Application of circWRNIP1 and Its Related Reagents in Regulating Chicken Follicle Development

Abstract

The present invention relates to the technical field of poultry breeding, and specifically provides the application of circWRNIP1 and its related reagents in regulating chicken follicular development. Taking primary chicken granulosa cells cultured in vitro as the experimental object, the present invention identifies and analyzes the structure of circWRNIP1 in chicken granulosa cells cultured in vitro, determines the effects and mechanisms of circWRNIP1 on the proliferation and apoptosis of chicken granulosa cells. The present invention helps to discover functional circRNAs that can directly regulate chicken follicular development, providing a new theoretical reference for future molecular marker-targeted breeding.

Description

Technical Field

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

[0002] Different from the periodic ovulation of mammals after sexual maturity, the ovaries of laying hens are simultaneously at different developmental stages. This continuous egg-laying pattern is crucial for laying hens to maintain high egg production. However, not all follicles can be successfully ovulated, and they must undergo a strict selection process. Follicle selection is a periodic process in which only a few dominant follicles are selected to become preovulatory follicles, while most of the other follicles are eliminated and eventually atresia. The regulation of follicle selection can extend or shorten the egg-laying sequence, directly affecting egg production. Granulosa cells (GCs) are an important part of follicles and play a crucial role in ovarian follicle formation. The proliferation and differentiation of granulosa cells are crucial for initiating the growth of primordial follicles, while the apoptosis of granulosa cells is considered the main cause of follicular atresia after the initiation of development. Importantly, both follicle selection and granulosa cell development are key biological processes in ovarian follicle development and are profoundly affected by various regulatory factors.

[0003] Circular RNAs (circRNAs) are a class of RNA molecules lacking 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 participate in regulating transcription, encoding proteins, and interacting with proteins as miRNA molecular sponges. 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 have been found to be dynamically expressed during chicken follicular development, marking the starting point for further research in this field. For example, aplacirc_13267 has been shown to accelerate duck granulosa cell apoptosis by binding to the appla - mir -1-13 / THBS1 axis. However, the research on circRNAs in poultry ovarian development, especially in chickens, is still limited. The present invention aims to further broaden the related research on circRNAs in chicken ovarian development. Summary of the Invention

[0004] The present invention performed circRNA RNA-seq and RNA-seq analyses to identify circular RNAs (DECs) differentially expressed between healthy and atretic chicken follicles, and several functional DECs were discovered. Subsequently, the present invention identified a novel circRNA from the back-spliced transcripts of exons 2 and 3 of the Werner helicase interacting protein 1 (WRNIP1) gene, named circWRNIP1.

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

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

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] In the first aspect, the present invention provides the application of a reagent that enhances or promotes the expression of the circWRNIP1 gene in the preparation of a product for promoting chicken follicular development, and the reagent has the sequence shown in SEQ ID NO. 19.

[0009] Preferably, the circWRNIP1 gene has the sequence shown in SEQ ID NO. 46.

[0010] Preferably, the target miRNA of the circWRNIP1 gene is miR-129-5p.

[0011] Preferably, the target gene of miR-129-5p is IGF2.

[0012] In the second aspect, the present invention provides the application of a reagent that reduces or inhibits the expression of miR-129-5p or a reagent that enhances the adsorption of the miR-129-5p in the preparation of a product for promoting chicken follicular development, and the reagent has the sequence shown in SEQ ID NO. 17.

[0013] In the present invention, the product includes but is not limited to a kit, a drug, or a pharmaceutical preparation.

[0014] In the third aspect, the present invention provides a product for promoting chicken follicular development, including: the sequences shown in SEQ ID NO. 19 and / or SEQ ID NO. 17, and necessary cell transfection tools.

[0015] Fourth aspect, the present invention provides a method for promoting chicken follicle development for non-therapeutic and non-diagnostic purposes, including: transfecting the sequence shown in SEQ ID NO. 19 into chicken granulosa cells.

[0016] Fifth aspect, the present invention provides a method for promoting chicken follicle development for non-therapeutic and non-diagnostic purposes, including: transfecting the sequence shown in SEQ ID NO. 17 into chicken granulosa cells.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention takes the in vitro cultured primary chicken granulosa cells as the experimental object, identifies and analyzes the structure of circWRNIP1 in the in vitro cultured chicken granulosa cells, determines the influence and mechanism of action of circWRNIP1 on the proliferation and apoptosis of chicken granulosa cells. The present invention helps to discover functional circRNAs that can directly regulate chicken follicle development, providing a new theoretical reference for future molecular marker-targeted breeding. Brief Description of the Drawings

[0019] The drawings described herein are used to provide a further understanding of the present invention, form a part of the present invention, and the illustrative embodiments and descriptions thereof are used to explain the present invention without unduly limiting the present invention. In the drawings:

[0020] Figure 1 It is the result of the structure identification and expression profile detection of circWRNIP1 in chicken ovarian follicles in Example 1 of the present invention. Among them, A is the sequencing result of circWRNIP1, B is the RNase R digestion resistance of circWRNIP1, C is the expression level result of circWRNIP1 amplification, D is the expression level of circWRNIP1 detected by RNA-seq, E is the expression level of circWRNIP1 detected by qPCR, and F is the enrichment result of circWRNIP1 in follicle cells and granulosa cells.

[0021] Figure 2Results of circWRNIP1 promoting the proliferation of chicken ovarian follicular granulosa cells (granulosa cells) in Example 1 of the present invention. Among them, A is the expression result of the circWRNIP1 overexpression plasmid in granulosa cells, B is the expression result of the small interfering RNA knocking down circWRNIP1 in granulosa cells, C is the relative expression level of proliferation-related mRNAs after overexpression of circWRNIP1, D is the relative expression level of proliferation-related mRNAs after knocking down circWRNIP1, E is the change in the granulosa cell ratio after overexpression of circWRNIP1, F is the change in the granulosa cell ratio after knocking down circWRNIP1, G is the change in the ratio of EdU-positive cells after overexpression of circWRNIP1, and F is the change in EdU-positive cells after knocking down circWRNIP1.

[0022] Figure 3 Results of circWRNIP1 inhibiting the apoptosis of chicken ovarian follicular granulosa cells in Example 1 of the present invention. Among them, A is the change in the relative expression level of mRNA after overexpression of circWRNIP1, B is the change in the relative expression level of mRNA after knocking down circWRNIP1, C is the change in the relative abundance of protein after overexpression of circWRNIP1, D is the change in the relative abundance of protein after knocking down circWRNIP1, E is the protein immunoblotting result after overexpression and knockdown of circWRNIP1, F is the TUNEL staining detection result after overexpression of circWRNIP1, and G is the TUNEL staining detection result after knocking down circWRNIP1.

[0023] Figure 4 Results of miR-129-5p being targeted by circWRNIP1 and inhibiting the proliferation of chicken granulosa cells in Example 1 of the present invention. Among them, A is the change in the relative expression level of mRNA after overexpression of circWRNIP1, B is the change in the relative expression level 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 dual-luciferase reporter gene detection result, F is the association result of miR-129-5p with miR-129-5p mimics and miR-129-5p inhibitors, G is the qPCR analysis result of the relative expression level of mRNA of miR-129-5p mimics, H is the qPCR analysis result of the relative expression level of mRNA 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.

[0024] Figure 5 This is the result of miR-129-5p promoting apoptosis of chicken ovarian follicular granulosa cells in Example 1 of the present invention. Among them, A shows the change in the expression level of apoptosis-related genes after transfection with miR-129-5p mimic, B shows the change in the expression level of apoptosis-related genes after transfection with miR-129-5p inhibitor, C shows the change in relative protein abundance after transfection with miR-129-5p mimic, D shows the change in relative protein abundance after transfection with miR-129-5p inhibitor, E shows the Western blot result after transfection with miR-129-5p mimic and miR-129-5p inhibitor, F shows the apoptosis cell staining result after transfection with miR-129-5p mimic, and G shows the apoptosis cell staining result after transfection with miR-129-5p inhibitor.

[0025] Figure 6 This is the result of confirming that IGF2 is an ideal target gene of miR-129-5p in chicken granulosa cells in Example 1 of the present invention. Among them, A shows the change in the level of certain specific proteins after transfection with miR-129-5p mimic, B shows the change in the level of certain specific proteins after transfection with miR-129-5p inhibitor, C shows the predicted binding site between IGF2 and miR-129-5p, D shows the result of dual-luciferase reporter gene detection, E shows the relative expression level of IGF2 overexpression plasmid in follicles, F shows the relative expression level of three siRNAs of IGF2 in follicles, G shows the Western blot detection result of pcDNA3.1-IGF2, H shows the Western blot detection result of si-IGF2, and I shows the Western blot result after transfection with pcDNA3.1-IGF2 and si-IGF2.

[0026] Figure 7 This is the result of IGF2 promoting the proliferation of chicken follicular granulosa cells and inhibiting apoptosis in Example 1 of the present invention. Among them, A shows the result of the relative mRNA expression level in cells after IGF2 overexpression, B shows the result of the relative mRNA expression level in cells after IGF2 interference, C shows the cell ratio result after IGF2 overexpression, D shows the cell ratio result after IGF2 interference, E shows the cell staining result after IGF2 overexpression, F shows the cell staining result after IGF2 interference, G shows the mRNA level of Caspase3 in cells after IGF2 overexpression, H shows the mRNA level of Caspase3 in cells after IGF2 interference, I shows the result of relative protein abundance after IGF2 overexpression, J shows the result of relative protein abundance after IGF2 interference, K shows the Western blot result after IGF2 overexpression and IGF2 interference, and L shows the cell staining result after IGF2 overexpression and IGF2 interference.

[0027] Figure 8This is the regulatory result of the circWRNIP1 / miR-129-5p axis on the proliferation and apoptosis of granulosa cells in Example 1 of the present invention. Among them, A is the phosphorylation levels 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 knockdown of IGF2, C is the change of p-ERK / ERK after knockdown of IGF2, D is the levels of p-AKT and p-ERK1 / 2 after co-transfection of pcDNA3.1-IGF2 + miR-129-5p mimics and miR-129-5p mimics + pCD25-circWRNIP1 into granulosa cells, E is the effect of miR-129-5p mimics on p-AKT / AKT, F is the effect of miR-129-5p mimics on p-ERK1 / 2 / ERK1 / 2, G is the effect of miR-129-5p mimics + pCD25-circWRNIP1 on p-AKT / AKT, and H is the effect of miR-129-5p mimics + pCD25-circWRNIP1 on p-ERK1 / 2 / ERK1 / 2. Detailed implementation manners

[0028] In the description of the present invention, it should be noted that for those not specified in the examples, the operations are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0029] In the following examples, the experimental subjects used are: Tianfu broiler TF-01 pure-line paternal breeding chickens cultivated in the Poultry Breeding Farm of Sichuan Agricultural University, which have good egg-laying performance. The present invention selects Tianfu broiler TF-01 pure-line paternal hens that are raised under the same conditions, have basically the same body weight and laying start time, and are between 200 and 250 days old (peak egg-laying period) as experimental animals. The experimental chickens are taken from the Poultry Breeding Farm of the Institute of Animal Genetics and Breeding of Sichuan Agricultural University. All animal experiments in this study have passed the review of the Experimental Animal Ethics Committee of Sichuan Agricultural University, conform to the principles of animal protection, and comply with the relevant regulations of national animal welfare ethics.

[0030] According to the egg-laying situation of Tianfu broiler chickens, chickens that did not lay eggs for a consecutive week, had fluffy feathers and were in a resting state were selected and judged as broody hens. The ovaries of normal egg-laying hens are plump, while those of broody hens are significantly atrophied, with obvious characteristics and easy to distinguish. Three normal egg-laying chickens and three broody chickens of Tianfu broiler chickens at 200 - 250 days old were selected respectively. Small white follicles (SWF), large white follicles (LWF), small yellow follicles (SYF), large yellow follicles (LYF) of normal egg-laying chickens and broody hens were collected, and each level up to the F1 follicle was classified (the F1 follicle retained the egg membrane layer and granulosa layer) to detect the circRNA expression profile. The egg yolk was extruded, rinsed with PBS, quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator for quantitative verification.

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0032] The embodiments of the present invention provide the application of circWRNIP1 and its related reagents in regulating chicken follicle development. The specific test methods are as follows:

[0033] 1. Cell culture and transfection

[0034] Hens at the peak egg-laying period were sacrificed by cervical bleeding. Hierarchical follicles F1 - F5 were isolated from the ovaries, and the blood vessels and connective tissues on the surface of the follicles were removed. After rinsing with sterile phosphate buffered saline (PBS), they were transferred to the cell room for further separation of the membrane layer and granulosa layer. The separated granulosa cell layer was washed clean with sterile PBS and minced until almost invisible to the naked eye. 5 - 10 mL of 0.1% type II collagenase was added, and digestion was carried out at 37°C for 5 - 10 min (shaking several times during the period until there were no visible tissue blocks in the tube, and always paying attention to the cell state to avoid over-digestion). Then, M199 basal medium was added to terminate digestion, and centrifugation was carried out at 2000 r / min for 5 min, and the supernatant was discarded. The cells were resuspended with 20 mL of complete medium (M199, 10% FBS, 1% double antibody), then filtered through a 200-mesh (70μm) cell sieve. After collecting the filtrate, centrifugation was carried out at 2000 r / min for 5 min, and the supernatant was discarded. The cells were resuspended with complete medium and plated. Then, it was placed in a constant temperature incubator and cultured under the conditions of 5% CO2, saturated humidity, and 37°C for 3 h. After the cells adhered, the medium was changed to remove the non-adherent cells and continue the culture.

[0035] The present invention designs RNA oligonucleotides and overexpression plasmids based on the circWRNIP1 sequence (SEQ ID NO.46), miR-129-5p, and IGF2. The circWRNIP1 overexpression plasmid and the relative negative control (pCD25-ciR-NC) were constructed using the pCD25-ciR vector (pCD25-circWRNIP1). pcDNA3.1 was used for the construction of the IGF2 overexpression plasmid (pcDNA3.1-IGF2) and the relative negative control (ov-NC). All overexpression plasmids were constructed by Tsingke 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 sequences of the constructed RNA oligonucleotides and overexpression plasmids are shown in Table 1 and Table 2 respectively:

[0036] Table 1 Sequences of circWRNIP1, miR-129-5p, and IGF2 RNA oligonucleotides

[0037]

[0038] Note: In the sequence listing, replace 'u' with 'w' in the sequences of Table 1 and Table 3.

[0039] Table 2 Sequences of circWRNIP1 and IGF2 overexpression plasmids

[0040]

[0041] 2. Total RNA extraction and real-time quantitative PCR

[0042] Total RNA was extracted from follicular tissues and granulosa cells using RNAiso Plus (Takara, Dalian, China) according to the manufacturer's instructions. After extraction, the purity and concentration of the RNA were measured. Subsequently, the concentration-normalized RNA was reverse-transcribed. miRNA was synthesized using the One-Step miRNA cDNA Synthesis Kit, and cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China). qPCR detection was performed using TB Green PCR Master Mix (Takara).

[0043] 3. CircRNA identification

[0044] First, the circWRNIP1 PCR products amplified using divergent primers were sent to Sangon Biotech Co., Ltd. for Sanger sequencing. For RNase R (Lucigen, Middleton, WI, USA) treatment, total RNA from chicken ovarian tissue was extracted, 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. The RNase R-treated RNA and untreated RNA were reverse-transcribed separately, and then the resistance of circWRNIP1 and β-actin to RNase R was detected by RT-qPCR. Random primers (N6) and oligo-d(T)18 primers (Tsingke) were used for reverse transcription respectively, and the abundance of circWRNIP1 in the two cDNA templates was detected by qPCR. Combining the above treatments, the circular structure of circWRNIP1 was effectively demonstrated.

[0045] 4, 5-Ethyl-2'-deoxyuridine (EdU) assay

[0046] The EdU detection was performed using the Cell-Light EdU Apollo567 In Vitro Kit (RiboBio, Guangzhou, China). After labeling granulosa cells with the EdU reagent A for 3 h, the cells were fixed with 4% paraformaldehyde and infiltrated with a permeabilizer (PBS diluted with 0.5% TritonX-100) to improve the membrane permeability and promote the adsorption of the EdU dye. Finally, the cells were incubated with DAPI for 30 min for fluorescence detection. The results were analyzed using Image-Pro Plus software.

[0047] 5, Flow cytometry cell cycle analysis

[0048] The granulosa cells 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. The PI / RNase staining buffer (BD Biosciences, Franklin Lakes, USA) was prepared according to the instructions in proportion, and then the granulosa cells were incubated at 37 °C for 15 min. Subsequently, cell cycle analysis was performed.

[0049] 6, Western blot

[0050] At 48 h after transfection, total protein was extracted from granulosa cells using a total protein extraction kit (BestBio, Shanghai, China). Before performing gel electrophoresis, the protein samples were quantified using a BCA protein assay kit (BestBio). The denatured proteins were separated by SDS-PAGE to achieve the separation of the target protein. Subsequently, these proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane and then blocked with a blocking solution. After that, the membrane was washed with PBS and then incubated with a specific primary antibody overnight at 4°C. The next day, the membrane was washed and incubated with the corresponding secondary antibody for 1 hour at 4°C. Signal detection was performed using an enhanced chemiluminescence (ECL) reagent (Beyotime, Shanghai, China).

[0051] 7. TUNEL cell apoptosis staining

[0052] The apoptosis rate of granulosa cells was evaluated using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit (Beyotime). At 48 h after 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 the TUNEL detection reagent in the dark at 37°C for 60 min. The TUNEL detection results were analyzed using Image-Pro Plus software.

[0053] 8. Dual-luciferase reporter assay

[0054] The binding sites of the miR-129-5p seed sequence on circWRNIP1 and IGF2 were cloned into the pmirGLO vector to construct dual-luciferase reporters, including wild-type reporters (pmirGLO-circWRNIP1-wt, pmirGLO-IGF2-wt) and corresponding mutant reporters (pmirGLO-circWRNIP1-mt, pmirGLO-IGF2-mt). The relative luciferase activity was detected using a dual-luciferase reporter assay kit (Beyotime) according to the manufacturer's protocol.

[0055] 9. Statistical analysis

[0056] The results of two independent groups were evaluated using a t-test, and one-way analysis of variance was applicable for comparisons of three or more groups. The statistical results were expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using SPSS 22 (SPSS, Inc., Chicago, USA). Data visualization was completed using 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 was statistically significant in this study.

[0057] Next, for the relevant experimental procedures described above, the following will be further illustrated by 3 examples.

[0058] Example 1: The role of circWRNIP1 in regulating chicken follicular development is as follows:

[0059] (1-1) Structural identification and expression profile detection of circWRNIP1 in chicken ovarian follicles.

[0060] The circWRNIP1 sequence was compared with the Ensembl database, and it was confirmed that circWRNIP1 originated from exons 2 and 3 of the WRNIP1 gene, and its splicing site was successfully verified by Sanger sequencing ( Figure 1 A). Further analysis showed that circWRNIP1 had significantly higher resistance to RNase R digestion compared with linear β-actin ( Figure 1 B). In addition, the expression level of circWRNIP1 amplified with Oligo d(T) primers was significantly lower than that amplified with random primers, indicating that circWRNIP1 lacked a poly(A) tail ( Figure 1 C). These findings provided strong evidence that circWRNIP1 is a typical covalently closed circular RNA. Detection by RNA-seq and qPCR found that circWRNIP1 expression was more abundant in healthy follicles (LF) than in atretic follicles (BF) ( Figure 1 D and 1E). In addition, during the entire follicular development process, circWRNIP1 was significantly enriched in pre-hierarchical follicles and F1 granulosa cells ( Figure 1 F). These results indicate that circWRNIP1 plays an important role in promoting chicken ovarian follicular development.

[0061] (1-2) circWRNIP1 promotes the proliferation of chicken ovarian follicular granulosa cells (granulosa cells).

[0062] 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 the upregulation of circWRNIP1 and decreased by the knockdown of circWRNIP1 ( Figure 2 C and 2D). In addition, when the level of circWRNIP1 was 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 caused the cell cycle to arrest in the G2 / M+S phase ( Figure 2 F). Similarly, after transfection with pCD25-circWRNIP1, the proportion of EdU-positive cells was upregulated, while after transfection with si-circWRNIP1-3, the proportion of EdU-positive cells was decreased ( Figure 2 G and 2H). The results clearly demonstrated that circWRNIP1 could promote the proliferation of chicken ovarian follicular granulosa cells.

[0063] (1 - 3) circWRNIP1 inhibits apoptosis of chicken ovarian follicular granulosa cells.

[0064] After transfection with pCD25-circWRNIP1 and si-circWRNIP1-3, downstream qPCR, Western blot, and TUNEL assays were performed respectively. On the one hand, at the mRNA transcription level, circWRNIP1 significantly inhibited the expression of the cysteine aspartic acid-specific protease 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 protein level of Caspase3 and upregulated the protein level of BCL2 ( Figure 3 C - 3E). TUNEL staining detected that pCD25-circWRNIP1 significantly reduced the number of apoptotic granulosa cells, while si-circWRNIP1-3 did the opposite ( Figure 3 F - 3G). Therefore, circWRNIP1 can inhibit apoptosis of chicken granulosa cells. Using this gene, a series of products for promoting chicken follicle development, such as kits, drugs, and pharmaceutical preparations, can be developed.

[0065] Example 2: miR-129-5p is targeted by circWRNIP1 and its role in regulating chicken follicle development is as follows:

[0066] (2-1)Inhibit the proliferation of chicken granulosa cells.

[0067] Refer to the circRNA RNA-seq data published in the SRA database (accession number: PRJNA721929), which shows the potential target miRNAs of circWRNIP1. miR-96-5p, miR-129-5p, miR-212-5p, miR-490-3p, and miR-757 were randomly selected for subsequent detection. The relative expression verification results of miRNAs showed that the expression of miR-129-5p was most consistent with the regulatory 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 the atretic ovarian follicles of incubated chickens than in the healthy ovarian follicles of laying hens ( Figure 4 C). To reveal the interaction relationship between circWRNIP1 and miR-129-5p, first, the RNAhybrid method was used to predict the binding site of miR-129-5p on the circWRNIP1 sequence, and wild-type and mutant dual-luciferase vectors were constructed ( Figure 4 D). The dual-luciferase reporter gene assay showed that miR-129-5p mimics significantly reduced the firefly / renilla luciferase ratio of pmirGLO-circWRNIP1-WT, while having little effect on the firefly / renilla luciferase ratio of pmirGLO-circWRNIP1-MT ( Figure 4 E). Therefore, miR-129-5p was determined to be targeted by circWRNIP1. Further verification 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 could correspondingly increase the relative expression levels of CDK2 and CCND1 ( Figure 4 H). Flow cytometry analysis found 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 4K-4L). In summary, miR-129-5p is a downstream member of the circWRNIP1 molecular sponge and can inhibit the proliferation of chicken ovarian follicular granulosa cells.

[0068] (2-2)miR-129-5p promotes apoptosis of chicken ovarian follicular granulosa cells.

[0069] 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. By comprehensively analyzing the changes in the expression levels of apoptosis-related genes, it is easy to clarify that miR-129-5p mimics enhance granulosa cell apoptosis, while miR-129-5p inhibitor weakens granulosa cell apoptosis ( Figure 5 A and 5B). The abundance of BCL2 and Casepase3 proteins was detected by western blot. The proliferative expression of miR-129-5p decreased the protein gray scale of BCL2, while the knockdown of miR-129-5p increased the protein gray scale of BCL2 ( Figure 5 C-5E). On the contrary, overexpression of miR-129-5p relatively promoted the protein level of Caspase3, while miR-129-5p interference inhibited the protein level of Caspase3 ( 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 decreased the number of apoptotic granulosa cells (Figure 5G). In summary, these results indicate that miR-129-5p is a positive regulator of apoptosis of chicken ovarian follicular granulosa cells. Therefore, by reducing or inhibiting the expression of miR-129-5p, or enhancing miR-129-5p adsorption, a series of products promoting chicken follicle development can be developed, such as kits, drugs and pharmaceutical preparations.

[0070] (2-3)IGF2 is an ideal target gene of miR-129-5p in chicken granulosa cells.

[0071] circRNAs always compete with mRNAs for binding to miRNAs and relieve the inhibitory effect of miRNAs on mRNAs. In this study, the target mRNAs of miR-129-5p were queried on the miRDB website to improve the circRNAs / miRNAs / mRNA interaction network, and mRNAs that had been reported to be related to ovarian follicle development were preferentially selected for subsequent verification. After transfection with miR-129-5p mimics and miR-129-5p inhibitors, the mRNA levels of neurotrophic tyrosine kinase receptor type 2 (NTRK2), high mobility group box 1 protein (HMGB1), autophagy related 14 like protein (ATG14), autophagy related 7 like protein (ATG7), insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), and insulin-like growth factor 2 (IGF2) were detected in sequence ( Figure 6 A and 6B). The results confirmed that miR-129-5p mimics only downregulated the IGF2 mRNA level, and miR-129-5p inhibitors also upregulated the IGF2 mRNA level ( Figure 6 A and 6B). Subsequently, the binding site between IGF2 and miR-129-5p was predicted by RNAhybrid software ( Figure 6 C). According to the sequences of the miR-129-5p and IGF2 binding sites, a dual-luciferase reporter gene was constructed, and the detection showed that miR-129-5p mimics strongly inhibited the Firefly / Renilla luciferase ratio ( Figure 6 D). These results indicate that IGF2 is a downstream target of the circWRNIP1 / miR-129-5p axis.

[0072] Example 3: The role of IGF2 in regulating chicken follicle development is as follows:

[0073] To accurately study the key function of IGF2 in chicken ovarian follicle development, an overexpression plasmid (pcDNA3.1-IGF2) was constructed ( Figure 6 E). At the same time, three siRNAs (si-IGF2-161, si-IGF2-239, si-IGF2-344) were synthesized, and it was found that si-IGF2-161 (subsequently named si-IGF2) was the most effective siRNA ( Figure 6 F). In addition, Western blot detection confirmed that pcDNA3.1-IGF2 could effectively promote the IGF2 protein level, and si-IGF2 could significantly downregulate the IGF2 protein level ( Figure 6 G-6I).

[0074] (3-1)IGF2 promotes the proliferation of chicken follicular granulosa cells and inhibits cell apoptosis.

[0075] To investigate the regulatory effect of IGF2 on the proliferation of chicken granulosa cells, we employed qPCR, flow cytometry cell cycle analysis, and EdU assay. First, it was found that overexpression of IGF2 promoted the expression levels of proliferation-related genes such as CDK2, CCND1, and CCND2, while interference with IGF2 inhibited their expression levels ( Figure 7 A and 7B). Second, pcDNA3.1-IGF2 accelerated the cell cycle of granulosa cells, while si-IGF2 blocked 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 E and 7F). Finally, IGF2 was confirmed to be a positive regulator of chicken granulosa cell proliferation.

[0076] To analyze the regulatory effect of IGF2 on apoptosis of chicken ovarian follicular granulosa cells, qPCR, Western blot, and TUNEL assay were respectively performed, and the primer information of the genes involved is shown in Table 3.

[0077] Table 3 Primer information

[0078]

[0079] On the one hand, overexpression of IGF2 effectively decreased the level of Caspase3 mRNA and increased the level of BCL2 mRNA ( Figure 7 G), while knockdown of IGF2 significantly upregulated the levels of Caspase3 and Caspase8 mRNA and inhibited the level of BCL2 mRNA ( 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 transfection of pcDNA3.1-IGF2 into granulosa cells, the protein level of BCL2 showed an upward trend, while after transfection of si-IGF2, the protein level of BCL2 decreased ( Figure 7 H -7J). Further TUNEL staining confirmed that overexpression of IGF2 reduced the number of apoptotic granulosa cells ( Figure 7 L), while interference with IGF2 increased the number of apoptotic granulosa cells ( Figure 7Therefore, in this study, IGF2 was considered as an inhibitor of chicken granulosa cell apoptosis. Similarly, using this gene, a series of products promoting chicken follicle development, such as kits, drugs and pharmaceutical preparations, can be developed.

[0080] (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.

[0081] To understand the mechanism by which circWRNIP1 regulates chicken ovarian follicle development, the present invention explored the downstream signaling pathways related to IGF2. First, the signaling pathways enriched by IGF2 were analyzed through KEGG (ORTHOLOGY: K13769), such as the MAPK signaling pathway (map04010), the Ras signaling pathway (map04014), and the PI3K-AKT signaling pathway (map04151). AKT and ERK are two important elements in these signaling pathways, and they have been widely reported to be involved in cell proliferation and programmed cell death. In the present invention, after overexpressing or interfering with IGF2 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 knockdown of IGF2 decreased the ratios of p-AKT / AKT and p-ERK / ERK ( Figure 8 B and 8C). In addition, we measured the levels of p-AKT and p-ERK1 / 2 after co-transfecting granulosa cells with 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 the phosphorylation of p-AKT and p-ERK1 / 2 ( Figure 8 E and 8F). In contrast, the inhibitory effect of miR-129-5p mimics on the phosphorylation of p-AKT and p-ERK1 / 2 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.

[0082] In summary, the present invention confirmed that circWRMIP1 is a covalently closed circular RNA, which is upregulated in healthy follicles compared with atretic follicles. CircWRNIP1 exhibits a spatiotemporal specific expression pattern during follicular development, especially enriched in pre-hierarchical follicles and F1 granulosa cells. In addition, circWRNIP1 promotes granulosa cell proliferation and inhibits apoptosis through the classical competitive endogenous RNA (ceRNA) mechanism. Finally, a molecular interaction network of the circWRNIP1 / miR-129-5p / IGF2 axis is established, involving PI3K-AKT and ERK1 / 2 signaling, providing new insights into the role of circRNAs in regulating ovarian follicular development.

[0083] In addition, in the present invention, the upregulation of circWRNIP1 in healthy follicles was shown to increase the survival rate of granulosa cells, thus playing a positive regulatory role in follicular development. Chicken follicular development involves recruitment, selection, and dominance. Follicle selection is a cyclic process through which developing follicles are selected to become preovulatory follicles and continue their maturation until ovulation. The present invention demonstrated that circWRNIP1 was significantly upregulated in pre-hierarchical follicles and F1 granulosa cells compared with other follicular stages, indicating its activity in regulating follicle selection and ovulation.

[0084] In the present invention, IGF2 was identified as a target gene of the circWRNIP1 / miR-129-5p axis, promoting chicken follicular development. The present invention demonstrated 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, and finally realizing its function of improving chicken follicular development.

[0085] Therefore, the newly discovered circWRNIP1, miR-129-5p, and IGF2 in chicken follicular development are expected to become potential molecular targets for improving chicken reproductive performance through molecular breeding strategies.

[0086] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents. The above-described embodiments merely represent several embodiments of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the present invention. It should be noted that for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. Use of an agent for reducing or inhibiting the expression of miR-129-5p or an agent for enhancing 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.

2. A method for promoting the development of chicken follicles 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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