In this study, we found that miR-214-3p plays an important role in GCs proliferation and estradiol synthesis. Specifically, miR-214-3p agomir promotes GCs proliferation and inhibits estradiol synthesis while miR-214-3p antagomir inhibits proliferation and promotes estradiol synthesis. Our findings may provide workable information regarding the regulation of GCs function by miR-214-3p.
GCs function has become a popular research topic in recent years as it concerns follicle growth, follicle, development, and female reproductive disorders. Ovarian GCs, as the main somatic cells in the follicle, play a significant role in the growth and development of follicles, atresia, oocyte maturation, and ovulation (30). GCs functions such as proliferation and estradiol synthesis are affected by many regulatory factors (31–33). miRNA plays an important part in these processes (34, 35). The results of this study suggest that miR-214-3p is highly conserved among species (Fig. 1B), which is consistent with previously published observations (22).
miR-214-3p is expressed to greatest extent in porcine ovarian tissue among other body tissues (24). Previous research has shown that miR-214-3p regulates the proliferation of breast cancer cells by targeting survivin protein (36) and can promote smooth muscle cell proliferation (37), which indicates that miR-214-3p is indeed involved in cell proliferation processes. We transfected miR-214-3p agomir and antagomir into porcine ovarian GCs in this study to explore the effects of miR-214-3p on GCs proliferation. We found that miR-214-3p promotes proliferation by upregulating the mRNA and protein levels of Cyclin B, Cyclin D, Cyclin E, and CDK4 (Figs. 2G-I and 3G-I).
We also used flow cytometry to detect the percentages of cells in the S phase and found that miR-214-3p agomir and antagomir promoted and inhibited them, respectively (Figs. 2B,C and 3B,C). Our EdU staining and CCK-8 assays also showed that miR-214-3p upregulated EdU labeled positive cells and cell viability (Figs. 2D-F and 3D-F). Cyclin B is a marker of immunohistochemical proliferation (37) and CDK4 is a kinase that regulates the transition from the G1 to S phases of the cell cycle (39). We found that due to miR-214-3p agomir and antagomir, compared to our NC, Cyclin B and CDK4 had the most significant differential expression of mRNA and protein levels.
It is commonly known that miRNA binds to the 3’UTR region of the target genes to inhibit their transcription or translation (13). Here, Mfn2 was used as a target gene of miR-214-3p as it not only reduces the Ras signaling pathway protein but also is a proliferation inhibitor. It can limit the expression of Cyclin D protein to inhibit the proliferation process (40). Feng et al. reported that miR-93 regulates vascular smooth muscle cell proliferation by targeting Mfn2 (41). Additionally, miR-497 promotes cardiomyocyte proliferation by downregulating the expression of Mfn2 (42).
There have been relatively few previous studies on the relationship between miR-214 and Mfn2 in cell proliferation processes. miR-214 mediates proliferation via inhibition of Mfn2 in cardiac fibroblasts (43) – a process that is relevant to Huntington’s disease (44). In the present study, we found that miR-214-3p can repress the mRNA and protein levels of Mfn2 (Fig. 4D-I). Via dual-luciferase reporter assay, we also found that Mfn2 is a direct target gene of miR-214-3p in GCs (Fig. 4C).
Interestingly, we found that although miR-214-3p promoted GCs proliferation, it also inhibited the synthesis of estradiol. Similarly, addition of 150, 300, or 500 microM of stearicacid inhibits cell proliferation but stimulates estradiol-17beta production in bovine GCs (45). In human GCs, proanthocyanidin B2 can increase steroidogenesis without affecting cell proliferation (46).
Our ElisA data showed that miR-214-3p agomir reduced the concentration of E2, while miR-214-3p antagomir enhanced the concentration of E2 (Figs. 5B and 6B). During the synthesis of E2, StAR can transport cholesterol from the outer to the inner mitochondrial membrane, where it is converted to pregnenolone by CYP11A1 (47). Aromatase (CYP19A1) in GCs transforms testosterone into estradiol (47, 48). We found that miR-214-3p attenuated the transcription and translation levels of Star, Cyp11a1, and Cyp19a1 (Figs. 5C-E and 6C-E). There have been no such results regarding the synthesis of estradiol by miR-214-3p published previously.
In order to further study the molecular mechanism of miR-214-3p regulating E2 synthesis in GCs, we selected NR5A1 as the target gene and performed a dual luciferase reporter assay, RT-PCR, and Western blot experiments. Our results proved that miR-214-3p attenuates the mRNA and protein levels of NR5A1 (Fig. 7D-I), which suggested that NR5A1 may be a target gene of miR-214-3p in GCs.
Our double luciferase reporter assay also showed that miR-214-3p agomir attenuated luciferase activity, which indicates that NR5A1 is the direct target gene of miR-214-3p (Fig. 7C). NR5A1 is also, as mentioned above, the orphan receptor Steroidogenic Factor-1 (SF-1). A member of the nuclear receptor superfamily, is present in fetal and adult steroidogenic tissues and participates in the regulation of ovarian function (49). NR5A1 plays an important role in E2 synthesis. It can bind to SF-1 response elements on the promoter of target genes such as Star, Cyp11a1, and Cyp19a1 to regulate their transcription processes (50, 51).
It is worth noting that many previous researchers have reached conclusions consistent with ours. For example, in mouse ovaries, miR-320 and miR-764-3p regulate estradiol synthesis by targeting SF-1 (52–54).