MiR-26a promotes apoptosis of porcine granulosa cells by targeting the 3β-hydroxysteroid-Δ24-reductase gene

Objective Apoptosis of ovarian granulosa cells (GCs) affects mammalian follicular development and fecundity. This study aimed to explore the regulatory relationship between microRNA-26a (miR-26a) and the 3β-hydroxysteroid-Δ24-reductase gene (DHCR24) gene in porcine follicular granular cells (pGCs), and to provide empirical data for the development of methods to improve the reproductive capacity of pigs. Methods The pGCs were transfected with miR-26a mimic, miR-26a inhibitor and DHCR24-siRNA in vitro. The cell apoptosis rate of pGCs was detected by the flow cytometry. The secretion levels of estradiol (E2) and progesterone (P) in pGCs were detected by enzyme-linked immunosorbent assay. Double luciferase validation system was used to detect the binding sites between miR-26a and DHCR24 3′-UTR region. Qualitative real-time polymerase chain reaction and Western blotting were used to verify the DHCR24 mRNA and protein expression in pGCs, respectively, after transfecting with miR-26a mimic and miR-26a inhibitor. Results Results showed that enhancement of miR-26a promoted apoptosis, and inhibited E2 and P secretion in pGCs. Meanwhile, inhibition of DHCR24 also upregulated the Caspase-3 expression, reduced the BCL-2 expression, promoted pGCs apoptosis, and inhibited E2 and P secretion in pGCs. There were the binding sites of miR-26a located within DHCR24 3′-UTR. Up-regulation of miR-26a inhibited DHCR24 mRNA and protein expression in pGCs. Conclusion This study demonstrates that miR-26a can promote cell apoptosis and inhibit E2 and P secretion by inhibiting the expression of DHCR24 in pGCs.


INTRODUCTION
The ovary is an important reproductive organ in mammals, and ovulation and hormonal regulation are closely related to reproductive performance [1,2]. The estrous cycle of sow is 21 days on average and can be divided into follicular phase and luteal phase based on ovarian morphological changes and hormone secretion [3]. Apoptosis of porcine ovarian granulosa cells (pGCs) is a physiological phenomenon that occurs during the transition from follicular phase to luteal phase in porcine ovaries, and the percentage of apoptotic granulosa cells (GCs) increases significantly as follicular atresia progress. Previous studies have indicated that miRNAs play important roles in the development of GCs. For example, miR23 and miR27a could promote apoptosis of human ovarian GCs [4]. MiR22 could inhibit apop tosis of mouse ovarian GCs by targeting the sirtuin 1 gene [5]. Liu et al [6] reported that the hyaluronan synthase 2 (HAS2) gene is a direct target of miR26b in pGCs, and miR26b positively regulates pGCs apoptosis via a HAS2HACD44 Caspase3 pathway by targeting the HAS2 gene. MiR26a has the same seed sequence with the miR26b and has been re ported as a tumor suppressor in liver cancer cells [7,8], osteosarcoma cells [9] and papillary thyroid cancer cells [10]. Studies have also shown that miR26a regulates the os teogenic differentiation of bone marrow mesenchymal stem cells [11], the proliferation of mouse hepatocyte [12], the apoptosis of endothelial cells [13] and the autophagy of swine Sertoli cells [14]. However, the regulation mechanism of miR26a in ovarian function and follicular development is still rarely reported. Only one research found that miR 26a/b might play a significant role in follicular development by targeting the mothers against decapentaplegic homolog 2 (SMAD2) gene [15]. Our previous researches indicated that the 3βhydroxysteroidΔ24reductase (DHCR24), which en codes for the 24dehydrocholesterol reductase protein, was a candidate target gene of miR26a in porcine ovaries [16,17]. The DHCR24 gene is the final catalytic enzyme involved in cholesterol synthesis, which catalyzes the reduction of strep tavidin to cholesterol [18]. The DHCR24 gene also may play an important role in stress signaling pathways and apoptosis [1921]. Therefore, in this study, we studied the effects of abnormal expression of miR26a and DHCR24 on apoptosis and hormone secretion of pGCs and elucidated the regulatory relationship between miR26a and DHCR24. The results show that miR26a promotes apoptosis of pGCs by targeting the 3′UTR of DHCR24 and regulates the post transcriptional expression of DHCR24. The empirical data reported in this paper can provide genetic information for elucidating the apoptotic mechanism of ovarian GCs.

Ethics statement
Experimental pigs were allowed access to feed and water ad libitum under normal condition and were sacrificed humanely to minimize suffering. All experimental procedures and sam ple collection were approved by the Institutional Animal Care and Use Committee of Anhui Agricultural University, Anhui, China under permit No. 20160523.

Cell culture
Fresh porcine ovaries were obtained from a commercial slaugh ter house and transported back to the laboratory within 1 h. The pGCs were collected from porcine ovarian follicles (3 to 6 mm diameter). The cells were seeded into a 60 mm dish and cultured at 37°C and 5% CO 2 in Dulbecco's modified eagle medium (DMEM)/F12 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA), 100 units/mL penicillin, and 100 mg/mL streptomycin (Gibco, USA). The 293T cells were incubated at 37°C and 5% CO 2 in DMEM containing 10% FBS.

Oligonucleotide transfection
The pGCs were collected at 48 h after transfection. pGCs were transfected with miR26a mimic, nontargeting control oli gonucleotide (NC mimic), miR26a inhibitor, nontargeting inhibitor oligonucleotide (NC inhibitor), DHCR24siRNA and NCsiRNA. These oligonucleotide sequences were de signed based on the porcine miR26a mature sequence in the miRBase database (http://www.mirbase.org) and the DHCR24 sequence in the GenBank database (https://www.ncbi.nlm. nih.gov/genbank) and were synthesized from Ribobio Co. Ltd (Ribobio, Guangzhou, China) ( Table 1). Transfection was performed using Lipofectamine 3000 reagent (Invitrogen, Waltham, MA, USA). Briefly, pGCs were seeded in 12well or 6well plates at 1 d prior to transfection. When the cells reached 60% to 70% coverage of one well, miRNAs and siRNAs were transfected into the cells at different final concentra tions. The final concentrations of miR26a mimic, NC mimic, DHCR24siRNA, and NCsiRNA were 100 nM. The final concentrations of miR26a inhibitor and NCinhibitor were 200 nM. All experiments were performed in triplicate.

Quantitative real-time polymerase chain reaction
Total RNA was extracted by RNA extraction kit (OMEGA, Norcross, GA, USA) and then reverse transcribed using a TransScript Green miRNA FirstStrand cDNA Synthesis Su perMix kit (TransGen Biotech, Beijing, China) for miRNA and a TransScript OneStep DNA Removal and cDNA Syn thesis SuperMix kit (TransGen Biotech, China) for mRNAs. Quantitative realtime polymerase chain reaction (qPCR) was performed using SYBR Premix Ex Taq (TaKaRa, Osaka, Japan) and a CFX96 realtime PCR Detection System (BioRad, Hercules, CA, USA), following the manufacturer' s instructions. Relative gene expression values were determined using the 2 -ΔΔCt method. The primers used are listed in Table 2. U6 small nuclear RNA and βactin were used as endogenous internal controls for miRNA and mRNA expression, respec tively. Briefly, a 10% SDSPAGE gel was prepared and 20 μg of pro tein was loaded per sample. After electrophoresis for 1 h, the proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked in 5% nonfat milk and then incubated at 4°C overnight with a diluted (1:500) monoclonal antiDHCR24 antibody (Bioss, Beijing, China) or antiβactin antibody (as an internal loading control) (Bioss, China), followed by in cubation with secondary antibody (1:2,000) for 2 h at room temperature. The specific complexes were visualized using the SuperSignal West Pico chemiluminescent substrate. Densito metric analysis was performed to quantify the signal intensity.

Apoptosis analysis
After cells were transfected and incubated for 48 h, cells were dissociated with trypsin and resuspended in 500 μL binding buffer containing 5 μL annexin Vfluorescein isothiocyanate and 10 μL propidium iodide (Bestbio, Shanghai, China). The counts of stained cells were determined using a FACSCalibur flow cytometry instrument (BD Biosciences, Franklin Lakes, NJ, USA). All experiments were performed at least three times.

Enzyme-linked immunosorbent assay
After 48 hours transfection, pGC culture medium containing 10% FBS was collected by centrifugation at 2,000×g for 20 min to measure estradiol (E2) and progesterone (P) concentrations using pig E2 and P ELISA kits (Ji Yin Mei, Wuhan, China), re spectively, according to manufacturer's instructions.

Statistical analyses
Differential analysis was performed using IBM SPSS Statistics v20.0 (SPSS Inc., Chicago, IL, USA). Unpaired twosided Student's ttests and oneway analysis of variance tests were used to evaluate the significance of the statistics. Statistical significance is defined when p values are less than 0.05.

MiR-26a promotes apoptosis and decreases E2 and P secretion in porcine ovarian granulosa cells
To determine whether miR26a plays a role in controlling apoptosis in pGCs, miR26a mimic and miR26a inhibitor were transfected into cultured pGCs. Apoptosis was evaluated in transfected pGCs using annexin V FITC/PI staining and flow cytometry analysis. Compared with NC mimic and NC inhibitor, the expression of miR26a was significantly upreg ulated and downregulated after transfecting with miR26a mimic and miR26a inhibitor, respectively (p<0.01) ( Figure  1A). Cell apoptosis was significantly higher in pGCs trans fected with miR26a mimic than in pGCs transfected with NC mimic (p<0.01). Likewise, apoptosis in pGCs transfected with miR26a inhibitor was significantly lower than pGCs transfected with NC inhibitor (p<0.05) ( Figure 1B). These results indicate that miR26a promotes apoptosis and is a proapoptotic factor in pGCs. Meanwhile, miR26a mimic significantly decreased E2 and P release, and miR26a in hibitor significantly promoted E2 and P release in cultured pGCs ( Figure 1C, 1D).

Inhibition of DHCR24 induces apoptosis and decreases E2 and P secretion in porcine ovarian granulosa cells
To explore the function of DHCR24 in apoptosis of pGCs, RNA interference was used to inhibit DHCR24 expression in pGCs cultured in vitro. Successful knockdown of DHCR24 confirmed by qPCR analysis; compared with pGCs transfect ed with the NCsiRNA, those transfected with the DHCR24 siRNA had significantly lower DHCR24 mRNA expression (p<0.05) (Figure 2A). Consistent with the mRNA expression, the expression level of DHCR24 protein was also downreg ulated significantly as a result of the specific siRNA treatment (p<0.01) ( Figure 2B). Flow cytometry analysis revealed that the rate of apoptosis in the DHCR24siRNA group was signifi cantly higher than in the NCsiRNA group (p<0.01) ( Figure  2C). In addition, inhibition of DHCR24 expression reduces the secretion of E2 and P in pGCs ( Figure 2D). Compared with the NCsiRNA group, the expression level of the pro apoptotic Caspase3 gene was increased significantly in the DHCR24siRNA group (p<0.05), while that of the antiapop totic BCL2 gene was not changed ( Figure 2E). These results indicate that inhibition of DHCR24 expression enhances apoptosis of pGCs in porcine ovaries.

DHCR24 is a direct target of miR-26a
To determine whether miR26a is able to regulate DHCR24 gene expression, the putative miR26a target sites in the porcine DHCR24 3′UTR were cloned downstream of the luciferase gene in the pmirGLO dualluciferase reporter vector to gen erate pmirGLODHCR243′UTR ( Figure 3A). 293T cells were transiently cotransfected with the reporter plasmid and with miR26a mimic or NC mimic oligos. Overexpression of exogenous miR26a repressed the activity of the luciferase reporter fused to the DHCR24 3′UTR (p<0.01) ( Figure 3B), however, the luciferase activity was not altered significantly when the cells were cotransfected with the miR26a mimic and a DHCR24 reporter 3′UTR construct containing a mu tation in the putative miR26a binding site ( Figure 3C). These results indicate that miR26a can regulate the expression of the porcine DHCR24 gene by binding to conserved sites in the DHCR24 3′UTR.

MiR-26a inhibits DHCR24 expression in porcine ovarian granulosa cells
To confirm that miR26a promotes apoptosis in pGCs by targeting the DHCR24 gene, the DHCR24 mRNA and protein levels were measured after transfection of cultured pGCs with miR26a mimic and miR26a inhibitor. The qPCR analysis revealed that DHCR24 mRNA expression was significantly lower in the pGCs transfected with the miR26a mimic than with the NC mimic (p<0.01) ( Figure 4A). Expression of DHCR24 mRNA in pGCs transfected with miR26a inhibi tor was significantly higher than those transfected with the NC inhibitor (p<0.05) ( Figure 4A). Meanwhile, trends in DHCR24 protein expression were consistent with changes in DHCR24 mRNA expression ( Figure 4B). These results suggest that miR26a accelerates apoptosis in pGCs by in hibiting the mRNA and protein expression of DHCR24.

DISCUSSION
The mammalian ovary is a dynamic organ. Follicular re cruitment, selection and ovulation coordination, and timely development are essential for functional ovaries and fertility [22]. With the apoptosis of ovarian GCs, follicular atresia gradually occurred. Previous studies have shown that apop tosis of ovarian GCs is the direct cause of follicular atresia [23], and once follicles enter the atresia process, it will be irreversible [24]. In recent years, the regulation of miRNAs in mammalian ovarian development has attracted much attention. miRNA can affect apoptosis of ovarian GCs, oocyte development, and hormone secretion, which are closely associated with mam malian reproductive traits [25,26]. MiR26a shares the same seed sequence as miR26b, which has also been reported to be involved in the regulation of animal reproduction. MiR26b can regulate the apoptosis of porcine follicular GCs by tar geting the Smad4 gene [27]; likewise, miR26a/b can target the Smad2 gene and regulate bovine follicular development [15]. MiR26a plays a role in regulating cell proliferation and apoptosis in embryonic stem cells of dairy goats by directly regulating the phosphatase and tensin homolog gene, and miR26a can indirectly regulate the PI3K/AKT pathway in endometrial epithelium cells [28]. However, studies on miR 26a in pGCs have not been reported. In this study, we found that transfection of miR26a mimics promoted apoptosis of pGCs, and inhibition of miR26a expression suppressed apop tosis of pGCs. These results are consistent with the observed functions of miR26a in other cells. These results suggest that miR26a is a proapoptotic factor in pGCs.
The DHCR24 gene encodes for the 24dehydrocholesterol reductase protein, which is the final catalytic enzyme involved in cholesterol synthesis, which catalyzes the reduction of strep tavidin to cholesterol [18]. Previous studies have shown that DHCR24 plays an important role in stress signaling pathways and apoptosis. Upregulation of DHCR24 protein expression can inhibit Caspase3 initiation during cellular stress respons es, thereby acting as an antiapoptotic agent and displaying neuroprotective effects [29]. Overexpression of DHCR24 in neurons cells has antiapoptotic effects and counters oxida tive stress by scavenging free radicals [30]. Previous studies also showed that 17β estradiol can promote DHCR24 ex pression and can increase intracellular cholesterol content, which protects neurons cultured in vitro [31]; however, this protection disappears after knocking out DHCR24, indicat ing that DHCR24 may be a sex hormonemediated regulator of neuroprotection [32]. Meanwhile, the estrogen signaling plays a critical role in the development of the female repro ductive system, and the generation of a primordial follicle may be dependent on both estrogen and ERα signaling path ways [33]. In this study, we found that inhibition of DHCR24 expression can significantly increase Caspase3 expression, Figure 4. Effects of miR-26a on the expression of DHCR24 in pGCs. (A) NC mimic, miR-26a mimic, NC inhibitor, or miR-26a inhibitor were transfected into pGCs. DHCR24 mRNA levels were detected by qPCR analysis. (B) NC mimic, miR-26a mimic, NC inhibitor, or miR-26a inhibitor were transfected into pGCs. DHCR24 protein expression was detected by western blotting. β-Actin was used as an internal control. NC means negative control. Average results from three independent experiments are shown. * p<0.05, ** p<0.01. (C) A model for miR-26a regulates pGCs apoptosis by targeting DHCR24. MiR-26a inhibits the expression of DHCR24 gene, which leads to an increase in expression of the proapoptotic Caspase-3 gene and inhibition in the secretion of E2/P and promotion of apoptosis level in pGCs. DHCR24, 3β-hydroxysteroid-Δ24reductase; pGCs, porcine ovarian granulosa cells; qPCR, qualitative real-time polymerase chain reaction.
promote apoptosis, and inhibit estradiol and progesterone secretion in pGCs. Because of DHCR24 is the final catalytic enzyme involved in cholesterol synthesis, which catalyzes the reduction of 24dehydrocholesterol to cholesterol, we pre dicted that inhibition of DHCR24 could promote apoptosis by inhibiting the secretion of estradiol and progesterone in pGCs. However, the accurate regulation mechanism still needs to be further studied.
MiRNAs are widely involved in the regulation of gene ex pression by destabilizing mRNA transcripts and interfering with posttranscriptional protein translation [34,35]. An es tablished approach to elucidate the function of a miRNA is to identify genes that are predicted to be regulated by the miRNA [36]. In this study, we predicted the binding sites of miR26a in the 3′UTR region of DHCR24 by the RNAbybird software. Although there were two mismatches (GU) in the seed se quences, they did not affect the results of the double luciferase reporter gene assay. Several previous studies also showed there was the mismatch of GU in RNA sequence [37,38]. The re sults indicate that miR26a can regulate the expression of the porcine DHCR24 gene by binding to conserved sites in the DHCR24 3′UTR.
In conclusion, our data provide direct evidence that miR 26a can induce apoptosis of pGCs and inhibit the secretion of estrogen and progesterone by inhibiting the expression of DHCR24 gene ( Figure 4C). These findings provide novel in sights into the mechanisms underlying apoptosis of GCs, follicular atresia, and development in mammalian ovaries.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.