Circ_0008219 regulates proliferation and apoptosis in goat granulosa cells via a feedback loop with the transcription factor YY1

Abstract

Background

Circular RNAs (circRNAs) are a class of competitive endogenous RNAs (ceRNAs) widely found in animals, they can directly or indirectly affect the proliferation and apoptosis of granulosa cells (GCs) by regulating microRNA (miRNA) pathways. Preliminary research findings indicate that circ_0008219 is differentially expressed in pre-ovulatory follicles between Boer goats and Macheng black goats, suggesting its potential critical role in goat ovulation. As a potential target, its specific functions and regulatory mechanisms warrant further investigation.

Results

A feedback loop of the YY1/circ_0008219/miR-130b-5p/FZD7 axis was constructed to elucidate the function and regulatory mechanism of circ_0008219 promoting GCs proliferation and suppressing apoptosis. Subsequent validation experiments were conducted in vitro using GCs. In our experiments, we identified circ_0008219 as a ceRNA in goat GCs that promoted proliferation. Through bioinformatics prediction and dual-luciferase reporter assays, we confirmed that circ_0008219 acts as a sponge for miR-130b-5p, preventing its binding to FZD7 mRNA and thereby increasing the expression of FZD7. This interaction also influenced the proliferation and apoptosis of goat GCs. Moreover, the transcription factor YY1 was found to form a feedback loop with circ_0008219 via the linkage of miR-130b-5p.

Conclusions

Our study validated the biological function of the TF-circRNA-miRNA-mRNA feedback loop in goat GCs and revealed the potential regulatory mechanism by which circ_0008219 functions as a ceRNA in goat GCs, thus provide new insights into the mechanisms of regulated ovarian follicles development in female goats.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-026-12552-x.

Background

The low fecundity of goats is mainly affected by genetic factors [1]. Due to their low heritability and low ovulation rate in the estrus cycle, reproductive efficiency in goats remains suboptimal, which limits the production capacity of goat and affects the economic viability of goat industry [2]. The fertility potential of female animals is often determined by the developmental dynamics of ovarian follicles [3]. Generally, mammalian species with more mature ovarian follicles and higher ovulation rates exhibit superior fecundity. Ovarian follicular development critically influences overall animal growth [4, 5].

Granulosa cells (GCs) development is a key determinant of ovulation in female livestock, with ovulation number directly influencing litter size [6–8]. The interaction between GCs and oocytes represents the most critical mechanism for fine-tuned regulation of follicular development [9, 10]. GCs deliver small molecules, nutrients, and second messengers to oocytes via paracrine pathways, and through these complex signaling transduction and metabolic exchange processes, thereby directly regulating oocyte developmental competence [11, 12]. During the proliferation and apoptosis of granulosa cells, the interaction between anti-apoptotic and pro-apoptotic factors, as well as regulation by multiple hormones and cytokines, are involved [13]. Additionally, transcription factors [14], miRNAs [15], and circRNAs [16] play crucial roles in this process by extensively participating in transcriptional and post-transcriptional mRNA regulation.

Circular RNA (circRNA) is a class of endogenous non-coding RNAs (ncRNAs) with covalently closed loops, which is widely present in the transcription of different species and tissues [17]. Compared with linear RNA, circRNAs possess greater structural stability and higher evolutionary conservation. Additionally, circRNAs function as miRNA sponge to participate in the regulation of linear RNA expression in different ways, encode proteins, and produce pseudogenes [18, 19]. These properties have established their critical roles in diverse physiological and pathological processes, including the regulation of GCs proliferation and apoptosis.

CircRNAs are produced via the back-splicing of pre-mRNA, and three circularization modes have been identified: intron-derived reverse complementary sequences-driven circularization, RNA-binding proteins (RBPs)-mediated circularization, and lariat-driven circularization [20, 21]. CircRNAs further regulate gene transcription by interacting with transcription factors [22]. Recent studies have demonstrated that the transcription factor Meis1 binds to the super-enhancer at the circNfix locus to upregulate its expression [23]. Additionally, circPTPN4 is positively regulated by the transcription factor forkhead box A2 (FOXA2) and promotes the fast-muscle phenotype [24].

Circ_0008219 was upregulated in the preovulation follicles of multi-lamb Macheng black goats (Log₂FC = 2.52, P < 0.01). Preliminary findings indicated that the differentially expressed circ_0008219 may play a critical role in goat ovulation [25]. The present study aimed to investigate the functions and mechanisms of circ_0008219 in regulating the proliferation and apoptosis of goat GCs. The results demonstrated that circ_0008219 acts as a ceRNA by sequestering miR-130b-5p, thereby alleviating its repression of FZD7 mRNA, upregulating FZD7 expression, and ultimately promoting GCs proliferation while suppressing apoptosis.

Method

Ethics approval and consent to participate

All animal experimental protocols were conformed to “The Instructive Notions with Respect to Caring for Laboratory Animals” issued by the Ministry of Science and Technology of the People’s Republic of China, and approved by the Animal Care and Use Committee of Hubei Academy of Agricultural Sciences (HBAAS-2023-014). Animals were humanely sacrificed as necessary to minimize suffering.

Animals and cells

All the experimental goats were from the lower goat farm of Hubei Academy of Agricultural Sciences. The Macheng black goats were required to be one-year-old healthy ewes that had only given birth once. They were raised under the same conditions, with an average weight of 37.08 ± 3.70 kg, and detailed production performance records were kept. All registered participants completed the study, meaning there was no dropout. The two tissues (i.e., ovary and ovarian follicle) of the Macheng black female goats were immediately frozen in liquid nitrogen and stored at -80 ℃. The newly collected ovaries of Macheng black goats were placed in saline solution at 37 ℃ and brought back to the laboratory within 1 h. Ovarian tissues were washed in PBS supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL), then transferred to culture dishes. Large antral follicles were punctured using a hypodermic needle to release goat GCs into the culture medium, followed by further digestion with type II collagenase (1 mg/mL, 10 min, 37 °C) and subsequent culture in the medium [26]. GCs and dermal fibroblasts were maintained in DMEM/F12 medium (Gibco, Grand Island, USA) or high-glucose DMEM (Gibco, Grand Island, USA), respectively. All media were supplemented with 10% fetal bovine serum (FBS), 2% penicillin (100 U/mL) and streptomycin (100 µg/mL). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

RNA extraction, cDNA synthesis, and quantitative real-time PCR (RT-qPCR)

Nuclear and cytoplasmic RNA fractionation was performed by using the PARIS Kit (Life Technologies, USA) as recommended by the supplier. Total RNA preparation and cDNA synthesis were performed using HP Total RNA Kit (Omega Bio-Tek Inc., USA) and First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA), respectively. RT-qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, USA) on a LightCycler 96 system (Roche, Switzerland), with all procedures conducted strictly in accordance with the manufacturer’s instructions. All PCRs were performed in triplicate and gene expression levels were quantified relatively to the expression of β-actin by employing 2^–ΔΔCt value. Primers used in the RT-qPCR are shown in Supplementary Table S1.

Western blotting

Total protein was extracted from cells with RIPA Lysis Buffer (Beyotime, China). Western blotting was performed as reported previously. Five µg proteins were boiled in 5× SDS buffer for 5 min, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore). Then, the membranes were blocked with skim milk and probed with Anti-YY1 (A12928, Abclonal, China), Anti-FZD7 (A4213, Abclonal, China). β-actin (AC026, Abclonal, China) was used as a loading control. All blots were performed in triplicate and protein expression levels were quantified relative to β-actin using the Image J (V.1.42q, Wayne Rasband).

Plasmid construction and cell transfection

The four deletion fragments of circ_0008219 promoter were amplified using Nest PCR. The purified PCR products were cloned into the pMD18-T vector (TaKaRa, China). Subsequently, the recombinant plasmids were double-digested with NheI and HindIII (Thermo, USA) and ligated into the pGL3-Basic vector (Promega, USA). The goat YY1 (XM_018066124.1) and FZD7 (XM_018060827.1) coding sequences (CDS) were amplified and double-digested with EcoRI and XhoI (Thermo, USA) and cloned into the pcDNA3.1(+) vector (Invitrogen, USA). The 3′ untranslated region (3’-UTR) of goat FZD7 were amplified, double-digested with MssI and XhoI, and then cloned into the pmirGLO vector (Promega, USA). The binding site mutants were generated using a MutanBEST Kit (R401, TaKaRa, China) and mutagenic primers. The plasmids were transfected into GCs using FuGENE^® HD Transfection Reagent (E2311, Roche, USA). The primers are described in Supplementary Table S1.

Bioinformatics analysis

Transcription factor binding sites in the promoter regions of goat circ_0008219 were predicted using the JASPAR 2022, AnimalTFDB3.0, PROMO, and TFBIND. The FZD7 3’-UTR sequence was acquired from NCBI (XM_018060827.1). Based on the goat-specific mature miRNA sequences, TargetScan, miRBase, RNAhybrid, PITA and RNA22V2 were used to predict the binding sites of FZD7 with miR-130b-5p.

Chromatin Immunoprecipitation (ChIP) assay

ChIP assay was performed in GCs (1 × 10⁷ cells) using the EZ-ChIP™ Kit (17–371, Millipore, USA). Briefly, after crosslinking the chromatin and neutralizing with glycine, samples were harvested, lysed, and sonicated. Nuclear lysates were sonicated 20 times for 10 s with 20 min intervals on ice water using an ultrasonic vibrator (VCX 130, Sonics & Materials, Inc., USA). An equal amount of chromatin was immunoprecipitated at 4 ℃ overnight with at least 1.5 µg of Anti-YY1 (A12928, Abclonal, China) and normal mouse IgG (12–371, Millipore, USA) antibodies. Immunoprecipitated products were collected after incubation with Protein A + G coated magnetic beads. The beads were washed, and the bound chromatin was eluted in ChIP elution buffer. The DNA was purified using the AxyPrep PCR Cleanup Kit (MAG-PCR-CL-50, Axygen, China). RT-qPCR was conducted according to the method described above. The experiment was conducted in triplicate. The primers used were described in Supplementary Table S1.

Cell proliferation using xCELLigence RTCA system

Cell proliferation assays were performed using the xCELLigence real-time cell analyzer (RTCA) system (Agilent Technologies, USA) as previously described [27]. Wells of the E-Plate 16 devices (Agilent Technologies, USA) were filled with 10% fetal bovine serum (FBS) and the background value was measured using the RTCA DP software. GCs were transfected with expression vectors using the FuGENE^® HD Transfection Reagent (Roche, USA). The slopes of the curves at various time points were calculated using an RTCA software (Agilent Technologies, USA).

CCK-8 assay

Cell proliferation of GCs was assessed using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan) in strict accordance with the manufacturer’s instructions. Briefly, GCs were seeded in 96-well plates at a density of 2 × 10⁴ cells per well and cultured for 24 h prior to transient transfection with expression vectors. CCK-8 solution was added at 12, 24 and 48 h and the cells were incubated in the cell incubator for 2 h at 37 °C, and the absorbencies at each time point were measured at 450 nm by a microplate reader. All experiments were biologically repeated at least three times.

Cell apoptosis analysis

Cells were harvested, and the cell concentration was adjusted to 1 × 10⁶ cells/mL. Cell apoptosis was analyzed using CytoFLEX Flow Cytometer (Beckman Coulter, China) using an Annexin V/FITC Apoptosis Detection Kit (AD10, Dojindo, Japan). All the data were analyzed using CytExpert software (Version 2.4).

Subcellular fractionation assay

To isolate cytoplasmic and nuclear RNAs from GCs of goat ovaries, PARIS kit (AM1921, Thermo Fisher Scientific) was used according to the manufacturer’s instructions. Cytoplasmic and nuclear RNA transcripts were first reversed into cDNA and evaluated by RT-qPCR. U6 (nuclear control) and GAPDH (cytoplasmic control) were used for normalization.

Fluorescence in situ hybridization (FISH)

An RNA-FISH experiment was performed to determine the subcellular distribution of circ_0008219 in GCs. The FISH probes were provided by Gene Pharma (Shanghai, China). Cyanine (Cy3)-labeled probes were used as markers for circ_0008219 (Table S1). A Fluorescence in Situ Hybridization Kit (Gene Pharma, Shanghai, China) was used to produce fluorescent signals, and the confocal laser scanning microscopy (CLSM, LSM 900, Zeiss) was used to capture cellular images.

Dual-luciferase reporter assay

The wild-type or mutant promoter sequence of circ_0008219 was cloned into the dual-luciferase reporter vector. After 48 h of transfection, dual-luciferase reporter assays were conducted using the dual-luciferase reporter assay system (E1910, Promega, USA). Relative luciferase activity was normalized to Renilla luciferase activity.

Statistical analysis

All data were analyzed using the SPSS (version 23.0; SPSS Inc., Chicago, IL, USA). When comparing just two groups, we relied on the t-test. However, for assessing disparities among multiple groups, we adopted one-way ANOVA followed by Tukey’s post hoc test. Statistical significance was defined as p < 0.05.

Results

Circ_0008219 was a novel circRNA

To further elucidate the regulation mechanisms of circRNA involved in proliferation and apoptosis of GCs, a novel differentially expressed circRNA, circ_0008219 in our published sequencing data (GEO accession number: GSE100773) was served as a candidate, sequences can be found in Supplementary text S1. The structure of circ_0008219, derived from partial of the first intron and the second exon of the ZCCHC24 gene on goat chromosome 28 and has a length of 2,045 bp (Fig. 1A). Divergent primers were used to amplify the junction site of circ_0008219 and confirmed using Sanger sequencing (Fig. 1B). Divergent and convergent primers for circ_0008219 amplified using PCR reaction, with amplification products analyzed by agarose gel electrophoresis. A single distinct band with the expected fragment size was only observed in cDNA but not genomic DNA (gDNA) (Fig. 1C). RNase R tolerance test showed that circ_0008219 exhibited better stability in cells compared to linear β-actin mRNA in GCs (Fig. 1D). In addition, the cytoplasmic localization of circ_0008219 was also confirmed by RT-qPCR and FISH in goat GCs, suggesting that circ_0008219 might regulate downstream gene expression by acting as ceRNA (Fig. 1E, F). The results of RT-qPCR revealed that the expression level of circ_0008219 in ovary and ovarian follicle was significantly higher than that of the expression level of ZCCHC24 (Fig. 1G). These results unveil that circ_0008219 is a novel circRNA and can enhance the expression level of its host gene ZCCHC24.

Fig. 1.

Identification of circ_0008219. (A) Schematic image of circ_0008219 derived from ZCCHC24. (B) Sanger sequencing confirmed the back-splicing junction sequence of circ_0008219. (C) Verification of circ_0008219 by amplifying with divergent primers. (D) Relative circ_0008219 and β-actin expression after treatment with RNase R. (E) Nuclear separation assay to detect the sublocalization of circ_0008219 in GCs. (F) FISH assay of circ_0008219 in goat GCs. (G)The expression levels of circ_0008219 and ZCCHC24 in ovarian follicles and ovary tissues of goats. Data were presented as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001

Circ_0008219 promotes GCs proliferation but suppresses apoptosis

To more comprehensively explore the effect of circ_0008219 on GCs proliferation and apoptosis, GCs were transfected with the circ_0008219 overexpression vector (pLCDH-8219). As shown by the xCELLigence RTCA system and CCK-8 assay, circ_0008219 overexpression significantly enhanced GCs proliferation (Fig. 2A and B). Flow cytometry showed that the apoptosis rate of GCs was significantly suppressed after pLCDH-8219 treatment (Fig. 2C). Quantitative analysis demonstrated that this change was statistically significant (P < 0.001) (Fig. 2D). Collectively, these findings indicate that circ_0008219 may contribute to the molecular regulation of follicular development by promoting GCs proliferation and inhibiting GCs apoptosis.

Fig. 2.

circ_0008219 regulated GCs proliferation and apoptosis. (A) GCs were transfected with pooled pLCDH-8219 or pLCDH vector when the cell index reached 1.0, and cell growth dynamics were then continuously monitored using an xCELLigence system. (B) The proliferation of GCs following indicated treatments were detected by CCK-8 assays. (C) (D) Cell apoptosis was determined by Annexin V/PI dual staining followed by flow cytometry after the cells were transfected with pLCDH-8219. Data were presented as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001

Circ_0008219 is positive regulated by transcription factor YY1

To elucidate the transcriptional regulation mechanism of circ_0008219, we identified its core promoter region, pGL3-D1 (− 1026 bp/−733 bp), using a dual-luciferase reporter assay (Fig. 3A). Potential transcription factors binding to this region were predicted through bioinformatic tools including JASPAR, and YY1 was selected for further validation based on its documented role in reproductive traits [28] (Fig. 3B). A specific YY1 binding site (− 901 bp/−898 bp) was identified within the pGL3-D1 region (Fig. 3C). An overexpression plasmid carrying YY1 (pc-YY1) was constructed and confirmed to be efficiently expressed (Fig. 3D, E). Mutation of the YY1 binding site (pGL3-D1-mut) abolished the responsiveness to YY1 overexpression, with no significant change in luciferase activity (Fig. 3F). Chromatin immunoprecipitation (ChIP) assays confirmed direct binding of YY1 to the pGL3-D1 region (Fig. 3G). Overexpression of YY1 upregulated the luciferase activity of pGL3-D1 (Fig. 3H) and specifically up-regulated the mRNA level of circ_0008219 (Fig. 3I). Collectively, these results demonstrate that YY1 positively regulates the expression of circ_0008219 through direct binding to its promoter.

Fig. 3.

The transcription factor YY1 upregulates the expression level of circ_0008219 by binding to its binding site in the promoter region of host gene ZCCHC24. (A) Luciferase assays show the activities of a series of deletion constructs in GCs. The left panel shows each deleted mutant linked with the luciferase gene in the pGL3-basic vector. The right panel indicates the relative luciferase activities of the deletion constructs. (B) Prediction of potential transcription factor for promoter region of host gene ZCCHC24 using PROMO, Jaspar, Animaltfdb3.0, TFBIND. (C) Schematic diagram of YY1 binding sequence in the ZCCHC24 promoter region, the predicted sequence was shown. (D) Western blot was used to detect YY1 protein expression level its overexpressed GCs. (E) RT-qPCR was used to detect YY1 mRNA expression level its overexpressed GCs. (F) Site-directed mutagenesis in the YY1 binding sites of the core promoter was analyzed using luciferase assays. (G) ChIP assay of YY1 binding to the ZCCHC24 promoter in GCs in vivo. DNA isolated from immunoprecipitated material was amplified by RT-qPCR total chromatin was used as the input. Normal IgG was used as a negative control. (H) Luciferase assays were used to detect the relative luciferase activities of D1 after GCs were transfected with pc-YY1 or pcDNA3.1. (I) RT-qPCR was used to detect endogenous circ_0008219 and ZCCHC24 expression level after GCs were transfected with pc-YY1 or pcDNA3.1. Data were presented as mean ± SD. * P < 0.05, *** P < 0.001, ns: no significant

Circ_0008219 serves as a sponge for miR-130b-5p

The circRNAs that are highly expressed in the cytoplasm tend to function as miRNA sponges [18]. Considering that circ_0008219 is predominantly highly expressed in the cytoplasm of GCs (Fig. 1E, F), we hypothesized that circ_0008219 might also sponge miRNAs in GCs. Therefore, ten candidate miRNAs (miR-485-5p, miR-423-3p, miR-483, miR-330-5p, miR-326-3p, miR-671-5p, miR-130b-5p, let-7 g-3p, miR-1343, and miR-29b-5p) were identified by overlapping the miRNA target prediction of miRanda, Targetscan, RNAhybrid, PITA, and RNA22V2 (Fig. 4A). miRanda predicted that circ_0008219 contained 13 putative binding sites of 10 miRNAs (Fig. 4B). Generally, the extent of contribution of sites were as follows: 8mer > 7mer-m8 > 7mer-A1 > 6mer [29]. Ten miRNAs classified according to inclusion of the following motifs in this order. The 8mer seed match type includes five miRNAs (miR-130b-5p, miR-326-3p, miR-330-5p, miR-423-3p and miR-671-5p) (Fig. 4C). The mimics of miR-130b-5p, miR-326-3p, miR-330-5p, miR-423-3p and miR-671-5p, and were co-transfected with the pmir-8219 dual-luciferase reporters into GCs. Compared with the negative control (NC), the miR-130b-5p mimic was able to significantly reduce the dual-luciferase reporter activities of pmir-8219 (P < 0.01) (Fig. 4D). Two miR-130b-5p binding sites of circ_0008219 were predicted on miRanda software. Then, we constructed dual-luciferase reporter vectors containing two mutant binding sites to analyze the interaction between miR-130b-5p and circ_0008219 (Fig. 4E). Individual mutation of site2 of circ_0008219 still markedly altered the luciferase activity. However, concurrent mutation of both sites of circ_0008219 did not show any obvious change in luciferase activity compared to the wild type (Fig. 4F). GC proliferation kinetics were evaluated by xCELLigence RTCA system. Cell index profiles revealed the reduced cell proliferation of the GCs expressing miR-130b-5p mimics (Fig. 4G). Analysis of cell apoptosis revealed that the early apoptosis rate of GCs increased in the miR-130b-5p mimics group (Fig. 4H and I). These results demonstrate that circ_0008219 may function as a sponge to miR-130b-5p by binding to the first binding site in the circ_0008219.

Fig. 4.

circ_0008219 functions as a ceRNA by sponging miR-130b-5p. (A) Prediction of potential miRNA for circ_0008219 using miRanda, Targetscan, RNAhybrid, PITA, RNA22V2 software. (B) Alignment of potential ten miRNAs base pairing with circ_0008219, as identified by the miRanda software. (C) The Mode diagram shows the target miRNA seed type (8mer, 7mer-m8). (D) Luciferase assays for the luciferase activity of circ_0008219 recombinant pmirGLO vectors in GCs transfected with five miRNA mimics or NC. (E) Binding sites for miR-130b-5p in the sequence of circ_0008219 were predicted by miRanda. Red characters indicate sequences that were mutated to abolish the interaction between miR-130b-5p and circ_0008219. (F) Site-directed mutagenesis in the miR-130b-5p binding sites of the circ_0008219 pmirGLO vector by luciferase assay. (G) Cell proliferation of GCs transfected with miR-130b-5p mimics or NC in real time was analyzed by the xCELLigence RTCA. (H) (I) Cell apoptosis was determined by Annexin V/PI dual staining followed by flow cytometry after the cells were transfected with miR-130b-5p mimics or NC. Data were presented as mean ± SD. ** P < 0.01, *** P < 0.001 and ns, no significance

Circ_0008219 interacts with miR-130b-5p to upregulate FZD7 expression

To explore the underlying functions and mechanisms of miR-130b-5p in GCs, we used miRanda, TargetScan, RNA22V2, and RNAhybrid software integrated analysis to identify 106 potential target genes (Fig. 5A). Based on the results, potential target genes were predicted and used pathway analysis. KEGG analysis identified the top six pathways involving miR-130b-5p target genes, including the HIF-1 signaling pathway, Pathways in cancer, Chronic myeloid leukemia, Hippo signaling pathway, Basal cell carcinoma, and mTOR signaling pathway (Fig. 5B). Since mTOR and Hippo pathways are critically involved in the regulation of ovarian follicle development, we selected seven genes (WWTR1, FZD7, ATP6V1A, PRKCI, GLI2, SESN2, EIF4E2) in these signaling pathways as the candidates for functional validation [30, 31]. Then, we constructed the dual-luciferase reporter vectors containing two mutant binding sites to analyze the interaction between miR-130b-5p and FZD7 (Fig. 5C). The results of qRT-PCR assays showed that transfection with the miR-130b-5p mimic resulted in a significant downregulation of FZD7 mRNA expression, with no observable effects on other candidate genes (Fig. 5D). Subsequently, we constructed two dual-luciferase reporter vectors containing the two binding sites of miR-130b-5p within the 3’-UTR of FZD7 (pmir-FZD7-1 and pmir-FZD7-2). These vectors were co-transfected with different concentrations (1, 3, and 6 µl) of miR-130b-5p mimics into GCs to analyze their interaction. The luciferase activity analysis showed that miR-130b-5p overexpression significantly decreased the luciferase activity of the wild-type reporter of pmir-FZD7-1 and pmir-FZD7-2 (Fig. 5E), but had no effect on that of the mutated reporter (Fig. 5F). The results showed that miR-130b-5p bind with the 3’-UTR of FZD7. We next evaluated the effect of miR-130b-5p and circ_0008219 on FZD7 expression in GCs, respectively. RT-qPCR and western blot analyses showed that miR-130b-5p overexpression significantly decreased the FZD7 mRNA level (Fig. 5G) and protein level (Fig. 5I). Nevertheless, circ_0008219 can promote FZD7 expression in GCs (Fig. 5H, J). Cell index profiles revealed the accelerated cell proliferation of the GCs expressing FZD7 (Fig. 5K). Analysis of cell apoptosis revealed that the apoptosis rate of GCs reduced in the pc-FZD7 group (Fig. 5L and M). Taking these results together, we proved that FZD7 is negatively regulated by miR-130b-5p and promotes the proliferation of GCs while inhibiting the apoptosis of GCs.

Fig. 5.

Identification of FZD7 gene as a direct target of miR-130b-5p. (A) Prediction of potential target genes for miR-130b-5p using miRanda, Targetscan, RNAhybrid, RNA22V2. (B) KEGG pathway analysis corresponds to the target gene of miR-130b-5p. The top 6 results are shown here. (C) Binding sites for miR-130b-5p in the 3’-UTR of the FZD7 were predicted by miRanda. Red characters indicate sequences that were mutated to abolish the interaction between miR-130b-5p and FZD7. (D) Luciferase assays for the luciferase activity of 7 recombinant pmirGLO vectors of target genes in GCs transfected with miR-130b-5p mimics or NC. (E) Luciferase assays were used to detect the relative luciferase activities of two pmirGLO vector of FZD7 after GCs were transfected with 1 µl, 3 µl, 6 µl mimics of miR-130b-5p. NC was used as a negative control. (F) Site-directed mutagenesis in the miR-130b-5p binding sites of the FZD7 pmirGLO vector by luciferase assay. (G) RT-qPCR analysis of FZD7 was shown in miR-130b-5p overexpressed GCs. (H) RT-qPCR analysis of FZD7 was shown in miR-130b-5p overexpressed GCs. (I) Western blot was used to detect FZD7 protein expression level in miR-130b-5p overexpressed GCs. (J) Western blot was used to detect FZD7 protein expression level in circ_0008219 overexpressed GCs. (K) Cell proliferation of GCs transfected with pc-FZD7 in real time was analyzed by the xCELLigence RTCA. (L) (M) Cell apoptosis was determined by Annexin V/PI dual staining followed by flow cytometry after the cells were transfected with pc-FZD7. Data were presented as mean ± SD. ** P < 0.01, *** P < 0.001

YY1-circ_0008219-miR-30b-5p constitute the feedback loop

During the analysis of miR-130b-5p target genes, we observed an intriguing phenomenon: the transcription factor YY1, which upregulates circ_0008219 expression, is itself a direct target of this miRNA. Furthermore, this study confirmed that circ_0008219 effectively sponges miR-130b-5p. These findings raise a critical question, whether miR-130b-5p, YY1, and circ_0008219 collectively form a regulatory feedback loop, warranting further mechanistic investigation. First, we used miRanda and TargetScan cross-analysis to identify 1054 potential target genes, among which the transcription factor YY1 was included (Fig. 6A). To verify that miR-130b-5p bind to 3’-UTR of transcription factor YY1, the pmiR-YY1 reporter vectors and two mutated binding site regions of pmirGLO vectors were constructed, details of which are shown in Fig. 6B. The results showed that the luciferase activity of the pmiR-YY1 + miR-130b-5p mimic group was significantly decreased (Fig. 6C), and there was no change compared to the luciferase activity associated with the mutD (Fig. 6D). Further analyses revealed that YY1 expression could be downregulated by overexpression of miR-130b-5p (Fig. 6E and F). Subsequently, RT-qPCR analysis revealed that miR-130b-5p overexpression significantly decreased the circ_0008219 mRNA level (Fig. 6G). We conclude that miR-130b-5p can form a feedback loop with YY1 and circ_0008219. Upon activation of circ_0008219, miR-130b-5p expression is reduced, thereby attenuating its inhibitory effect on the target gene YY1. The subsequent increase in YY1 expression enhances circ_0008219 transcriptional activity, forming a closed feedback loop.

Fig. 6.

MiR-130b-5p downregulates circ_0008219 expression through inhibits transcription factor YY1. (A) Prediction of potential target genes for miR-130b-5p using miRanda and Targetscan. (B) Binding sites for miR-130b-5p in the 3’-UTR of the YY1 were predicted by miRanda. Red characters indicate sequences that were mutated to abolish the interaction between miR-130b-5p and YY1. (C) Luciferase assays for the luciferase activity of recombinant pmirGLO vectors of YY1 in GCs transfected with miR-130b-5p mimics or NC. (D) Site-directed mutagenesis in the two miR-130b-5p binding sites of the YY1 pmirGLO vector by luciferase assay. (E) RT-qPCR analysis of YY1 was shown in miR-130b-5p overexpressed GCs. (F) Western blot was used to detect YY1 protein expression level in miR-130b-5p overexpressed GCs. (G) RT-qPCR was used to detect circ_0008219 expression level in miR-130b-5p overexpressed GCs. Data were presented as mean ± SD. *** P < 0.001 and ns, no significance

Discussion

CircRNAs, a class of non-coding RNAs ubiquitously present in eukaryotic cells, were once dismissed as “junk RNA“ [32]. Emerging evidence indicates, circRNAs have been found to exert increasingly important roles in diseases, gene regulation, and reproductive biology through various mechanisms, such as acting as microRNA sponges [18], encoding small peptides, and regulating RBPs [19]. Growing evidence has demonstrated that circRNAs play critical regulatory roles in ovarian development, and this conclusion has been validated in multiple animal species, such as pigs [33], bovine [34], sheep [35], goat [25] and mice [36]. Notably, circRNAs play significant roles in the proliferation and apoptosis of GCs in domestic animal follicles [37].

Here, we elucidated the functional roles of circ_0008219, a circRNA stably expressed in goat ovarian GCs. Key findings from this study include: First, circ_0008219 was demonstrated to promote GC proliferation and suppress apoptosis. GCs, the most critical somatic cells in ovarian follicles, form follicles together with oocytes. GCs communicate with oocytes via gap junctions, playing pivotal roles in determining the fate of follicular development and atresia, including whether follicles mature or undergo atresia [38], this finding highlights the pivotal role of circ_0008219 in regulating goat follicular growth. Second, subcellular localization analysis revealed that circ_0008219 is predominantly cytoplasmic in GCs, where it acts as a miRNA sponge for miR-130b-5p. This interaction modulates the expression of the target gene FZD7, thereby influencing biological processes such as GC proliferation and apoptosis. Third, we identified that the transcription factor YY1 binds to the core promoter region of ZCCHC24, the host gene of circ_0008219, and positively regulates circ_0008219 expression without affecting ZCCHC24 transcription. Notably, YY1 was also identified as a direct target of miR-130b-5p, establishing a closed-loop feedback regulatory circuit among YY1, circ_0008219, and miR-130b-5p. Despite the critical roles of circRNAs in follicular biology, their regulatory functions and molecular mechanisms in goat ovarian GCs remain poorly understood. This study provides novel insights into the regulatory roles of circRNAs during livestock folliculogenesis, advancing our understanding of non-coding RNA-mediated mechanisms in reproductive biology (Fig. 7).

Fig. 7.

Model of circ_0008219 functions as a ceRNA to regulate FZD7 expression by sponging miR-130b-5p, thus promoting GC proliferation and inhibiting GC apoptosis

In our previous study, transcriptome sequencing was used to compare the circRNA expression profiles in preovulatory follicles of three Boer goats and three Macheng black goats. A total of 13,950 circRNAs were detected, among which 37 circRNAs were preliminarily identified as significantly differentially expressed. Bioinformatics analysis was further performed to investigate the potential functions of these circRNAs. According to the RNA-seq results, circ_0008219 was upregulated in the pre-ovulatory follicles of Macheng goat and Boer goat (Log₂FC = 2.52, P < 0.0066). Preliminary findings indicated that the differentially expressed circ_0008219 may play a critical role in goat ovulation. As a potential target, its specific functions and regulatory mechanisms warrant further investigation [25]. Circ_0008219 arose from the ZCCHC24 gene and consisted of the head-to-tail splicing of intron 1 and exon 2 (2,045 bp). Subsequently, we confirmed the head-to-tail splicing in the RT–PCR product of circ_0008219 with expected size by Sanger sequencing.

According to the existing literature, circRNAs are classified into four categories based on the source of the sequence: The first category is circRNAs (Exonic circRNAs, EciRNAs) derived only from the exons of the host gene; The second type is the lasotype circRNAs (Intronic circRNAs, ciRNAs) derived only from introns of the host gene; The third category is circRNAs (Exon-intron circRNAs, EIciRNAs) derived from exons and introns of the host gene; The fourth category includes Antisense circRNAs derived from antisense strand transcripts and Intergenic circRNAs derived from intergenic sequences or other unannotated genomic sequences [17, 39, 40]. The vast majority of circRNAs belong to the exon type and have relatively high conservation. According to literature reports, eciRNAs are mainly detected in the cytoplasm, while ciRNAs are mainly located in the nucleus.

According to literature reports, eciRNAs represent the most extensively studied category of circRNAs, predominantly localized in the cytoplasm. They typically function as miRNA sponges by utilizing abundant miRNA-binding sites within their sequences, thereby enhancing the expression of miRNA-targeted genes [41]. In contrast, ciRNAs exhibit nuclear localization, accumulate proximal to host gene transcription start sites, and cis-regulate transcriptional activity through coordinated interactions with RNA polymerase II complexes [42]. Similar to ciRNAs, EIciRNAs are nuclear-localized and recruit U1 snRNP to interact with RNA polymerase II (Pol II) at the promoter regions of host genes, thereby promoting transcriptional activation of their parental genes [43]. Although circ_0008219 is annotated as an EIciRNA, our findings demonstrate its dual nuclear-cytoplasmic localization with predominant cytoplasmic accumulation in goat GCs. Mechanistically, it functions as a miRNA sponge, which contrasts with previous reports of nuclear-restricted EIciRNAs. This is not an isolated case, the EIciRNA circRNF217 promotes innate immunity and antibacterial activity in teleost fish by sponging miR-130-3p and attenuating its function [44]. This discrepancy may indicate species-specific regulatory divergence of circRNA in Goat.

Besides their non-coding roles in gene regulation, circRNAs can initiate protein translation in a cap-independent manner. This process is mediated by internal ribosomal entry sites (IRES), and recent studies have shown that m⁶A methylation modification of circRNAs can also drive protein synthesis. Additionally, circRNAs may utilize other sequence elements to facilitate translation [45]. And emerging evidence has confirmed that circRNAs can regulate GCs apoptosis and autophagy by encoding small peptides [46]. Bioinformatic prediction using ORFfinder revealed 16 putative open reading frames (ORFs) within circ_0008219, suggesting its potential as a dual-functional circRNA with both non-coding regulatory roles and protein-coding capacity.

This study has several limitations. The functional validation of circ_0008219 was not performed in vivo using goats, functional rescue experiments are lacking, and the protein-coding potential of circ_0008219 has not been verified. We plan to further investigate these aspects in our future studies.

Conclusion

In conclusion, our study systematically demonstrates that circ_0008219, a novel circRNA highly expressed in the cytoplasm of GCs, is positively regulated by the transcription factor YY1. Mechanistically, circ_0008219 functions as a ceRNA to regulate FZD7 expression by sponging miR-130b-5p, thus promotes proliferation and suppress apoptosis in goat GCs. Moreover, we identified a novel regulatory feedback loop in which YY1, in addition to promoting circ_0008219 expression, is itself a target of miR-130b-5p, thus forming a precise YY1/miR-130b-5p/circ_0008219 regulatory circuit (Fig. 7). Our findings provide new insights into the molecular mechanisms and regulatory networks underlying follicular development in the goat ovary, and establish a foundation for further functional studies on circRNA-mediated regulation in mammalian reproduction.

Abbreviations

circRNAs

circular RNAs

ceRNAs

competitive endogenous RNAs

GCs

Granulosa cells

miRNAs

microRNAs

ncRNAs

non-coding RNAs

RBPs

RNA-binding proteins

CDS

Coding sequences

ChIP

Chromatin immunoprecipitation

RTCA

Real-time cell analyzer

FBS

Fetal bovine serum

CCK-8

Cell Counting Kit-8

CLSM

Confocal laser scanning microscopy

gDNA

genomic DNA

EciRNAs

Exonic circRNAs

ciRNAs

Intronic circRNAs

EIciRNAs

Exon-intron circRNAs

IRES

Internal ribosomal entry sites

ORFs

Open reading frames

Authors’ contributions

H. T.and Q. X. conceived and designed the experiments. H. T. wrote the manuscript. F. Z. and A.S S.G. collection of the sample. Z. N., Y. S., and H. Z. performed the experiments. J. Y. analyzed the data. Q. X. revised the draft. All authors commented on the manuscript.

Funding

This work was supported financially by National Natural Science Foundation of China (No.31972995), Hubei Province's Special Program for Centrally Guided Local Sci-tech Development (2024BBB112, 2025BEB061), Funds for Supporting High-Quality Development of Seed and Breeding Industry Projects in Hubei Province (HBZY2023B008), JD Technology Research and Development Project of Hubei Province (2023BAA029) and Hubei Provincial Science and Technology Program Project (2025EBA004, 2025EBA026). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability

The data used in this study are from the articles we published previously [34] (doi: 10.1016/j.ygeno.2017.10.005). All RNA-seq data have been deposited in the NCBI Gene Expression Omnibus (GEO accession number: GSE100773). The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.