Chi-miR-4110 promotes granulosa cell apoptosis by targeting Sma- and Mad-related protein 2 (Smad2) in the caprine ovary

Follicular atresia mainly results from the apoptosis of granulosa cells (GCs). Whilst our previous investigations examined the role of chi-miR-4110 in regulating ovarian function, the present study detected the role of chi-miR-4110 in GC development. We transfected caprine GCs cultured in vitro with chi-miR-4110 mimics. Results revealed that chi-miR-4110 decreased mRNA and protein levels of Smad2 by targeting its 3′-untranslated region (3′UTR). FoxC1 and Sp1 mRNA and protein levels markedly increased, whereas those of bHLHe22 significantly decreased (P<0.01 or 0.05) in GCs transfected with the chi-miR-4110 mimics. Further studies revealed a significantly higher number of apoptotic cells in GCs transfected with the chi-miR-4110 mimics (P< 0.05) than in GCs transfected with mimics negative control. GCs transfected with the chi-miR-4110 mimics exhibited significantly increased mRNA and protein levels of the pro-apoptotic gene Bax (P<0.01) and significantly decreased expression levels of the anti-apoptotic gene BCL-2 (P<0.01). Smad2 interference (Si-1282) results were consistent with those of the chi-miR-4110 mimics. Previous reports and our results showed that chi-miR-4110 increases Sp1 expression by repressing Smad2. The increase in Sp1 induces p53-upregulated modulator of apoptosis, which increases the relative abundance of Bax and causes caprine GC apoptosis. Our findings may provide relevant data for the investigation of miRNA-mediated regulation of ovarian functions.


Introduction
Proliferation of ovarian granulosa cells (GCs) is essential in follicular development, maturation, atresia, ovulation and luteinisation [1]. Folliculogenesis is a complex process during which oocytes increase in size and develop into a mature form, accompanied by the proliferation and differentiation of the surrounding GCs [2]. This process is regulated by various extra-a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and intra-ovarian factors, such as Anti-mullerian hormone (AMH), Smads, Activins, Inhibins, Bone morphogenetic proteins (BMP) and Growth differentiation factor (GDF9) [3,4].
Smad proteins (Smad1, Smad2, Smad4, Smad3, Smad5 and Smad8) are the central mediators of the transforming growth factor beta (TGFβ) signalling pathway [5]. Previous studies found that TGFβ superfamily ligands and their receptors are expressed in oocytes, GCs and theca cells of ovarian follicles, functioning as intra-ovarian regulatory molecules involved in follicle recruitment, GC and theca cell proliferation/atresia, steroidogenesis, oocyte maturation and luteinisation [6,7]. Members of the TGFβ family ligands bind to types I and II serine/threonine kinase receptors to initiate signalling by activating Smads proteins, which are intracellular TGFβ signalling mediators [8]. Receptor-regulated Smads (R-Smads) are Smad molecules phosphorylated by type I receptors [9,10]. Smad2 and Smad3 are activated by GDF-9 and other TGFβ ligands [11]. Smad1, Smad5 and Smad8 are BMP-specific R-Smads. Upon activation, R-Smads interact with the common Smad (Co-Smad4), which acts as a common partner for all R-Smads [12]. Activated R-Smads form oligomeric complexes with Co-Smad4 and translocate into the nucleus, where they regulate target gene expression via interaction with a multitude of other transcription factors (FoxC1, bHLHe22 and Sp1), co-activators and corepressor [8,13,14].
MicroRNAs (miRNAs) regulate gene expression by targeting mRNAs for degradation or blocking mRNA translation by binding to the 3 0 -untranslated region (3 0 -UTR) [15]. Within the past decade, miRNAs have been recognised as important regulators in many biological and cellular processes, such as cell proliferation, differentiation, apoptosis and tumorigenesis [16][17][18]. Although miRNA functions have not been fully elucidated, emerging evidence shows that miRNAs are involved in GC proliferation, ovarian estradiol synthesis and oocyte maturation [19,20]. The regulatory mechanisms of chi-miR-4110 in GC apoptosis during follicle atresia have not been elucidated. Based on previous findings [21], we hypothesise that chi-miR-4110 has important roles in Smad2 expression and goat ovarian GC survival. To test this hypothesis, we investigated the influence of chi-miR-4110 on apoptosis-related gene expression, follicular GC proliferation, apoptosis and atresia in the goat ovary. Our results may help explain the potential role of chi-miR-4110 in GC apoptosis.

Ethics statement
All animals were maintained in accordance with proclamation No. 5 of the Ministry of Agriculture, China. Sample collection was approved by the Institutional Animal Care and Use Ethics Committee of Northwest A&F University and performed in accordance with the ''Guidelines for Experimental Animals" of the Ministry of Science and Technology (Beijing, China). The Institutional Animal Care and Use Ethics Committee of Northwest A&F University approved this study. then treated with specific reagents for the interval indicated in the text. At the end of each culture period, cells were collected for total RNA and protein isolation.

RNA isolation, reverse transcription and real-time PCR
Total RNA were extracted from GCs with RNAiso Plus (TaKaRa, Dalian, China) following the manufacturer's instructions. RNA concentration and purity were determined by measuring optical density (OD) at wavelengths of 260 and 280 nm with an Epoch microplate spectrophotometer (BioTek Instruments, Inc., USA). The OD 260/280 ratios were >1.8 and <2.1 for all samples. The reverse transcription reaction system are showed as follows: (1)Mixed 800 ng of total RNA, 2 μL of 5 × gDNA Eraser Buffer, 1μL of gDNA Eraser and RNase-Free dH 2 O to a final volume of 10 uL, and then incubated the mixture at 42˚C for 2 min. (2) added 4 μL of 5 × Prime Script Buffer 2, 1 μL of PrimeScript RT Enzyme Mix 1, 1 μL of RT Primer Mix and RNase-Free dH 2 O to a final volume of 20 uL, and then incubated the mixture at 42˚C for 15 min followed by 85˚C for 5 S using PrimeScrip RT reagent Kit (TaKaRa, Dalian, China). The cDNA products were stored at -20 o C until further analysis.
Real-time PCR was then performed with a 25 μL reaction volume containing 12.5 μL of SYBR Premix Ex Taq II (TaKaRa, Dalian, China), 2 μL of template cDNA and 1 μM of primers by the CFX Connect Real-Time PCR Detection System (Bio-Rad, CA, USA). Thermal cycling conditions were 95˚C for 10 min, followed by 40 cycles at 94˚C for 15 s, 60˚C for 30 s and 72˚C for 30 s. Primers are shown in Table 1. The GAPDH gene was used for normalisation. Each experiment was independently repeated at least three times, and the fold change in the expression of each gene was analysed via the 2 -ΔΔCt method. MiRNA binding sites were predicted according to the minimum free energy hybridisation of RNA sequences and miRNAs (ΔΔG value). The lower (more negative) the value of the energetic score ΔΔG, the stronger the expected binding of the miRNAs to the given site. The combined ΔΔG values of these approaches were considered to reduce possible false positives.
Plasmid construction and luciferase reporter gene assay

Annexin V-FITC assay
GCs transfected with chi-miR-4110 mimics, mimics NC, chi-miR-4110 inhibitor, inhibitor NC, Smad2 siRNAs (Si-907, Si-1151 and Si-1282) or siRNA negative control (Si-NC) were harvested 48 h after transfection. In transfected GCs, the final concentration of siRNAs is 40 nmol/L. The chi-miR-4110 inhibitor is the reverse complementary sequence of chi-miR-4110 mimics, which can be competitive binding to mature chi-miR-4110 sequence and reduce the gene silencing effect of endogenous chi-miR-4110.

Statistical analysis
All data were presented as mean ± standard deviation. Results were analysed by one-or twoway analysis of variance, followed by a least significant difference Tukey's test. P < 0.05 was considered statistically significant. All statistical analyses were performed by SPSS 16.0.

Results
Chi-miR-4110 directly targets the 3 0 UTR of Smad2 mRNA The genes targeted by chi-miR-4110 were predicted by bioinformatics analytical tools. Results showed that chi-miR-4110 targeted the Smad2 gene, with putative binding sites at positions 1706-1727 of the Smad2 gene (XM_005697184.2; Fig 1A). To validate the identified direct binding site, luciferase activity was analysed by a luciferase reporter system. The wild-type psi-CHECK-2-Smad2-3 0 UTR (WT) vector and mutant psiCHECK-2-Smad2-3'UTR (Mutant) vector were constructed (Fig 1B), with the latter containing six mutant nucleotides (Fig 1C). The WT and Mutant vectors were co-transfected with either chi-miR-4110 mimics or mimics NC. The WT group exhibited significantly inhibited luciferase activity (P< 0.05) compared with mimics NC and mutation groups (Fig 1D). Chi-miR-4110 promotes granulosa cell apoptosis To further determine whether chi-miR-4110 actually targeted the Smad2 gene, goat GCs were transfected with mimics NC, chi-miR-4110 mimics, chi-miR-4110 inhibitor or inhibitor NC. The level of chi-miR-4110 in chi-miR-4110 mimics group was more than 680 times higher than that of endogenous chi-miR-4110 (P<0.01; S1 Fig). Real-time PCR and western blot analysis revealed that Smad2 mRNA and protein levels were significantly lower in GCs transfected with chi-miR-4110 mimics than in GCs transfected with mimics NC (P<0.01; Fig 2). In addition, Smad2 mRNA and protein levels were markedly higher in the chi-miR-4110 inhibitor group than in the inhibitor NC group (P< 0.05 or 0.01).

Chi-miR-4110 regulates Sp1, FoxC1 and bHLHe22 expression by targeting Smad2
To explore the effects of chi-miR-4110 on Sp1, FoxC1 and bHLHe22 expression, the expression level of chi-miR-4110 was increased or decreased with chi-miR-4110 mimics or inhibitor in GCs. The mRNA and protein expression levels of FoxC1 and Sp1 markedly increased in the chi-miR-4110 mimics group compared with that of mimics NC group (P<0.01 or 0.05; Fig 3A and  3B and Fig 4A and 4B). In addition, bHLHe22 mRNA and protein levels significantly decreased in the chi-miR-4110 mimics group compared with that of mimics NC group (P<0.01 or 0.05; Fig 3C and Fig 4A and 4B). By contrast, FoxC1 and Sp1 mRNA and protein levels decreased in the chi-miR-4110 inhibitor group, whereas bHLHe22 mRNA and protein levels increased in the inhibitor NC group (P<0.01 or 0.05; Fig 3A-3C and Fig 4C and 4D).
To detect the effects of Smad2 on Sp1, FoxC1 and bHLHe22 expression, three siRNAs (Si-907, Si-1151and Si-1282) and Si-NC were transfected into GCs. Smad2 mRNA and protein levels significantly decreased in the Si-1151 and Si-1282 groups (P<0.01 or 0.05, Fig 5) compared with the Si-NC group. Given that Smad2 had the lowest expression level in the Si-1282 group, GCs were transfected with Si-1282 to confirm the effects of Smad2 on Sp1, FoxC1 and bHLHe22. The results showed that Sp1 and FoxC1 mRNA and protein levels were higher in the Si-1282 group than in the Si-NC group (P<0.01 or 0.05, Fig 6A and 6B and Fig 7A and  7B). In addition, bHLHe22 mRNA and protein levels were lower in the Si-1282 group than in the Si-NC group (P<0.01 or 0.05, Fig 6C and Fig 7A and 7B). Si-1282 interference results were consistent with experimental results of the chi-miR-4110 mimics, but contrary to those of the chi-miR-4110 inhibitor (Fig 3A-3C and Fig 4A-4D). These results suggested that chi-miR-4110 regulates Sp1, FoxC1 and bHLHe22 expression by targeting Smad2. Increasing chi-miR-4110 level promotes GC apoptosis GC apoptosis is a key factor of goat follicular atresia. To confirm the effects of chi-miR-4110 on GC apoptosis in vitro, cultured GCs were transfected with chi-miR-4110 mimics and inhibitor. The apoptosis rate of GCs transfected with chi-miR-4110 mimics or inhibitor was examined by flow cytometry (Fig 8A-8D). The number of apoptotic cells was significantly higher in Chi-miR-4110 promotes granulosa cell apoptosis GCs transfected with chi-miR-4110 mimics (P<0.05) than in GCs transfected with mimics NC (Fig 8E). In addition, the number of apoptotic cells was lower inthechi-miR-4110 inhibitor group than in the inhibitor NC group (P<0.01). The relative expression levels of apoptosisassociated genes, including Bcl2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2), were determined. Bax mRNA and protein expression levels (P<0.01) significantly increased, whereas BCL-2 expression levels (P<0.01) significantly decreased in GCs transfected with chi-miR-4110 mimics than in GCs transfected with mimics NC (Fig 9A-9D). The results of chi-miR-4110 inhibitor were contrary to those of the chi-miR-4110 mimics. Our results showed that increasing chi-miR-4110 level strongly promotes apoptosis.

Smad2 knockdown in GCs increases apoptosis
To evaluate the effect of Smad2 on GC apoptosis in vitro, Smad2 expression in cultured GCs was knocked down by RNA interference (Si-1282). The apoptosis rate of Smad2-knockdown GCs was examined by flow cytometry (Fig 10A and 10B). The number of apoptotic cells was significantly higher in the Si-1282 group (P<0.05) than in the Si-NC-transfected GCs ( Fig   Fig 5. Smad2 siRNA treatment decreased Smad2 mRNA expression. Real-time PCR (A) and western blot analysis (B and C) showed Smad2 mRNA and protein expression in GCs from goat ovaries cultured with Si-NC, Si-907, Si-1151 and Si-1282. Data are presented as means ± standard deviation. P<0.05 is shown as *, and P<0.01 is shown as **. "ns" represents no significant difference.

Discussion
Cell proliferation is critical in normal reproductive processes [24], including ovarian development, follicle progression from the primordial stage to the primary, secondary and tertiary or Graafian stages and ovulation [25]. However, because more than 99% of ovarian follicles undergo degeneration and atresia, less than 1% of the ovarian follicles reach the ovulation stage in mammals [26]. Previous studies demonstrated that GC apoptosis triggers follicular atresia [27], but the molecular mechanisms involved in this process are still unclear. In domestic animals, promoting follicular development, maturation and ovulation, whilst inhibiting GC apoptosis and follicular atresia may improve animal fertility, specifically ovulation and litter size. Therefore, GC apoptosis and follicular atresia analyses have become hotspots in animal science research [28][29][30]. Zhou et al. (2015) demonstrated that let-7g regulates GCs apoptosis Chi-miR-4110 promotes granulosa cell apoptosis in pig ovaries by targeting TGFBR1 and down regulating the TGF-β signalling pathway [20]. MiR-320 regulates GC proliferation and the production of testosterone, progesterone and estradiol by targeting E2F1 and SF-1 during follicular development [25]. Grossman et al. (2015) demonstrated that human chorionic gonadotropin (hCG) decreases miR-125a-3p expression towards ovulation, enabling mural GC migration. This process is partly mediated by Fyn and may be important for proper ovulation [31]. In this study, we demonstrated that chi-miR-4110 promotes GC apoptosis by targeting Smad2 in the caprine ovary.
Predicting miRNA target genes by computational analysis is efficient, but can result in tens or hundreds of target genes with high false-positive rates [32]. To avoid this issue, this study executed a luciferase assay to identify a chi-miR-4110binding site in the 3 0 UTR of Smad2 mRNA, confirming that the Smad2 mRNA 3 0 UTR from 1706bp to 1727bp is the chi-miR-4110 binding site. Initial reports posited that the extent of complementarity between miRNA and its target mRNA governs either translational mRNA cleavage or repression [33]. In plants, nearly perfect complementarity leads to the degradation of target miRNAs [34], whereas in animals, partial complementarity results in a translational block [35]. However, recent studies have reported that miRNAs can also induce mRNA degradation in animals and, conversely, translational repression in plants [36]. Furthermore, the state of the complementarity between target mRNA and miRNA possibly affects mRNA degradation [37,38]. In this study, because the chi-miR-4110 inhibitor has a sequence reversed with chi-miR-4110, and binds specifically to the endogenous chi-miR-4110 in the cells to knock out the endogenous chi-miR-4110, attenuate its effect on target gene silencing and enhance target protein expression, the Smad2 mRNA and protein levels of the chi-miR-4110 inhibitor group were markedly higher than those of the inhibitor NC group. In addition, the results presented in this study demonstrated that chi-miR-4110 decreased Smad2 protein production by altering Smad2 mRNA levels. It is arguable whether Smad2 protein reduction by chi-miR-4110 is significant for physiological functions in the ovary. Previous reports implicated Activin, which signals through Smad2 and Smad3, in the regulation of early oocyte development [39]. Treating newborn pups with Activin increases the survival of germ cells and assembly into primordial follicles [40]. In the canonical pathway, the downstream signalling molecules, Smad2 and Smad3, are phosphorylated and activated when the ligand binds to the receptor. In combination with common Smad4, they subsequently translocate into the nucleus where these molecules mediate TGF-β1-regulated gene expression in GCs [41]. In this study, chi-miR-4110 repressed Smad2, increasing Sp1 expression. The increase in Sp1 induces p53-upregulated modulator of apoptosis (PUMA), which is essential to p53-dependent apoptosis following DNA damage (Fig 12), subsequently increasing the relative abundance of Bax and causing caprine GC apoptosis [42]. Chi-miR-4110 promotes granulosa cell apoptosis

Conclusions
We demonstrate that chi-miR-4110 promoted GC apoptosis in the caprine ovary by directly targeting and down regulating the Smad2 gene, showing therapeutic implications in the control of fertility. We note that our results do not exclude the possibility that other ovarian functions, such as folliculogenesis, oocyte maturation and ovulation, are regulated by chi-miR-4110. However, our results show that ovarian GC apoptosis is sensitive to chi-miR-4110. Although the precise mechanism regulating GC apoptosis is not elucidated, our findings may provide relevant data for those investigating miRNA-mediated regulation of ovarian functions.