Application of circWRNIP1 and related reagents thereof in regulation and control of follicular development of chicken

Abstract

The invention relates to the technical field of poultry breeding, and particularly provides application of circWRNIP1 and related reagents thereof in regulation and control of follicular development of chicken. Chicken primary granular cells cultured in vitro are used as test objects, circWRNIP1 is subjected to identification and structural analysis in the chicken granular cells cultured in vitro, the influence and action mechanism of circWRNIP1 on chicken granular cell proliferation and apoptosis are determined, functional circRNA capable of directly regulating chicken follicular development can be excavated, and the method has the advantages that the method is simple and convenient to implement, and the application value of the circWRNIP1 in chicken follicular cell proliferation and apoptosis can be increased. And a new theoretical reference is provided for developing molecular marker targeted breeding in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of poultry breeding, and in particular to application of circWRNIP1 and related reagents in regulating chicken follicle development. Background Art

[0002] Unlike mammals, which ovulate periodically after sexual maturity, the ovaries of laying hens are in different stages of development at the same time. This continuous egg-laying pattern is essential for laying hens to maintain high egg production. However, not all follicles can be successfully released, and they must go through a strict selection process. Follicle selection is a cyclical process in which only a few dominant follicles are selected to become preovulatory follicles, while most other follicles are eliminated and eventually atresia. The regulation of follicle selection can extend or shorten the egg-laying sequence and directly affect egg production. Granulosa cells (GCs) are an important component of follicles and play a vital role in ovarian follicle formation. The proliferation and differentiation of granulosa cells are essential for initiating the growth of primordial follicles, while the apoptosis of granulosa cells is considered to be the main reason for the atresia of follicles after the initiation of development. Importantly, both follicle selection and granulosa cell development are key biological processes in the development of ovarian follicles, which are profoundly affected by multiple regulatory factors.

[0003] Circular RNAs (circRNAs) are a class of RNA molecules that lack a 5' cap and a 3' poly (A) tail, which makes them more stable than linear RNAs in eukaryotes. Since the discovery of CDR1as, the first circRNA with regulatory potential, circRNAs have been found to generally act as molecular sponges for miRNAs to regulate transcription, encode proteins, and interact with proteins. Given these different functions, circRNAs are involved in a variety of biological processes, including tumor metastasis, lipid metabolism, and muscle formation. CircRNAs have been shown to play an important role in mammalian ovarian dysfunction. Recently, the key role of circRNAs in poultry ovarian development has been preliminarily established, and some circRNAs with regulatory potential were found to be dynamically expressed during chicken follicle development, marking the starting point for further research in this field. For example, aplacirc_13267 was shown to accelerate duck granulosa cell apoptosis by binding to the apla-miR-1-13 / THBS1 axis. However, research on circRNAs in poultry ovarian development, especially in chickens, is still limited. The present invention aims to further broaden the relevant research on circRNAs in chicken ovarian development. Summary of the invention

[0004] We performed circRNA RNA-seq and RNA-seq analysis to identify differentially expressed circRNAs (DECs) between healthy and atretic chicken follicles and found several functional DECs. We then identified a novel circRNA from the back-spliced ​​transcripts of exon 2 and exon 3 of the Werner helicase interacting protein 1 (WRNIP1) gene, named circWRNIP1.

[0005] CircWRNIP1 has a higher expression abundance in healthy follicles than in atretic follicles. By analyzing its expression during follicular development, it was found that circWRNIP1 is highly expressed in pre-fractional follicles and F1 GCs. Based on these findings, the present invention speculates that circWRNIP1 plays an important role in follicle selection and maturation, thereby regulating chicken follicle development.

[0006] Based on this, the present invention provides the use of circWRNIP1 and related reagents in regulating chicken follicle development.

[0007] In order to achieve the above object, the technical solution of the present invention is: In a first aspect, the present invention provides the use of an agent for enhancing or promoting the expression of the circWRNIP1 gene in the preparation of a product for promoting chicken follicle development, wherein the agent has a sequence as shown in SEQ ID NO. 19.

[0008] Preferably, the circWRNIP1 gene has the sequence shown in SEQ ID NO.46.

[0009] Preferably, the target miRNA of the circWRNIP1 gene is miR-129-5p.

[0010] Preferably, the target gene of miR-129-5p is IGF2.

[0011] In a second aspect, the present invention provides an agent for reducing or inhibiting the expression of miR-129-5p or an agent for enhancing the adsorption of miR-129-5p for use in the preparation of a product for promoting chicken follicle development, wherein the agent has a sequence as shown in SEQ ID NO. 17.

[0012] In the present invention, the product includes but is not limited to a kit, a drug or a drug preparation.

[0013] In a third aspect, the present invention provides a product for promoting the development of chicken follicles, comprising: a sequence shown in SEQ ID NO. 19 and / or SEQ ID NO. 17, and necessary cell transfection tools.

[0014] In a fourth aspect, the present invention provides a method for promoting chicken follicle development for non-therapeutic and non-diagnostic purposes, comprising: transfecting a sequence as shown in SEQ ID NO. 19 into chicken granulosa cells.

[0015] In a fifth aspect, the present invention provides a method for promoting chicken follicle development for non-therapeutic and non-diagnostic purposes, comprising: transfecting a sequence as shown in SEQ ID NO. 17 into chicken granulosa cells.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses in vitro cultured chicken primary granulosa cells as experimental objects, identifies and structurally analyzes circWRNIP1 in in vitro cultured chicken granulosa cells, and determines the effects and mechanism of circWRNIP1 on the proliferation and apoptosis of chicken granulosa cells. The present invention helps to discover functional circRNA that can directly regulate the development of chicken follicles, and provides a new theoretical reference for future molecular marker targeted breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 The structural identification and expression profile detection results of circWRNIP1 in chicken ovarian follicles in Example 1 of the present invention, wherein A is the sequencing result of circWRNIP1, B is the RNase R resistance of circWRNIP1, C is the expression result of circWRNIP1 amplification, D is the expression amount of circWRNIP1 detected by RNA-seq, E is the expression amount of circWRNIP1 detected by qPCR, and F is the enrichment result of circWRNIP1 in follicular cells and granulosa cells.

[0018] Figure 2The results of circWRNIP1 promoting the proliferation of chicken ovarian follicle granulosa cells (granulosa cells) in Example 1 of the present invention, wherein A is the expression result of circWRNIP1 overexpression plasmid in granulosa cells, B is the expression result of small interfering RNA knocking down circWRNIP1 in granulosa cells, C is the relative expression level of proliferation-related mRNA after circWRNIP1 overexpression, D is the relative expression level of proliferation-related mRNA after circWRNIP1 knockdown, E is the change in granulosa cell ratio after circWRNIP1 overexpression, F is the change in granulosa cell ratio after circWRNIP1 knockdown, G is the change in EdU-positive cell ratio after circWRNIP1 overexpression, and F is the change in EdU-positive cell ratio after circWRNIP1 knockdown.

[0019] Figure 3 These are the results of circWRNIP1 inhibiting apoptosis of chicken ovarian follicle granulosa cells in Example 1 of the present invention, wherein A is the change in relative mRNA expression after circWRNIP1 overexpression, B is the change in relative mRNA expression after circWRNIP1 knockdown, C is the change in relative protein abundance after circWRNIP1 overexpression, D is the change in relative protein abundance after circWRNIP1 knockdown, E is the protein immunoblotting result after circWRNIP1 overexpression and knockdown, F is the TUNEL staining detection result after circWRNIP1 overexpression, and G is the TUNEL staining detection result after circWRNIP1 knockdown.

[0020] Figure 4 The results of miR-129-5p being targeted by circWRNIP1 and inhibiting the proliferation of chicken granulosa cells in Example 1 of the present invention, wherein A is the change in the relative expression of mRNA after circWRNIP1 overexpression, B is the change in the relative expression of mRNA after knocking down circWRNIP1, C is the expression level of miR-129-5p in atretic ovarian follicles and healthy ovarian follicles, D is the prediction of the binding site of miR-129-5p on the circWRNIP1 sequence, E is the result of dual luciferase reporter gene detection, and F is the result of miR-129-5p and miR- 129-5p mimics and the association results of miR-129-5p inhibitors, G is the qPCR analysis result of the mRNA relative expression of miR-129-5p mimics, H is the qPCR analysis result of the mRNA relative expression of miR-129-5p inhibitors, I is the flow cytometry detection result of miR-129-5p mimics, J is the flow cytometry detection result of miR-129-5p inhibitors, K is the EdU staining detection result of miR-129-5p mimics, and L is the EdU staining detection result of miR-129-5p inhibitors.

[0021] Figure 5 The results of miR-129-5p promoting apoptosis of chicken ovarian follicle granulosa cells in Example 1 of the present invention, wherein A is the change in expression level of apoptosis-related genes after transfection with miR-129-5p mimics, B is the change in expression level of apoptosis-related genes after transfection with miR-129-5p inhibitors, C is the change in relative protein abundance after transfection with miR-129-5p mimics, D is the change in relative protein abundance after transfection with miR-129-5p inhibitors, E is the protein immunoblotting results after transfection with miR-129-5p mimics and miR-129-5p inhibitors, F is the staining result of apoptotic cells after transfection with miR-129-5p mimics, and G is the staining result of apoptotic cells after transfection with miR-129-5p inhibitors.

[0022] Figure 6 The results of confirmation that IGF2 is an ideal target gene of miR-129-5p in chicken granulosa cells in Example 1 of the present invention, wherein A is the change in the level of certain specific proteins after transfection of miR-129-5p mimics, B is the change in the level of certain specific proteins after transfection of miR-129-5p inhibitors, C is the prediction of the binding site between IGF2 and miR-129-5p, D is the result of dual luciferase reporter gene detection, E is the relative expression of IGF2 overexpression plasmid in follicles, F is the relative expression of three siRNAs of IGF2 in follicles, G is the Western blot detection result of pcDNA3.1-IGF2, H is the Western blot detection result of si-IGF2, and I is the protein immunoblotting result after transfection of pcDNA3.1-IGF2 and si-IGF2.

[0023] Figure 7 The results of IGF2 promoting the proliferation of chicken follicle granulosa cells and inhibiting cell apoptosis in Example 1 of the present invention, wherein A is the relative expression result of mRNA in cells after IGF2 overexpression, B is the relative expression result of mRNA in cells after IGF2 interference, C is the cell ratio result after IGF2 overexpression, D is the cell ratio result after IGF2 interference, E is the cell staining result after IGF2 overexpression, F is the cell staining result after IGF2 interference, G is the Caspase3 mRNA level of cells after IGF2 overexpression, H is the Caspase3 mRNA level of cells after IGF2 interference, I is the relative protein abundance result after IGF2 overexpression, J is the relative protein abundance result after IGF2 interference, K is the protein immunoblotting result after IGF2 overexpression and IGF2 interference, and L is the cell staining result after IGF2 overexpression and IGF2 interference.

[0024] Figure 8The results of the regulation of IGF2 on granulosa cell proliferation and apoptosis by the circWRNIP1 / miR-129-5p axis in Example 1 of the present invention, wherein A is the phosphorylation level of AKT (p-AKT) and ERK1 / 2 (p-ERK1 / 2) proteins after overexpression or interference of IGF2 in granulosa cells, B is the change of p-AKT / AKT after IGF2 knockdown, C is the change of p-ERK / ERK after IGF2 knockdown, D is the level of p-AKT and p-ERK1 / 2 after pcDNA3.1-IGF2 + miR-129-5p mimics and miR-129-5p mimics + pCD25-circWRNIP1 co-transfection of granulosa cells, E is the effect of miR-129-5p mimics on p-AKT / AKT, and F is the effect of miR-129-5p mimics on p-ERK1 / 2 / The effect of miR-129-5p mimics + pCD25-circWRNIP1 on p-AKT / AKT, and H shows the effect of miR-129-5p mimics + pCD25-circWRNIP1 on p-ERK1 / 2 / ERK1 / 2. DETAILED DESCRIPTION

[0025] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0026] In the following examples, the test subjects used are: Tianfu broiler TF-01 pure paternal breeder chickens bred in the poultry breeding farm of Sichuan Agricultural University, which have good egg-laying performance. The present invention selects Tianfu broiler TF-01 pure paternal hens raised under the same conditions, with basically the same weight and laying time, and between 200-250 days of age (peak egg-laying period) as test animals. The test chickens were taken from the poultry breeding farm of the Animal Genetics and Breeding Research Institute of Sichuan Agricultural University. All animal experiments in this study have passed the review of the Experimental Animal Ethics Committee of Sichuan Agricultural University, in line with the principles of animal protection, and in line with the relevant provisions of the national animal welfare ethics.

[0027] According to the egg-laying situation of Tianfu broilers, chickens that have not laid eggs for a week, have fluffy fur and are in a resting state are selected as nesting chickens. The ovaries of normal egg-laying hens are plump, while the ovaries of nesting chickens are obviously atrophied, with obvious characteristics and easy to distinguish. Three normal laying hens and nesting chickens of Tianfu broilers aged 200-250 days were selected, and the small white follicles (SWF), large white follicles (LWF), small yellow follicles (SYF), large yellow follicles (LYF) to F1 follicles of normal laying hens and nesting chickens were collected and classified (F1 follicles retain the egg membrane layer and granulosa layer) to detect the circRNA expression profile. The yolk was squeezed out, rinsed with PBS, quickly frozen in liquid nitrogen, and stored in a -80℃ refrigerator for quantitative verification.

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The present invention provides an application of circWRNIP1 and related reagents in regulating chicken follicle development. The specific test method is as follows: 1. Cell culture and transfection Take the hens at the peak of egg production, bleed the neck and kill them, separate the graded follicles F1-F5 from the ovaries, remove the blood vessels and connective tissue on the surface of the follicles, rinse with sterile phosphate buffered saline (PBS) and transfer to the cell chamber to further separate the membrane layer and granular layer, wash the separated granular cell layer with sterile PBS, and cut it into pieces until it is almost invisible to the naked eye. Add 5-10mL 0.1% type II collagenase, digest at 37℃ for 5-10 min (shake several times during the period until there are no visible tissue blocks in the tube, always pay attention to the cell state to avoid over-digestion), then add M199 basal medium to terminate digestion, centrifuge at 2000 r / min for 5 min, and discard the supernatant. Resuspend the cells with 20 mL complete medium (M199, 10% FBS, 1% double antibody), then filter with a 200 mesh (70μm) cell sieve, collect the filtrate and centrifuge at 2000 r / min for 5 min, and discard the supernatant. Add complete medium to resuspend 12 cells and plate them. Then place them in a constant temperature incubator and culture them for 3 h at 5% CO2, saturated humidity, and 37°C. After the cells are completely attached to the wall, change the medium, remove the cells that are not attached to the wall, and continue to culture.

[0030] The present invention designs RNA oligonucleotides and overexpression plasmids based on the circWRNIP1 sequence (SEQ ID NO.46), miR-129-5p and IGF2. The pCD25-ciR vector (pCD25-circWRNIP1) is used to construct the circWRNIP1 overexpression plasmid and the relative negative control (pCD25-ciR-NC). pcDNA3.1 is used to construct the IGF2 overexpression plasmid (pcDNA3.1-IGF2) and the relative negative control (ov-NC). All overexpression plasmids were constructed by Qingke Biotechnology Co., Ltd. (Beijing, China), and all RNA oligonucleotides were synthesized by GenePharma Co., Ltd. (Shanghai, China). Lipofectamine 3000 (Invitrogen, Carlsbad, CA) and Opti-MEM (Gibco, Grand Island, NY) were transfected according to the instructions. The constructed RNA oligonucleotides and overexpression plasmid sequences are shown in Table 1 and Table 2, respectively: Table 1 circWRNIP1, miR-129-5p, IGF2 RNA oligonucleotide sequences

[0031] Note: The sequences in Tables 1 and 3 have 'u' replaced by 'w' in the sequence listing.

[0032] Table 2 Sequences of circWRNIP1 and IGF2 overexpression plasmids

[0033] 2. Total RNA Extraction and Real-time Quantitative PCR Total RNA was extracted from follicular tissue and granulosa cells using RNAiso Plus (Takara, Dalian, China) according to the manufacturer's instructions. The purity and concentration of RNA were determined after extraction. The concentration-normalized RNA was then reverse transcribed. miRNA was synthesized using the One-Step miRNA cDNA Synthesis Kit, and cDNA was synthesized using the TransScript One-StepgDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China). qPCR detection was performed using TB Green PCR Master Mix (Takara).

[0034] 3. CircRNA Identification First, the circWRNIP1 PCR product amplified using divergent primers was sent to Sangon Biotech Co., Ltd. for Sanger sequencing. For RNase R treatment (Lucigen, Middleton, WI, USA), total RNA was extracted from chicken ovarian tissue, and the total RNA was treated with RNase R at 37°C for 10 min according to the kit instructions, and RNase R was inactivated at 90°C for 10 min. RNase R-treated RNA and untreated RNA were reverse transcribed separately, and then RT-qPCR was used to detect the resistance of circWRNIP1 and β-actin to RNase R. Random primers (N6) and oligo-d(T)18 primers (Qingke) were used for reverse transcription, and the abundance of circWRNIP1 in the two cDNA templates was detected by qPCR. Combined with the above treatments, the circular structure of circWRNIP1 was effectively demonstrated.

[0035] 4. 5-Ethyl-2'-deoxyuridine (EdU) assay EdU detection was performed using the Cell-Light EdU Apollo567 in vitro kit (RiboBio, Guangzhou, China). After labeling granulosa cells with EdU reagent A for 3 h, the cells were fixed with 4% paraformaldehyde and infiltrated with a permeabilizing agent (0.5% TritoX-100 diluted in PBS) to increase the permeability of the membrane and promote the adsorption of EdU dye. Finally, DAPI was incubated for 30 min and fluorescence detection was performed. The results were analyzed using Image-Pro Plus software.

[0036] 5. Flow cytometry cell cycle analysis Granulocytes cultured for flow cytometry cell cycle analysis were first digested with 0.25% trypsin-EDTA (diluted with an equal volume of PBS) at 37°C for 2 min, and then fixed with 70% ethanol at 4°C overnight. PI / RNase staining buffer (BD Biosciences, Franklin Lakes, USA) was prepared according to the instructions, and then the granulocytes were incubated at 37°C for 15 min. Cell cycle analysis was then performed.

[0037] 6. Western Blot 48 h after transfection, total proteins were extracted from granulosa cells using a total protein extraction kit (BestBio, Shanghai, China). Protein samples were quantified using a BCA protein assay kit (BestBio) before gel electrophoresis. Denatured proteins were separated by SDS-PAGE to achieve separation of target proteins. Subsequently, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and then blocked with a sealing solution. Afterwards, the membrane was washed with PBS and then incubated with specific primary antibodies overnight at 4 °C. The next day, the membrane was washed and incubated with the corresponding secondary antibodies for 1 h at 4 °C. Signal detection was performed using an enhanced chemiluminescence (ECL) reagent (Beyotime, Shanghai, China).

[0038] 7. TUNEL cell apoptosis staining The apoptotic rate of granulosa cells was evaluated using the terminal deoxynucleotidyl transferase dUTP nickel end labeling (TUNEL) assay kit (Beyotime). After 48 h of transfection, granulosa cells were fixed with 4% paraformaldehyde for 30 min and then permeabilized with 0.3% Triton X-100. The cells were incubated with TUNEL assay reagent at 37°C in the dark for 60 min. The TUNEL assay results were analyzed using Image-Pro Plus software.

[0039] 8. Dual luciferase reporter gene assay The binding sites of miR-129-5p seed sequence on circWRNIP1 and IGF2 were cloned into pmirGLO vector to construct dual luciferase reporter genes, including wild-type reporter genes (pmirGLO-circWRNIP1- wt, pmirGLO-IGF2- wt) and corresponding mutant reporter genes (pmirGLO-circWRNIP1- mt, pmirGLO-IGF2- mt). The relative luciferase activity was detected using the dual luciferase reporter assay kit (Beyotime) according to the manufacturer's protocol.

[0040] 9. Statistical analysis The results of two independent groups were evaluated by t-test, and one-way analysis of variance was applied to the comparison of three or more groups. Statistical results are expressed as mean ± standard error (SEM). Statistical analysis was performed using SPSS 22 (SPSS, Inc., Chicago, USA). Data visualization was performed by GraphPad Prism 8 (GraphPad Software, San Diego, USA). Each test had three or more replicates. * P <0.05, ** P <0.01,a-d P <0.05 indicated that the difference was statistically significant in this study.

[0041] The above-described experimental process is further described below through the following three examples.

[0042] Example 1: The role of circWRNIP1 in regulating chicken follicle development is as follows: (1-1) Structural identification and expression profile detection of circWRNIP1 in chicken ovarian follicles.

[0043] Comparison of the circWRNIP1 sequence with the Ensembl database confirmed that circWRNIP1 originated from exon 2 and exon 3 of the WRNIP1 gene, and its splicing sites were successfully verified by Sanger sequencing ( Figure 1 A). Further analysis showed that circWRNIP1 had significantly higher resistance to RNase R cleavage compared with linear β-actin ( Figure 1 B). In addition, the expression level of circWRNIP1 amplified using Oligo d(T) primers was significantly lower than that of circWRNIP1 amplified using random primers, indicating that circWRNIP1 lacks a poly(A) tail ( Figure 1 C). These findings provide strong evidence that circWRNIP1 is a typical covalently closed circular RNA. RNA-seq and qPCR detection revealed that circWRNIP1 expression was more abundant in healthy follicles (LF) than in atretic follicles (BF) ( Figure 1 D and 1E). In addition, circWRNIP1 was significantly enriched in pre-hierarchical follicles and F1 granulosa cells throughout follicular development ( Figure 1 F). These results suggest that circWRNIP1 plays an important role in promoting the development of chicken ovarian follicles.

[0044] (1-2) circWRNIP1 promotes the proliferation of chicken ovarian follicle granulosa cells (GCs).

[0045] The present invention successfully constructed the circWRNIP1 overexpression plasmid pCD25-circWRNIP1. Among the small interfering RNAs that knocked down circWRNIP1, si-circWRNIP1-3 had the highest efficiency ( Figure 2A and 2B). These small interfering RNAs and overexpression vectors were used in subsequent experiments. The relative expression levels of proliferation-related mRNAs, including proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase 2 (CDK2), cyclin D1 (CCND1), and cyclin D2 (CCND2), were increased by upregulation of circWRNIP1 and decreased by knockdown of circWRNIP1 ( Figure 2 C and 2D). In addition, when circWRNIP1 levels were increased, the cell cycle of granulosa cells was significantly accelerated from the G0 / G1 phase to the G2 / M+S phase ( Figure 2 E), while the interference of circWRNIP1 would cause the cell cycle to remain in the G2 / M+S phase ( Figure 2 F). Similarly, after transfection with pCD25-circWRNIP1, the proportion of EdU-positive cells increased, while after transfection with si-circWRNIP1-3, the proportion of EdU-positive cells decreased ( Figure 2 G and 2H). The results clearly demonstrated that circWRNIP1 can promote the proliferation of chicken ovarian follicle granulosa cells.

[0046] (1-3) circWRNIP1 inhibits apoptosis of chicken ovarian granulosa cells.

[0047] After transfection of pCD25-circWRNIP1 and si-circWRNIP1-3, downstream qPCR, Western blot and TUNEL detection were performed respectively. On the one hand, at the mRNA transcription level, circWRNIP1 significantly inhibited the expression of the caspase family (Caspase3, Caspase8, Caspase9), but promoted the expression of the apoptosis inhibitor B-cell lymphoma-2 (BCL2) ( Figure 3 A and 3B). On the other hand, at the protein translation level, circWRNIP1 correspondingly inhibited the level of Caspase3 protein and upregulated the level of BCL2 protein ( Figure 3 C-3E). TUNEL staining revealed that pCD25-circWRNIP1 significantly reduced the number of apoptotic granulosa cells, while si-circWRNIP1-3 had the opposite effect ( Figure 3 F-3G). Therefore, circWRNIP1 can inhibit apoptosis of chicken granulosa cells. Using this gene, a series of products that promote chicken follicle development can be developed, such as kits, drugs and pharmaceutical preparations.

[0048] Example 2: miR-129-5p is targeted by circWRNIP1 and plays a role in regulating chicken follicle development, as follows: (2-1) Inhibit the proliferation of chicken granulosa cells.

[0049] Referring to the circRNA RNA-seq data published in the SRA database (accession number: PRJNA721929), potential target miRNAs of circWRNIP1 were displayed, and miR-96-5p, miR-129-5p, miR-212-5p, miR-490-3p, and miR-757 were randomly selected for subsequent detection. The relative expression validation results of miRNA showed that the expression of miR-129-5p was most consistent with the regulation results of circWRNIP1, indicating that miR-129-5p is the most promising downstream target of circWRNIP1 ( Figure 4 A and 4B). In addition, compared with circWRNIP1, the expression level of miR-129-5p was detected to be higher in atretic ovarian follicles of hatching chickens than in healthy ovarian follicles of laying hens ( Figure 4 C). In order to reveal the interaction between circWRNIP1 and miR-129-5p, the RNAhybrid method was first used to predict the binding sites of miR-129-5p on the circWRNIP1 sequence, and wild-type and mutant dual-luciferase vectors were constructed ( Figure 4 D). Dual luciferase reporter gene assay showed that miR-129-5p mimic significantly reduced the firefly / Renilla luciferase ratio of pmirGLO-circWRNIP1-WT, but had little effect on the firefly / Renilla luciferase ratio of pmirGLO-circWRNIP1-MT ( Figure 4 E). Therefore, miR-129-5p was determined by targeting circWRNIP1. Further validation confirmed that miR-129-5p was specifically upregulated by miR-129-5p mimics and specifically downregulated by miR-129-5p inhibitors ( Figure 4 F). Subsequently, qPCR analysis showed that miR-129-5p mimics significantly reduced the expression levels of proliferation-related genes, such as CDK2 and CCND1 ( Figure 4 G). In contrast, miR-129-5p inhibitors can increase the relative expression levels of CDK2 and CCND1 accordingly ( Figure 4 H). Flow cytometry analysis revealed that miR-129-5p could inhibit the progression of the cell cycle ( Figure 4 I-4J). Similarly, EdU staining detected that miR-129-5p mimics significantly reduced the number of EdU-positive cells, while miR-129-5p inhibitors significantly increased the proportion of EdU-positive cells ( Figure 4 K-4L). In conclusion, miR-129-5p is a downstream member of the circWRNIP1 molecular sponge and can inhibit the proliferation of chicken ovarian granulosa cells.

[0050] (2-2) miR-129-5p promotes apoptosis of chicken ovarian granulosa cells.

[0051] To evaluate the effect of miR-129-5p on apoptosis of chicken granulosa cells, we transfected miR-129-5p mimics and miR-129-5p inhibitor, respectively. Comprehensive analysis of the changes in the expression levels of apoptosis-related genes easily revealed that miR-129-5p mimics enhanced apoptosis of granulosa cells, while miR-129-5p inhibitor attenuated apoptosis of granulosa cells ( Figure 5 A and 5B). Western blot was used to detect the abundance of BCL2 and Casepase3 proteins. The proliferation expression of miR-129-5p decreased the protein gray level of BCL2, while the knockdown of miR-129-5p increased the protein gray level of BCL2 ( Figure 5 C-5E). In contrast, overexpression of miR-129-5p relatively promoted the level of Caspase3 protein, but miR-129-5p interference inhibited the level of Caspase3 protein ( Figure 5 C-5E). The results were consistent with those of qPCR analysis. In addition, apoptotic cells were stained with TUNEL reagent and it was found that miR-129-5p mimics increased the number of apoptotic granulosa cells ( Figure 5 F), while miR-129-5p inhibitor reduced the number of apoptotic granulosa cells (Figure 5G). Taken together, these results indicate that miR-129-5p is a positive regulator of apoptosis in granulosa cells of chicken ovarian follicles. Therefore, by reducing or inhibiting miR-129-5p expression, or enhancing miR-129-5p adsorption, a series of products that promote chicken follicle development, such as kits, drugs, and pharmaceutical preparations, can be developed.

[0052] (2-3) IGF2 is an ideal target gene of miR-129-5p in chicken granulosa cells.

[0053] circRNAs always compete with mRNA for binding to miRNAs and alleviate the inhibitory effect of miRNAs on mRNA. In this study, the target mRNAs of miR-129-5p were queried on the miRDB website to improve the interaction network of circRNAs / miRNAs / mRNA, and mRNAs reported to be related to ovarian follicle development were preferentially selected for subsequent verification. After transfection of miR-129-5p mimics and miR-129-5p inhibitors, the mRNA levels of neurotrophin receptor kinase 2 (NTRK2), high mobility group box 1 protein (HMGB1), autophagy-related protein 14-like protein (ATG14), autophagy-related protein 7-like protein (ATG7), insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), insulin-like growth factor 2 (IGF2) mRNA were detected in turn. Figure 6 A and 6B). The results confirmed that miR-129-5p mimics only downregulated IGF2 mRNA levels, and miR-129-5p inhibitors also upregulated IGF2 mRNA levels ( Figure 6 A and 6B). Subsequently, RNAhybrid software was used to predict the binding sites between IGF2 and miR-129-5p ( Figure 6 C). Based on the sequences of miR-129-5p and IGF2 binding sites, a dual luciferase reporter gene was constructed. The test showed that the miR-129-5p mimic strongly inhibited the luciferase ratio of Firefly / Renilla ( Figure 6 D). These results suggest that IGF2 is a downstream target of the circWRNIP1 / miR-129-5p axis.

[0054] Example 3: The role of IGF2 in regulating the development of chicken follicles is as follows: In order to accurately study the key function of IGF2 in the development of chicken ovarian follicles, an overexpression plasmid (pcDNA3.1-IGF2) was constructed ( Figure 6 E). Three siRNAs (si-IGF2-161, si-IGF2-239, and si-IGF2-344) were synthesized simultaneously, and it was found that si-IGF2-161 (later named si-IGF2) was the most effective siRNA ( Figure 6 F). In addition, Western blot analysis confirmed that pcDNA3.1-IGF2 could effectively promote the IGF2 protein level, while si-IGF2 could significantly downregulate the IGF2 protein level ( Figure 6 G-6I).

[0055] (3-1) IGF2 promotes the proliferation of chicken granulosa cells and inhibits cell apoptosis.

[0056] To investigate the regulatory effect of IGF2 on the proliferation of chicken granulosa cells, we used qPCR, flow cytometry cell cycle analysis, and EdU detection. First, we found that IGF2 overexpression promoted the expression levels of proliferation-related genes such as CDK2, CCND1, and CCND2, while IGF2 interference inhibited their expression levels ( Figure 7 A and 7B). Secondly, pcDNA3.1-IGF2 can accelerate the cell cycle of granulosa cells, while si-IGF2 can block the cell cycle of granulosa cells ( Figure 7 C and 7D). Similarly, IGF2 also increased the proportion of proliferating granulosa cells in chickens. pcDNA3.1-IGF2 upregulated the proportion of EdU-positive cells, while si-IGF2 downregulated the proportion of EdU-positive cells ( Figure 7 Finally, IGF2 was confirmed to be a positive regulator of chicken granulosa cell proliferation.

[0057] In order to analyze the regulatory effect of IGF2 on apoptosis of chicken ovarian follicle granulosa cells, qPCR, Western blot and TUNEL detection were performed, and the primer information of the genes involved is shown in Table 3.

[0058] Table 3 Primer information

[0059] On the one hand, IGF2 overexpression can effectively reduce Caspase3 mRNA levels and increase BCL2 mRNA levels ( Figure 7 G), while IGF2 knockdown significantly upregulated Caspase3 and Caspase8 mRNA levels and inhibited BCL2 mRNA levels ( Figure 7 H). On the other hand, pcDNA3.1-IGF2 also decreased the protein level of Caspase3, while si-IGF2 increased the protein level of Caspase3 ( Figure 7 I -7K). In contrast, after pcDNA3.1-IGF2 transfection of granulosa cells, the BCL2 protein level increased, while after si-IGF2 transfection, the BCL2 protein level decreased ( Figure 7 H -7J). Further TUNEL staining confirmed that IGF2 overexpression reduced the number of apoptotic granulosa cells ( Figure 7 L), while IGF2 interference increased the number of apoptotic granulosa cells ( Figure 7 M). Therefore, in this study, IGF2 was considered to be an inhibitor of apoptosis in chicken granulosa cells. Similarly, using this gene, a series of products that promote chicken follicle development can be developed, such as kits, drugs and pharmaceutical preparations.

[0060] (3-2) IGF2 is a key downstream element of the circWRNIP1 / miR-129-5p axis and regulates chicken follicle development by interacting with the PI3K-AKT and ERK1 / 2 signaling pathways.

[0061] In order to understand the mechanism by which circWRNIP1 regulates the development of chicken ovarian follicles, the present invention explored the downstream signaling pathways associated with IGF2. First, the IGF2-enriched signaling pathways, such as the MAPK signaling pathway (map04010), the Ras signaling pathway (map04014), and the PI3K-AKT signaling pathway (map04151), were analyzed by KEGG (ORTHOLOGY: K13769). AKT and ERK are two important elements in these signaling pathways, which are widely reported to be involved in cell proliferation and programmed cell death. In the present invention, after IGF2 was overexpressed or interfered in granulosa cells, the phosphorylation levels of AKT (p-AKT) and ERK1 / 2 (p-ERK1 / 2) proteins were detected ( Figure 8 A). The results showed that pcDNA3.1-IGF2 upregulated the ratios of p-AKT / AKT and p-ERK / ERK, while IGF2 knockdown decreased the ratios of p-AKT / AKT and p-ERK / ERK ( Figure 8 In addition, we measured the levels of p-AKT and p-ERK1 / 2 in granulosa cells after co-transfection of pcDNA3.1-IGF2 + miR-129-5p mimics and miR-129-5p mimics + pCD25-circWRNIP1 ( Figure 8 D). The results showed that miR-129-5p mimics alleviated the promoting effect of pcDNA3.1-IGF2 on p-AKT and p-ERK1 / 2 phosphorylation ( Figure 8 E and 8F). In contrast, the inhibitory effect of miR-129-5p mimic on p-AKT and p-ERK1 / 2 phosphorylation was alleviated by pCD25-circWRNIP1 ( Figure 8 G and 8H). Taken together, these results indicate that circWRNIP1 promotes granulosa cell proliferation and inhibits granulosa cell apoptosis by binding to the miR-129-5p / IGF2 / PI3K-AKT and miR-129-5p / IGF2 / ERK1 / 2 signaling pathways.

[0062] In summary, the present invention confirmed that circWRMIP1 is a covalently closed circular RNA that is upregulated in healthy follicles compared with atretic follicles. CircWRNIP1 exhibits spatiotemporal-specific expression patterns during follicular development, especially enriched in pre-grading follicles and F1 granulosa cells. In addition, circWRNIP1 promotes granulosa cell proliferation and inhibits apoptosis through a classic competitive endogenous RNA (ceRNA) mechanism. Finally, a molecular interaction network of the circWRNIP1 / miR-129-5p / IGF2 axis was established, involving PI3K-AKT and ERK1 / 2 signals, providing new insights into the role of circRNAs in regulating ovarian follicle development.

[0063] Furthermore, in the present invention, upregulation of circWRNIP1 in healthy follicles was shown to increase the survival rate of granulosa cells, thereby playing a positive regulatory role in follicular development. Chicken follicular development involves recruitment, selection and dominance. Follicular selection is a cyclical process by which developing follicles are selected to become preovulatory follicles and continue their maturation until ovulation. The present invention demonstrates that circWRNIP1 is significantly upregulated in pre-fractional follicles and F1 granulosa cells compared to other follicular stages, indicating its activity in regulating follicle selection and ovulation.

[0064] In the present invention, IGF2 is identified as a target gene of the circWRNIP1 / miR-129-5p axis, promoting chicken follicle development. The present invention proves that circWRNIP1 activates the PI3K-AKT and ERK1 / 2 signaling pathways through the miR-129-5p / IGF2 axis, thereby promoting granulosa cell proliferation and inhibiting apoptosis, ultimately achieving its function of improving chicken follicle development.

[0065] Therefore, the newly discovered circWRNIP1, miR-129-5p, and IGF2 in chicken follicle development are expected to become potential molecular targets for improving chicken reproductive performance through molecular breeding strategies.

[0066] Although the embodiments of the present invention have been shown and described, it is understood that a person of ordinary skill in the art may make various changes, modifications, substitutions and variations to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the attached claims and their equivalents. The above-described embodiments only express several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that a person of ordinary skill in the art may make several variations and improvements without departing from the concept of the present invention, and these all belong to the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the attached claims.

Claims

1. Use of an agent for enhancing or promoting the expression of circWRNIP1 gene in the preparation of a product for promoting chicken follicle development, wherein the agent has a sequence as shown in SEQ ID NO.

19.

2. The use according to claim 1, characterized in that: The circWRNIP1 gene has a sequence as shown in SEQ ID NO.

46.

3. The use according to claim 1, characterized in that: The target miRNA of the circWRNIP1 gene is miR-129-5p.

4. The use according to claim 3, characterized in that: The target gene of miR-129-5p is IGF2.

5. Use of an agent that reduces or inhibits the expression of miR-129-5p or an agent that enhances the adsorption of miR-129-5p in the preparation of a product for promoting chicken follicle development, wherein the agent has a sequence as shown in SEQ ID NO.

17.

6. A product for promoting the development of chicken follicles, characterized in that: include: The sequence shown in SEQ ID NO. 19 and / or SEQ ID NO. 17, and necessary cell transfection tools.

7. A method for promoting chicken follicle development for non-therapeutic and non-diagnostic purposes, characterized in that: include: The sequence shown in SEQ ID NO. 19 was transfected into chicken granulosa cells.

8. A method for promoting chicken follicle development for non-therapeutic and non-diagnostic purposes, characterized in that: include: The sequence shown in SEQ ID NO. 17 was transfected into chicken granulosa cells.

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