig. 2A, the expression level of miR-23a was lowest in cov434 cells and highest in KGN cells. Therefore, we chose cov434 cell line for subsequent experiments. Subsequently, miR-23a-specific-siRNA or mimic was transfected into cov434 cells to explore the role of miR-23a in human ovarian granulosa cells. As shown in Fig. 2B, the expression of miR-23a in cells was significantly increased by the transfection of miR-23a mimic (P < 0.001). Similarly, the expression of miR-23a in cells was significantly knocked down by the transfection of miR-23a inhibitor (Fig. 2C) (P < 0.05).
Then, CCK8 assay was performed to detect the effect of miR-23a on the proliferation of cov434 cells. As shown in Fig. 2D, compared with the control group, the transfection of miR-23a mimic significantly inhibited the proliferation of cov434 cells (P < 0.05); on the contrary, the transfection of miR-23a inhibitor significantly promoted the proliferation of cov434 cells (P < 0.05). These data proved that the expression level of miR-23a was involved in the regulation of cov434 cell proliferation.
MiR-23a induced cell cycle arrest on G0/G1 phase of cov434 cells
Next, flow cytometry was used to detect the effect of miR-23a on the cell cycle of cov434. As shown in Fig. 3, cells stagnated in G0/G1 phase after transfection of miR-23a mimic (P < 0.05), and the proportion of cells in S phase and G2/M phase decreased significantly (P < 0.05). The results were consistent with the inhibition of cell proliferation by over-expression of miR-23a, suggesting that miR-23a induced cell cycle arrest and thus inhibit cell proliferation in cov434 cells. On the contrary, the proportion of G2/M phase cells increased significantly in the miR-23a inhibitor group(P < 0.05), while that of G0/G1 and S phase cells decreased (P < 0.05). The results showed that low expression of miR-23a promoted cell cycle progression and thus cell proliferation.
MiR-23a promotes apoptosis of cov434 cells
Flow cytometry was performed to detect the effect of the expression of miR-23a on the apoptosis of cov434 cells. As shown in Figure 4, apoptotic cells increased significantly (P < 0.05) after the transfection of miR-23a mimic, and decreased significantly (P < 0.05) after the transfection of miR-23a inhibitor. These results suggested that overexpression of miR-23a promoted apoptosis, while low expression of miR-23a inhibited apoptosis.
FGD4 is the bind target of miR-23a in cov434 cells
Then, we predicted six novel potential target of miR-23a via the analysis on bioinformatics software Target Scan. Subsequently, the results of double luciferase reporter assay proved that only FGD4 could bind to miR-23a directly through predicted sites. The binding sites are shown in Fig. 5A. Co-transfection of miR-23a mimic inhibited the luciferase activity of FGD4-WT plasmid (P < 0.01), but had no effect on the luciferase activity of FGD4-Mut plasmid (Fig. 5B). The results showed that miR-23a and FGD4 bind directly through predictive sites.
The effect of miR-23a on the expression of FGD4 in cov434 cells was investigated using qPCR and western blot. As shown in Fig. 6A, the expression of FGD4 was significantly decreased by the transfection of miR-23a mimic (P < 0.01), whereas the transfection of miR-23a inhibitor significantly increased the mRNA expression of FGD4 in cov434 cells (P < 0.05). As shown in Fig. 6B and 6C, the protein level of FGD4 was significantly decreased by the transfection of miR-23a mimic (P < 0.01), whereas the protein level of FGD4 was significantly increased by miR-23a inhibitor (P < 0.05). Combining with the double Luciferase Report experiment, these results indicated that miR-23a physically bind to the 3'-UTR region of FGD4, thereby regulating the level of FGD4 in cov434 cells.
MiR-23a induces the activation of CDC42/PAK-1 signaling pathway in cov434 cells
CDC42 is a member of the Rho GTPase protein family. FGD4 is responsible for activating CDC42 through GTP exchange of GDP. PAK-1, a serine/threonine kinase, was initially identified as a protein interacting with CDC42 [19]. CDC42/PAK-1 signaling pathway involved in the regulation of cell proliferation, apoptosis and cell cycle [19]. As shown in Fig. 6D, the protein expression of activated CDC42 (GTP bround) was significantly increased by the transfection of miR-23a mimic (P < 0.01), and significantly decreased by the transfection of miR-23a inhibitor (P < 0.05). The effect of miR-23a on the expression of p-PAK-1 protein was similar to that of CDC42 protein (Fig. 6F).