DOI: https://doi.org/10.21203/rs.3.rs-20342/v1
Background: Granulosa cells proliferation and estradiol synthesis significantly affect follicular development. The miR-214-3p expression in the ovarian tissues of high-yielding sows is higher than that in low-yielding sows, indicating that miR-214-3p may be involved in sow fertility. However, the functions and mechanisms of miR-214-3p on granulosa cells are unclear. In this study, miR-214-3p was transfected into porcine ovarian granulosa cells to investigate its functions in terms of proliferation and estradiol synthesis via flow cytometry, CCK-8 assay, EdU staining, ElisA, Real-Time PCR, and Western blot analyses. We also identified the targets of miR-214-3p via Luciferase Reporter Assay.
Results: Our findings revealed that miR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine granulosa cells. We also found that miR-214-3p up-regulates the expression of cell cycle genes including Cell cycle protein B (Cyclin B), Cell cycle protein D (Cyclin D), Cell cycle protein E (Cyclin E), and Cyclin-dependent kinase 4 (CDK4) at the transcription and translation levels, while down-regulating the mRNA and protein levels of cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 19 subfamily A member 1 (CYP19A1), and steroidogenic acute regulatory protein (StAR) (i.e., the key enzymes in estradiol synthesis). On-line prediction, bioinformatics analysis, a luciferase reporter assay, RT-qPCR, and Western blot results showed that the target genes of miR-214-3p in proliferation and estradiol synthesis are Mfn2 and NR5A1, respectively.
Conclusions: Our findings suggest that miR-214-3p plays an important role in the functional regulation of porcine granulosa cells and therefore may be a target gene for regulating follicular development.
Granulosa cells, as the largest cell population in mature follicles, are the body’s primary source of estrogen and progesterone. The morphology and function of granulosa cells is altered by primordial follicle growth initiation, proliferation, differentiation, atresia, ovulation, and luteum formation. Granulosa cells also can regulate the development of oocytes and follicles by secreting cytokines and hormones, which further affects female reproductive performance (1, 2). Thus, the proliferation and hormone secretion of granulosa cells are closely related to the growth and development of follicles (3).
Follicle development in the ovary requires recruitment, selection, and dominance processes. The original follicles gradually develop into primary follicles, secondary follicles, antral follicles, and preovulatory follicles (4) accompanied by the transformation of granulosa cells from a monolayer to a cubic shape of 2–3 layers, followed by multiple layers and cavities (5). Follicle growth is, to this effect, inseparable from granulosa cell (GC) proliferation (6).
There are three types of estrogen, the most active of which is estradiol (7). During the synthesis of estradiol, FSH (follicle-stimulating hormone) receptors produced by GCs bind to FSH from the pituitary gland, which activates the FSH signaling pathway and increases the expression of related enzymes (e.g., CYP11A1, a cytochrome P450) while promoting estradiol synthesis (8, 9). FSH can also interact with receptors in the surface membranes of GCs, activate adenylyl cyclase, and subsequently increase intracellular cAMP levels. The expression of aromatase (CYP19A1) corresponds to the increase of E2 secretion. In addition, StAR can transport the cholesterol from the outer to the inner mitochondrial membrane, where it is converted to pregnenolone by CYP11A1. Estradiol promotes the formation of follicles and gonadotropin receptors in the ovary (10, 11), inhibits the apoptosis of GCs (12), facilitates the formation of corpus luteum, and maintains the corpus luteum and regulates steroid synthesis, among other functions.
MicroRNA (miRNA) is a short (20-24nt) non-coding RNA, which mainly binds to the 3’UTR of the target gene’s mRNA sequence to stimulate degradation of mRNA to regulate mRNA expression at the post-transcriptional level and inhibit its translation (13, 14). Many previous studies have demonstrated that miRNA regulates the biological function of GCs by its targets. For example, in mouse GCs, miRNA-746-3p targets steroidogenic factor-1 (SF-1) to regulate 17β-estradiol synthesis (15). MiR-202-5p induces apoptosis in goat GCs by targeting TGFβR2 (16). Another research proved that miR-1275 controls GCs apoptosis and estradiol synthesis by impairing LRH-1/CYP19A1 axis (17). However, certain phenotypes and mechanisms that other miRNAs regulate porcine ovarian GCs proliferation and estradiol synthesis yet merit further research.
MiR-214 is transcribed from Dynamin3 and forms a vertebrate-specific conserved cluster with miR-199 (18, 19). Research on miR-214-3p tends to center on oncology, skeletal muscle development, adipogenesis, and similar applications (20–22). Sequencing results from the ovarian tissue of Yorkshire pigs has shown that miR-214 expression significantly differs between large and small litter sizes (23). Studies have also shown that miR-214 may regulate steroids by targeting low-density lipoprotein receptor genes in rat GCs (24). We used Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses to find that miR-214-3p is enriched in the TGF-beta signaling pathway and mTOR signaling pathway, and further, is involved in the physiological processes of cell proliferation and estradiol synthesis. In short, the literature suggests that miR-214 is involved in the biological functions of GCs. However, the specific effects of miR-214 on GCs remain unclear and are worth further analysis.
In this study, we demonstrated that miR-214-3p can promote the proliferation of porcine ovarian GCs by targeting Mfn2 and can inhibit estradiol synthesis by targeting NR5A1 in the GCs. The results presented here may provide new insight into the mechanisms by which miR-214-3p regulates GCs biological functions.
Landrace ovaries (n = 20) from cyclic sows (Sus scrofa) were obtained immediately after slaughter, soaked in saline solution, and stored at 37 ℃. The ovaries were shipped back to the laboratory within 2 h and were dissected and cleaned in thermostatic saline solution. The antral follicles (3–5 mm diameter) situated on the ovarian surface were punctured by needles to release the follicular fluid and flushed with culture medium DMEM/F12 containing 3% BSA, 1 IU FSH (SHUSHENG, China), and 1 IU LH (SHUSHENG, China) (25). The culture medium with GCs and cumuluse oocyte complexes was filtered through a 70-mm cell strainer. The cumuluse oocyte complexes were filtered out and the filtrate with GCs was centrifuged at 1000 × g for 10 min. The GCs were then suspended with DMEM/F12 containing 3% BSA, inoculated in a cell culture well, and cultured in a cell incubator with 5% CO2 at 37 ℃ (3).
An agomir is a type of specially labeled and chemically modified double-stranded microRNA which can regulate the biological function of a target gene by mimicking endogenous microRNA. An antagomir is a type of specially labeled and chemically modified single-stranded microRNA, designed based on the mature microRNA sequence, which can inhibit the expression of endogenous microRNA. The miR-214-3p agomir, antagomir, and respective nonspecific control (NC) materials used in this study were purchased from GenePharma (Shanghai, China) and were transfected into GCs with X-treme GENE HP DNA Transfection Reagent (Roche, Mannheim, Germany) at a final concentration of 50 nM according to the manufacturer’s protocol. The medium was changed once after 24 h of transfection (22).
Total RNA samples were isolated using Trizol (TakaRa, Otsu, Japan). The final concentrations were measured by NanoDrop 2000 (Thermo, Waltham, MA, USA). The cDNA was synthesized using a reverse transcription kit (TakaRa, Otsu, Japan). We used quantitative real-time PCR (RT-qPCR) for mRNA analysis. Every reaction was performed in triplicate with SYBR Premix (Vazyme, Nanjing, China) on a StepOne Real-Time PCR Machine (ABI, Carlsbad, CA, USA) (26). The relative mRNA level was normalized to that of Gapdh and calculated using the 2−∆∆Ct algorithm. The primer sequences we used for the RT-qPCR are listed in Table 1.
The cell total protein was isolated using RIPA (Applygen Technologies Inc., Beijing, China). Protease inhibitor (CWBIO, Shanghai, China) was added into the RIPA at a ratio of 1:100. After adding RIPA to the cell culture plate, we collected the cells and centrifuged (12,000 rpm) the material at 4 ℃ for 10 min (27). Protein concentrations were measured on a Thermo Scientific Pierce BCA protein assay kit (Thermo Fisher, USA) with 1/4 volume of 5 × loading buffer added to the supernatant. A total of 20 µL of protein was blotted using 10% SDS-polyacrylamide gel, then transferred to a polyethylene difluoride (PVDF) membrane (CST, Boston, MA, USA). After blocking with 5% defatted milk for 2 h, the membranes were incubated overnight at 4 ℃ with antibodies (1:1000) against StAR, CYP19A1, CYP11A1, Mfn2, NR5A1 (Abcam, Cambridge, UK) and against Cyclin B, Cyclin D, Cyclin E, CDK4 (Santa Cruz, TX, USA). The membrane HRP goat anti-mouse IgG, goat anti-rabbit IgG, and rabbit anti-goat IgG secondary antibodies (BOSTER, China) were diluted 1:3000 according to the instructions and incubated for 1 h. Detection was performed using chemiluminescence Western blotting substrate (Santa Cruz, CA, USA) in Image Lab analysis software (Image Lab™, Bio-Rad, Berkeley, CA, USA).
Porcine GCs were cultured in a 6-well culture plate at a density of 4 × 105 per well. The cells were treated with miR-214-3p-agomir or antagomir for 48 h. The cells were digested with 0.25% trypsin and terminated with DMEM containing 10% FBS, then collected and fixed in cold 70% ethanol overnight at 4 ℃ (28). Afterwards, the cells were washed twice and stained with 50 mg/mL propidium iodide (PI) for 30 min. Finally, the cell cycles of the porcine subcutaneous preadipocytes were analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).
GCs were seeded in 96-well plates at a concentration of 2 × 103 per well. The granulosa cells were then treated with miR-214-3p agomir and antagomir for 48 h and incubated with 50 µM EDU (RiboBio, Guangzhou, China) for 2 h. The cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, neutralized with 2 mg/mL glycine for 5 min, then permeabilized with 0.5% Trixon-100 for 5 min. At the end of each step, the cells were washed twice with PBS for 5 min. According to the kit, the cells were incubated in a mixture of Reagents B, C, D, and E for 30 min. The cells were then washed three times with 0.5% Trixon-100, then twice with methanol. The nuclei were stained with Hoechst for 30 min. The stained cells were finally observed on a Nikon TE2000 microscope (Nikon, Tokyo, Japan) and the data were analyzed in Image J.
Porcine GCs were seeded in 96 well plates with 2,000 cells per well. After 48 h of rHhip treatment, 10 mL CCK8 reagent (Vazyme) was added into each well away from light, then the cells were incubated at 37 ℃ for 2–4 h. Finally, the plate absorbance was measured at 450 nm.
Luciferase reporter plasmids (psi-CHECK2) containing the wild-type 3’UTRs of Mfn2/NR5A1(WT-Mfn2/NR5A1) and mutant 3’UTRs of MfnFN2/NR5A1 (Mut-Mfn2/NR5A1) were obtained as manufactured by General Biosystems Co., Ltd. (Anhui, China). HEK293T was seeded in a 48-well plate. X-treme GENE HP DNA Transfection Reagent was used to co-transfect the HEK293T cells with the wild-type or mutant 3’UTR luciferase reporter plasmids (60) and the miR-214-3p agomir or the negative control, respectively. The cells were harvested 24 h after transfection. Luciferase activities were measured on a Dual-Glo Luciferase AssaySystem (Promega; Madison, WI, USA) following the manufacturer’s instructions. Firefly luciferase was used as a normalization control.
E2 existing in the follicular fluid and medium supernatant was detected using a porcine E2 ElisA Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) operated according to the manufacturer’s instructions (tolerance within batch: CV < 10%; tolerance between batches: CV < 12%; sensitivity: 20e6000 ng/L).
We performed a bioinformatics analysis using TargetScan, miRBase and miRTarBase. Many thousands of potential target genes were predicted. The common target gene associated with myogenes was predicted by at least these three programs. We also used KOBAS 3.0 to complete a gene ontology (GO) analysis and the Kyoto encyclopedia of genes and genomes (KEGG) for further analysis.
Statistical analyses were performed in GraphPad Prism 6 software. One-way analysis of variance (ANOVA) and a Newman-Keuls test were used to compare the groups. A paired Student’s test was used for comparison between any two groups. Data are expressed here as the mean ± SE and statistical significance is * = P < 0.05; **= P < 0.01.
We detected the expression level of miR-214-3p in the ovarian tissue of Yorkshire × Landrace sows with high-litter and low-litter characteristics in this study. We observed a higher expression in high-litter sows than in low-litter sows (Fig. 1A). The mature sequence of miR-214-3p is highly conserved across multiple species (e.g., mouse, pig, human, rat) (Fig. 1B). We also performed GO analysis on the target of miR-214-3p to find that it may indeed be involved in follicular growth processes such as cell proliferation and steroid synthesis (Fig. 1C). The TGF-beta and mTOR signaling pathways play important roles in the process of follicular growth. Our KEGG pathway analysis showed that miR-214-3p participates in these signaling pathways (Fig. 1D).
In order to determine the effect of miR-214-3p on the proliferation of porcine ovarian GCs, we transfected the GCs samples with miR-214-3p agomir, antagomir, and the negative control. The expression of miR-214-3p increased significantly after transfection into agomir (Fig. 2A). Flow cytometry analysis indicated that miR-214-3p increased the percentage of S-phage cells (Fig. 2B,C). The EdU staining assay showed that cells were labeled positive in the miR-214-3p agomir group, unlike the negative control group (Fig. 2D,E). The CCK-8 assay also upregulated cell viability (Fig. 2F). In addition, cell cycle-related genes (Cyclin B, Cyclin E, and CDK4) showed remarkably higher mRNA and protein levels but there was no such effect in Cyclin D (Fig. 2G-I).
To further explore the effect of miR-214-3p on GCs proliferation, we next treated the cells with antagomir-NC and antagomir. The expression of miR-214-3p in the treatment group was dramatically reduced below the negative control group (Fig. 3A). The flow cytometry results indicated down-regulation of the S-phage cells after suppressing the expression of miR-214-3p (Fig. 3B,C). Our EdU staining assay showed that inhibition of miR-214-3p can markedly increase the number of EdU labeled positive cells (Fig. 3D,E). Our CCK-8 assay also verified the knock-down of miR-214-3p induced cell viability (Fig. 3F). RT-qPCR and Western blot data showed that miR-214-3p inhibition depressed the expression of cell cycle genes (Fig. 3G-I). In summary, miR-214-3p was found to promote GCs proliferation.
The experiments described above showed that miR-214-3p can promote porcine GCs proliferation (Figs. 2 and 3). To better understand the regulatory mechanism of this process, we used TargetScan7.2 and miRTarBase to predict potential target genes. We detected Mfn2 as a candidate gene from thousands of target genes (Fig. 4A) and constructed wild-type Mfn2 3’UTR and mutant Mfn2 3’UTR dual luciferase reporter vectors accordingly (Fig. 4B). We found that the dual-luciferase activity of wild-type Mfn2 3’UTR and agomir co-transfected into GCs was higher than that of co-transfected wild-type Mfn2 3’UTR and NC, while mutant dual-luciferase activity with NC and agomir appears to have no effect (Fig. 4C). Our RT-qPCR and Western blot data also suggest that Mfn2 mRNA and protein levels were reduced and increased, respectively, in the miR-214-3p-agomir/antagomir groups (Fig. 4D-I). Altogether, our tests demonstrated that miR-214-3p promotes GCs proliferation by directly targeting Mfn2.
One of the most important functions of GCs is the secretion of estradiol. We detected the E2 concentration in our culture medium accordingly. The expression of miR-214-3p increased or decreased sharply after transfection with agomir or antagomir (Figs. 5A and 6A). The ElisA results demonstrated that E2 concentration was markedly down-regulated or up-regulated in different treatment groups (Fig. 5B and 6B). E2 synthesis-related genes including Star, Cyp11a1, and Cyp19a1 were also suppressed in mRNA and protein levels in the agomir group (Fig. 5C-E). The results in the antagomir treatment group were consistent with this (Fig. 6C-E). We infer that miR-214-3p inhibits GCs estradiol synthesis.
NR5A1 is also referred to as “steroidogenic factor 1” and is known to regulate estradiol synthesis by regulating the transcription of Cyp11a1 and Cyp19a1 genes via binding to the nuclear receptor motifs. To explore the mechanism by which miR-214-3p regulates estradiol synthesis, we forecasted the target genes of miR-214-3p with TargetScan7.2 and miRTarBase.
Coincidentally, NR5A1 is one of the candidate target genes of miR-214-3p. This caught our attention over the course of our analysis, so we tested it specifically as a target gene of miR-214-3p (Fig. 7A). Similar to the results reported in Sect. 3.3, we constructed a dual luciferase reporter vector for assay (Fig. 7B); the assay revealed that miR-214-3p markedly suppressed the dual-luciferase activity (Fig. 7C). NR5A1 mRNA and protein levels were also attenuated and both increased in the miR-214-3p agomir/antagomir groups (Fig. 7D-I). These observations suggest that miR-214-3p inhibits GCs estradiol synthesis by targeting NR5A1.
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).
In summary, as shown in Fig. 8, our results show that miR-214-3p promotes GCs proliferation by targeting Mfn2 and inhibits GCs estradiol synthesis by targeting NR5A1. The results presented here may provide workable insight into regulating the GCs functions, and follicular growth, and follicular development.
GCs: granulosa cells; StAR: steroidogenic acute regulatory protein; CYP11A1: cytochrome P450 family 11 subfamily A member 1; CYP19A1: aromatase; Cyclin B: Cell cycle protein B; Cyclin D: Cell cycle protein D; Cyclin E: Cell cycle protein E; CDK4: Cyclin-dependent kinase 4; mmu: Mus musculus; ssc: Sus scrofa; hsa: Homo sapiens; rno: Rattus norvegicus; mml: Macaca mulatta; mdo: Monodelphis domestica; oan: Ornithorhynchus anatinus; tgu: Taeniopygia guttata; aca: Anolis carolinensis
Ethics approval and consent to participate
These studies were approved by Northwest Agriculture and Forestry University Animal Research Ethics Committee (Yangling, Shaanxi, China).
Consent for publication
Not applicable
Availability of data and material
The data sets used and analysed during the current study are available from the corresponding author on reasonable request.
Competing interests
The authors declare they have no competing interest.
Funding
This work was supported by grants from the National Natural Science Foundation (No.31802047), the National Science and Technology Major Project of China (No. 2016ZX08006003) and Shanxi Provincial Key Research and Development Project (CN)(No. 2018ZDXM-NY-035)
Authors’ contribution
SSJ and CGY conceived and designed the experiments; SSJ, ZXG and LJJ performed the experiments; HYM and ZLT contributed reagents/materials/analysis tools; YGS managed the project; SSJ wrote the manuscript and CGY modified the manuscript.
Acknowledgements
The authors acknowledged all the teachers and students in Laboratory of Animal Fat Deposition & Muscle Development.
Authors' information
1Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, Yangling 712100, China
2Laboratory of Animal Fat Deposition & Muscle Development, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
Due to technical limitations, Table 1 is provided in the Supplementary Files section.