Application of miR-103-3p in regulating chicken follicle development

Abstract

The present invention relates to the technical field of poultry breeding, and the purpose is to provide the application of miR-103-3p in regulating the development of chicken follicles. The present invention screens out lncRNAXR_003071938.1 with differential expression in the group and translation ability through joint analysis of transcriptome and translation group sequencing results. The seed sequence GCAGCATT of miR-103-3p can be combined with the sequence of XR_003071938.1, and the expression of miRNA-103-3p is increased or the adsorption is reduced, which can promote the proliferation of chicken follicle granulosa cells and inhibit the apoptosis of chicken follicle granulosa cells. The present invention has a good application value for studying follicle development and the like mechanism by exploring the application of reagents for promoting or promoting miR-103-3p expression or reducing miR-103-3p adsorption reagents in follicle granulosa cells.

Description

Technical Field

[0001] The invention relates to the technical field of poultry breeding, and in particular to the application of miR-103-3p in regulating the development of chicken follicles. Background Art

[0002] Egg-laying performance is one of the most important production performances of hens, which is mainly affected by factors such as genetics, nutrition and environmental conditions. These factors mainly act on ovarian development by regulating the dynamic development of follicles in the chicken ovaries, thereby affecting egg-laying performance. In the process of poultry reproduction, follicular atresia occurs at various stages of follicular development and is a common physiological phenomenon. Most follicles in the chicken ovaries stop growing and become atretic, and only a few dominant follicles continue to develop until ovulation. The selection of dominant follicles largely determines the reproductive capacity of animals, and this dynamic process is regulated by multiple factors.

[0003] A complete follicle cell type includes three types: oocyte cavity, granulosa cells and theca cells. The formation of theca cells around granulosa cells marks the entry of primary follicles into the secondary follicle stage, while the obvious feature of follicular atresia is the formation of oligonucleosomes in the granulosa cell layer, while theca cells in the membrane layer undergo less apoptosis. Chicken follicle atresia starts from the granulosa cell layer. The proliferation and apoptosis of granulosa cells in normal growing follicles coexist, while only the apoptosis of granulosa cells can be observed in atretic follicles. Therefore, the biological state of granulosa cells determines whether the follicles continue to grow or atresia occurs. Granulosa cells can secrete growth factors such as estrogen and insulin. If these growth factors are missing, granulosa cells will lose their regulatory function or even apoptosis, leading to cell apoptosis. Therefore, regulating factors related to differentiation, proliferation, and apoptosis in granulosa cells is the key to solving cell apoptosis and ensuring the development of dominant follicles.

[0004] lncRNA is a type of non-coding RNA with a length of more than 200 nt, which was previously considered to have no biological activity. With the continuous deepening of research, the biological activity of lncRNA has been continuously revealed, for example, lncRNA can exert biological activity by translating small peptides. In poultry, there are few studies on lncRNA related to ovarian development. lnc_13814 is significantly downregulated in the yellow follicles of female ducks, and acts as a molecular sponge of apla-miR-145-4 to release the inhibitory effect of miRNA on the DDIT3 gene, thereby promoting the apoptosis of duck granulosa cells; lncRNAMSTRG.7894.4 regulates the differentially expressed coding gene FOXK2 in the ovaries of domestic pigeons, thereby regulating the expression of estrogen receptors. Although many lncRNAs have been detected in the ovaries, different follicles and granulosa cells of Bashang Longtail Chicken, Hailan Brown Shell Laying Hen, Jinghai Yellow Chicken and Roman Laying Hen, the molecular mechanism of a specific lncRNA acting on the development of chicken follicles is still unclear. The present invention aims to carry out in-depth research in this regard to explore the regulatory mechanism of lncRNA in the development of chicken follicles. Summary of the invention

[0005] The purpose of the present invention is to provide an application of miR-103-3p in regulating chicken follicle development, and through the joint analysis of transcriptome and translation group sequencing results, 25 lncRNAs that are both differentially expressed within the group and have translation ability are screened, and lncRNAXR_003071938.1 is further screened to explore the mechanism of action of lncRNAXR_003071938.1 and its target miRNA on chicken follicle development. The technical solution of the present invention is:

[0006] In the first aspect, the present invention provides the use of a reagent for reducing or inhibiting lncRNA expression in the preparation of a reagent, composition or medicine for promoting the proliferation and development of chicken granulosa cells or promoting the development of chicken follicles. The lncRNA is XR_003071938.1, and its nucleotide sequence is shown in SEQ ID.1. The target miRNA of XR_003071938.1 is miR-103-3p, and the target gene of miR-103-3p is FBXW7. The reagent for reducing or inhibiting expression includes: a reagent for reducing or inhibiting the expression of the lncRNA and / or the expression of the protein encoded by the lncRNA.

[0007] Furthermore, the encoded amino acid sequence of XR_003071938.1 is shown in SEQ ID.2.

[0008] Furthermore, the target miRNA of XR_003071938.1 is miR-103-3p, the target gene of miR-103-3p is FBXW7, and the reagent for reducing or inhibiting expression also includes: a reagent for reducing or inhibiting the expression of the target gene FBXW7.

[0009] Preferably, the reagent has the sequence shown as SEQ ID.6 and SEQ ID.7.

[0010] Preferably, the reagent has the sequence shown as SEQ ID.18 and SEQ ID.19.

[0011] In a second aspect, the present invention also provides the use of reagents for enhancing or promoting miR-103-3p expression or reagents for reducing miR-103-3p adsorption in the preparation of reagents, compositions or drugs for promoting granulosa cell proliferation and development or promoting follicle development.

[0012] Preferably, the reagent has the sequence shown in SEQ ID.12.

[0013] In a third aspect, the present invention provides a method for promoting the proliferation and development of chicken granulosa cells, comprising: transfecting the sequences shown in SEQ ID.6 and SEQ ID.7 into primary chicken granulosa cells.

[0014] In a fourth aspect, the present invention provides a method for promoting the proliferation and development of chicken granulosa cells, comprising: transfecting the sequence shown in SEQ ID.12 into primary chicken granulosa cells.

[0015] In a fifth aspect, the present invention provides a method for promoting the proliferation and development of chicken granulosa cells, comprising: transfecting the sequences shown in SEQ ID.18 and SEQ ID.19 into primary chicken granulosa cells.

[0016] The present invention performs transcriptome and translationome sequencing on normal follicles and atretic follicles, and screens a total of 170 differentially expressed lncRNAs and 578 lncRNAs with sORFs, and obtains 25 lncRNAs that are both differentially expressed in the group and have translation ability through joint analysis. Then, through primer design, qPCR amplification and sequencing, and tissue expression verification, lncRNAXR_003071938.1 with differential expression in the group and translation ability is screened out, and it is found that by reducing the lncRNA, reducing its encoded protein, and / or increasing or reducing the expression of miRNA-103-3p to inhibit the expression of FBXW7, the proliferation of chicken follicle granulosa cells can be promoted and the apoptosis of chicken follicle granulosa cells can be inhibited. The present invention has a good application value for studying the mechanism of follicular development by exploring the application of reagents for reducing or inhibiting lncRNA expression, reagents for increasing or promoting miR-103-3p expression, or reagents for reducing miR-103-3p adsorption in follicular granulosa cells. 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:

[0018] Figure 1 This is the prediction and verification of the lncRNA-miRNA target relationship in Example 4 of the present invention, wherein Figure A shows the binding mode of the miR-103-3p seed sequence and the XR_003071938.1 sequence; Figure B shows the expression of gga-miR-103-3p in si-XR_003071938.1, si-NC, ov-XR_003071938.1, and ov-NC.

[0019] Figure 2 The interference efficiency and overexpression efficiency of miR-103-3P in Example 4 of the present invention.

[0020] Figure 3 This is the verification of the target relationship between XR_003071938.1 and miR-103-3p in Example 4 of the present invention, wherein A: the binding site of XR_003071938.1 and miR-103-3p; B: dual luciferase reporter result.

[0021] Figure 4 The effects of miR-103-3p on the expression of proliferation-related genes and proteins in granulosa cells in Example 4 of the present invention, wherein A: the expression of proliferation-related genes in primary granulosa cells of chickens after transfection with miR-103-3p inhibitor; B: the expression of proliferation-related genes in primary granulosa cells of chickens after transfection with miR-103-3p mimics; C: the expression of proliferation-related proteins in primary granulosa cells of chickens after transfection with miR-103-3p inhibitor; D: the expression of proliferation-related proteins in primary granulosa cells of chickens after transfection with miR-103-3pmimics.

[0022] Figure 5 The effect of miR-103-3p on granulosa cell proliferation detected by EdU in Example 4 of the present invention (200×), wherein A and B: EdU detection of cell proliferation after transfection with miR-103-3 inhibitor; C and D: EdU detection of cell proliferation after transfection with miR-103-3p mimic. EdU (red) fluorescent labeling indicates the number of proliferating cells; Hoechst (blue) fluorescent labeling indicates cell nuclei.

[0023] Figure 6 The flow cytometry analysis of the effect of miR-103-3p on granulosa cell proliferation in Example 4 of the present invention, wherein A and B: interference with miR-103-3p; C and D: overexpression of miR-103-3p.

[0024] Figure 7 The effect of miR-103-3p on the mRNA expression of apoptosis-related genes in granulosa cells detected by qPCR in Example 4 of the present invention, wherein A: interference with miR-103-3p; B: overexpression of miR-103-3p.

[0025] Figure 8 The Western Blot analysis of Example 4 of the present invention shows the effect of miR-103-3p on the expression of apoptosis-related gene proteins in granulosa cells, wherein A and B: interference with miR-103-3p; C and D: overexpression of miR-103-3p.

[0026] Fig. 9The effect of miR-103-3p on granulosa cell apoptosis detected by flow cytometry in Example 4 of the present invention, wherein A: interference with miR-103-3p; B: overexpression of miR-103-3p.

[0027] Fig.10 This is the effect of qPCR detection of miR-103-3p on the mRNA expression of genes related to steroid hormone synthesis in granulosa cells in Example 4 of the present invention, wherein A: interference with miR-103-3p; B: overexpression of miR-103-3p.

[0028] Fig.11 This is the effect of miR-103-3p on the secretion of E2 and PROG hormones in granulosa cells detected by ELISA in Example 4 of the present invention, wherein A: interference with miR-103-3p; B: overexpression of miR-103-3p.

[0029] Fig.12 This is the effect of miR-103-3p on the expression of proteins related to steroid hormone synthesis in granulosa cells in Example 4 of the present invention, wherein A and B: interference with miR-103-3p; C and D: overexpression of miR-103-3p.

[0030] Fig.13 This is the prediction and qPCR verification of the miRNA-mRNA target relationship in Example 5 of the present invention, wherein A: prediction of miR-103-3p target genes; B: qPCR verification of the miRNA-mRNA target relationship.

[0031] Fig.14 This is the dual luciferase reporter verification of miR-103-3p targeting FBXW7 in Example 5 of the present invention, wherein A: construction of FBXW7 wild-type and mutant dual luciferase reporter plasmids; B: dual luciferase reporter results.

[0032] Fig.15 The interference efficiency of FBXW7 detected by qPCR in Example 5 of the present invention

[0033] Fig.16 The effect of FBXW7 interference on granulosa cell proliferation in Example 5 of the present invention, wherein A: expression levels of CCND1, CCND2, CDK2 and PCNA; B: PCNA protein expression level; C, D: proliferation cycle of granulosa cells.

[0034] Fig.17The cell proliferation number (200×) was detected by EdU 36 hours after the chicken granulosa cells were transfected with si-FBXW7 and si-NC in Example 5 of the present invention, wherein Figure A is the staining result and Figure B is the data statistical result; EdU (red) fluorescently marks the number of proliferating cells; Hoechst (blue) fluorescently marks the cell nucleus.

[0035] Fig.18 The effects of FBXW7 on granulosa cell apoptosis in Example 5 of the present invention, wherein A: qPCR detection of the effect of FBXW7 on the expression of genes related to granulosa cell apoptosis; B, C: Western Blot detection of the effect of FBXW7 on the expression of proteins related to granulosa cell apoptosis; D: flow cytometry detection of the effect of FBXW7 on cell apoptosis.

[0036] Fig.19 The effects of FBXW7 interference on granulosa cell steroid hormones in Example 5 of the present invention, wherein A: qPCR detection of CYP11A1sCYP19A1 and STAR mRNA expression levels; B, C: ELISA detection of granulosa cell E2 and PROG hormone secretion; D, E: Western Blot detection of granulosa cell steroid hormone synthesis related gene protein expression. DETAILED DESCRIPTION

[0037] The Daheng 799 broiler chicken matching system used in the embodiment of the present invention is a three-line matching green-legged and hemp-feathered high-quality broiler chicken new variety bred by Sichuan Daheng Poultry Breeding Co., Ltd. and Sichuan Academy of Animal Husbandry Sciences (certificate number: Nong 09 New Variety Certificate No. 84). It was approved by the National Committee for Livestock and Poultry Genetic Resources in 2020 and was included in the "National Livestock and Poultry Genetic Resources Variety List (2021 Edition)".

[0038] 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.

[0039] In the following examples, EdU labeling technology, staining, flow cytometry, Western Blot detection, ELISA detection, etc. are all conventional detection techniques in the art.

[0040] Example 1

[0041] In this study, transcriptome and translationome sequencing were performed on normal follicles and atretic follicles of 43-week-old Daheng broiler chickens, and 170 differentially expressed lncRNAs, 47 differentially expressed miRNAs and 578 lncRNAs with translation ability were found, respectively.

[0042] According to the lncRNA base sequence obtained by transcriptome sequencing, PrimerPremier5.0 software was used for primer design. Because the primer design process of some lncRNA sequences did not meet the primer design principles, only 9 lncRNAs could be designed with primers in the end, and only 3 lncRNAs were amplified by PCR. The amplified products were recovered and Sanger sequencing was performed. The sequencing results were compared with the transcriptome sequencing sequences, and it was found that XR_003071938.1 was expressed at the highest level in atretic follicles. This lncRNA is located on chicken chromosome 27. The primer sequences of XR_003071938.1 and its amplified sequence are shown in Table 1 and Table 2 respectively:

[0043] Table 1 Primer sequence information

[0044]

[0045] Table 2 Amplified sequences of three lncRNAs

[0046]

[0047] Normal follicles and atretic follicles were collected and RNA was extracted for reverse transcription. The expression of lncRNAXR_003071938.1 in normal follicles and atretic follicles was verified by qPCR. The quantitative results showed that the expression levels of lncRNAXR_003071938.1 in atretic follicles and normal follicles were significantly different. The expression pattern of lncRNA XR_003071938.1 in different tissues was explored. XR_003071938.1 is expressed in heart, brain, muscle, liver, spleen, lung, kidney, intestine, gizzard, uterus and ovary, but its expression level in ovary is higher than that in other tissues; in addition, the expression level of lncRNAXR_003071938.1 gradually decreases from hierarchical follicles F1-F5 (F1-5 are hierarchical follicles at different stages); the expression level of lncRNAXR_003071938.1 in SYF (small yellow follicle), LYF (large yellow follicle), SWF (small white follicle) and LWF (large white follicle) shows that its expression level in small white follicle is higher. These results suggest that lncRNAXR_003071938.1 may have an important regulatory role in the ovarian development of chickens.

[0048] The results of translation group sequencing showed that the translation efficiency of lncRNA in normal follicles was lower than that in atretic follicles. According to the full-length sequence of XR_003071938.1, the open reading frame of XR_003071938.1 was predicted using NCBIORFFinder. The results showed that XR_003071938.1 contained an open reading frame (ORF), which was consistent with the sequencing data. The ORF started from the 199th base of XR_003071938.1, that is, the 199th base of XR_003071938.1 was the start codon ATG, and ended at the 567th base, that is, the 567th base was the stop codon TGA.

[0049] XR_003071938.1 encodes a small peptide of 122 amino acids, XR_003071938.1-122AA, the sequence of which is shown in SEQID.2. Based on the amino acid sequence, the molecular weight of the protein is predicted to be 12.43KD.

[0050] Example 2

[0051] To verify the protein encoding ability of XR_003071938.1, a 3×FLAG protein tag sequence was inserted after the start codon ATG sequence of the ORF of XR_003071938.1, and the overexpression plasmid ov-XR_003071938.1-FLAG was further constructed. After mutating its initial codon ATG to CCC, another vector was constructed, named ov-XR_003071938.1-FLAG-MT, and after inserting the 3×FLAG sequence, the size of the XR_003071938.1-122aa-Flag protein after the tag insertion was calculated to be 15.58 kD based on the amino acid sequence. Western Blot detection was performed using β-tubulin antibody and FLAG antibody. The results showed that β-tubulin protein was detected in both the ov-XR_003071938.1-FLAG transfection group and the ov-XR_003071938.1-FLAG-MT transfection group; while the expression of Flag-tagged protein was detected in the ov-XR_003071938.1-FLAG transfection group, which was consistent with the theoretical size of XR_003071938.1-122aa-Flag, while the expression of Flag-tagged protein was not detected in the ov-XR_003071938.1-FLAG-MT transfection group.

[0052] The XR_003071938.1-122aa sequence carrying the Flag-tagged protein was inserted into the pcDNA3.1 vector to construct the ov-XR_003071938.1-Flag-122aa overexpression plasmid and Western Blot analysis was performed. The results showed that ov-XR_003071938.1-Flag-122aa could successfully express the Flag-tagged protein.

[0053] qPCR was used to detect the expression of XR_003071938.1 in the ov-XR_003071938.1-FLAG, ov-XR_003071938.1-FLAG-MT and ov-XR_003071938.1-Flag-122aa transfection groups. The results showed that compared with the ov-NC (i.e. pcDNA3.1) transfection group, the ov-XR_003071938.1-FLAG, ov-XR_003071938.1-FLAG-MT and ov-XR_003071938.1-Flag-122aa groups could increase the expression level of XR_003071938.1, among which ov-XR_003071938.1-Flag-122aa was the most significant.

[0054] The above results indicate that XR_003071938.1 can successfully encode a protein consisting of 122 amino acids.

[0055] Overexpression of XR_003071938.1-122aa in chicken primary granulosa cells, qPCR (purchased from SYBR Green RT-PCR kit, Takara Biotechnology (Dalian) Co., Ltd., the same below)

[0056] The expression of proliferation-related genes CCND1, CCND2, CDK2 and PCNA was detected. The results showed that the expression of CCND1, CCND2, CDK2 and PCNA was significantly reduced after overexpression of XR_003071938.1-122aa compared with the control group. Western Blot detection results showed that the abundance of proliferation-related protein PCNA in the XR_003071938.1-122aa transfection group was significantly lower than that in the control group. Flow cytometry analysis results showed that the number of cells in the G0 / G1 phase of the ov-XR-003071938.1-122aa group was significantly higher than that of ov-NC, and the number of cells in the S+G2 phase was significantly lower than that of ov-NC.

[0057] Chicken primary granulosa cells were obtained from 43-week-old large Heng broiler chickens with normal egg production. They were killed by cervical dislocation. The chickens with normal ovarian development were observed by autopsy. The pre-grade follicles were collected. After rinsing with PBS, the granulosa cell layer was gently separated in PBS buffer for standby use. This experiment was approved by the Animal Ethics Committee of Sichuan Agricultural University (the same as in the following examples). The culture of chicken primary granulosa cells and the overexpression of XR_003071938.1-122aa belong to routine operations in this field and are not described in detail here.

[0058] EdU staining results showed that the number of EdU-stained cells in the XR_003071938.1-122aa transfection group was significantly less than that in the control group. Flow cytometry analysis showed that overexpression of XR_003071938.1-122aa significantly reduced the number of granulosa cells in the S and G2 phases, but the number of cells in the G0 and G1 phases was significantly higher than that in the control group.

[0059] In summary, XR_003071938.1-122aa can inhibit the proliferation of chicken primary granulosa cells.

[0060] qPCR verified the mRNA levels of apoptosis-related genes Caspase3, Caspase8, Caspase9 and BCL-2. The results showed that after overexpression of XR_003071938.1-122aa, the mRNA levels of Caspase3, Caspase8 and Caspase9 were significantly upregulated compared with the control group, but the expression of BCL-2 was significantly downregulated. Western Blot detection results showed that XR_003071938.1-122aa could significantly increase the protein expression levels of Caspase3 and Caspase9. Flow cytometry cycle detection results showed that overexpression of XR_003071938.1-122aa led to an increase in the apoptosis rate of granulosa cells.

[0061] In summary, XR_003071938.1-122aa promotes the apoptosis of granulosa cells.

[0062] qPCR verified the mRNA levels of steroid hormone synthesis-related genes CYP11A1, CYP19A1 and STAR. The results showed that after overexpression of XR_003071938.1-122aa, the mRNA levels of CYP11A1, CYP19A1 and STAR were significantly reduced compared with the control group. ELISA test results showed that overexpression of XR_003071938.1-122aa significantly reduced the secretion levels of reproductive hormones E2 and PROG in granulosa cells. Western Blot results showed that overexpression of XR_003071938.1-122aa could significantly reduce the protein expression capacity of STAR and CYP19A1.

[0063] In conclusion, XR_003071938.1-122aa can inhibit the secretion and synthesis of steroid hormones in chicken primary granulosa cells.

[0064] Example 3

[0065] The overexpression plasmid of XR_003071938.1 was constructed using the pcDNA3.1-ciR vector. Chicken primary granulosa cells were transfected with siRNA (Table 3) and overexpression plasmids in vitro. Total cell RNA samples were collected 24 hours after treatment, and the cell RNA was extracted and reverse transcribed to obtain a cDNA library. qPCR was used to detect the changes in the expression level of XR_003071938.1 in granulosa cells after interference and overexpression.

[0066] The results showed that after transfection of XR_003071938.1 siRNA in granulosa cells, the expression level of XR_003071938.1 was significantly inhibited compared with the control group, but after transfection of overexpression plasmid, the expression level of XR_003071938.1 was significantly increased compared with the control group.

[0067] Table 3 siRNA sequence information

[0068]

[0069]

[0070] In the sequence listing, U is changed to W.

[0071] The expression of proliferation-related genes CyclinD1 (CCND1), CyclinD2 (CCND2), CyclinDependentKinase2 (CDK2) and ProliferatingCellNuclearAntigen (PCNA) in chicken primary granulosa cells transfected with XR_003071938.1 siRNA and overexpression plasmid was detected by qPCR, and it was found that XR_003071938.1 significantly inhibited the expression of CCND1, CCND2, CDK2 and PCNA. Western Blot detection showed that XR_003071938.1 inhibited the expression of proliferation-related protein PCNA.

[0072] Flow cytometry analysis showed that XR_003071938.1 could reduce the number of granulosa cells in S and G2 phases, and increase the number of granulosa cells in G0 and G1 phases. EDU staining revealed that XR_003071938.1 could reduce the number of EdU-positive cells.

[0073] The above results indicate that XR_003071938.1 can inhibit the proliferation of chicken primary granulosa cells, while reducing or inhibiting the expression of XR_003071938.1 can promote the proliferation of chicken primary granulosa cells.

[0074] The mRNA levels of apoptosis-related genes Caspase3, Caspase8, Caspase9 and BCL-2 in chicken primary granulosa cells transfected with XR_003071938.1 siRNA and overexpression plasmid were detected by qPCR, and it was found that XR_003071938.1 significantly increased the mRNA levels of Caspase3, Caspase8 and Caspase9, while the mRNA level of BCL-2 was significantly reduced. Western Blot detection results showed that XR_003071938.1 increased the expression of apoptosis-related proteins Caspase3 and Caspase9.

[0075] The cells were stained with annexin V antibody and propidium iodide (PI), and then flow cytometry was used to analyze cell apoptosis. The results showed that XR_003071938.1 could increase the apoptosis rate of granulosa cells.

[0076] The above results indicate that XR_003071938.1 can promote apoptosis of granulosa cells, while reducing or inhibiting the expression of XR_003071938.1 can promote the development of primary granulosa cells in chickens.

[0077] The expression of steroid synthesis-related genes CYP11A1, CYP19A1 and STAR in chicken primary granulosa cells transfected with XR_003071938.1 siRNA and overexpression plasmid was detected by qPCR, and it was found that XR_003071938.1 reduced the mRNA levels of CYP11A1, CYP19A1 and STAR. The secretion levels of reproductive hormones E2 and PROG were detected by ELISA, and it was found that XR_003071938.1 could inhibit the secretion of reproductive hormones E2 and PROG. Western Blot detection results showed that XR_003071938.1 could inhibit the expression levels of steroid hormone synthesis-related proteins STAR and CYP19A1.

[0078] In addition, XR_003071938.1 can be modified through gene editing such as CRISPR-Cas9 technology to reduce or inhibit its expression, thereby promoting the proliferation and development of chicken primary granulosa cells.

[0079] Example 4

[0080] The target miRNA of XR_003071938.1 was predicted by RNAhybrid, and it was found that the seed sequence GCAGCATT of miR-103-3p could bind to the sequence of XR_003071938.1 ( Figure 1 ). Transcriptome sequencing results showed ( Figure 2 ), the expression level of miR-103-3p in normal follicles was higher than that in atretic follicles, the inhibitor / mimics reference gene sequence and inhibitor / mimics Ncontrol sequence of miR-103-3p were designed (Table 4), primary chicken granulosa cells were cultured in vitro and transfected with siRNA and overexpression plasmid of XR_003071938.1, total RNA was extracted and reverse transcribed for miRNA, and then qPCR was performed for detection. Figure 2 The results showed that miR-103-3p inhibitor significantly reduced the expression of miR-103-3p in chicken primary granulosa cells, but miR-103-3pmimic significantly increased the expression of miR-103-3p in granulosa cells; and compared with the control group, the expression of miR-103-3p mimic increased by about 100 times. At the same time, after inhibiting the expression of XR_003071938.1 in granulosa cells, the expression of miR-103-3p increased significantly, while after transfection of XR_003071938.1 overexpression plasmid, the expression of miR-103-3p decreased.

[0081] Table 4 miRNA inhibitor / mimics sequence information

[0082]

[0083] In the sequence listing, U is changed to W.

[0084] The predicted binding target site and its mutant sequence were constructed into a dual luciferase reporter gene vector, and the wild-type plasmid pmirGLO-XR_003071938.1-WT and mutant plasmid pmirGLO-XR_003071938.1-MT were constructed, in which the sequence of the binding site was mutated from "ATGCTGC" to "TACGACG". The dual luciferase reporter gene plasmid (WT / MT) and miR-103-3p mimic / mimic NC were co-transfected into chicken DF-1 cells, and then the dual luciferase reporter assay was performed. The results showed that ( Figure 3): Compared with the control group, co-transfection of lncRNA-WT and miR-103-3p mimics significantly reduced the ratio of firefly luciferase activity to Renilla luciferase activity, while the ratios of the other three groups did not change. The experimental results confirmed that miR-103-3p is the target miRNA of XR_003071938.1. The nucleic acid sequence of miR-103-3p is: 3'-AGTATCGGGACATGTTACGACGA-5'.

[0085] By transfecting miR-103-3p inhibitor and miR-103-3p mimic into chicken primary granulosa cells, qPCR was used to detect the expression levels of proliferation-related genes CCND1, CCND2, CDK2 and PCNA at the gene level. The results showed that miR-103-3p significantly increased the expression of CCND1, CCND2, CDK2 and PCNA ( Figure 4 A and B), Western Blot results showed that miR-103-3p could increase the expression of proliferation-related protein PCNA ( Figure 4 (C, D).

[0086] The EdU results showed that the number of EdU positive cells in the miR-103-3p mimic transfection group was significantly higher than that in the NC transfection group ( Figure 5 (Figure AD).

[0087] Flow cytometry analysis showed that miR-103-3p could increase the number of granulosa cells in the S and G2 phases and decrease the number of granulosa cells in the G0 and G1 phases. Figure 6 (Figure AD).

[0088] In summary, miR-103-3p can promote the proliferation of chicken primary granulosa cells.

[0089] qPCR verified the mRNA levels of apoptosis-related genes Caspase3, Caspase8, Caspase9 and BCL-2. The results showed that miR-103-3p could reduce the mRNA levels of Caspase3, Caspase8 and Caspase9, and increase the mRNA level of BCL-2 ( Figure 7 ).

[0090] Western Blot results showed that miR-103-3p significantly reduced the expression levels of apoptosis-related proteins Caspase3 and Caspase9 ( Figure 8 (Figure AD).

[0091] Flow cytometry analysis showed that the increase in miR-103-3p expression reduced the apoptosis rate of granulosa cells ( Fig. 9 ).

[0092] In summary, miR-103-3p inhibits the apoptosis of granulosa cells.

[0093] qPCR verified the mRNA levels of steroid hormone synthesis-related genes CYP11A1, CYP19A1, and STAR. The results showed that miR-103-3p increased the mRNA levels of CYP11A1, CYP19A1, and STAR ( Fig.10 ). The secretion levels of reproductive hormones E2 and PROG were detected by ELISA, and it was found that miR-103-3p could promote the secretion of E2 and PROG ( Fig.11 ).

[0094] Western Blot results showed that miR-103-3p could increase the expression levels of steroidogenesis-related proteins STAR and CYP19A1 ( Fig.12 (Figure AD).

[0095] Example 5

[0096] DIANA, miRDB and TargetScann software were used to predict the target genes of miR-103-3p, and a total of 14 target genes of miR-103-3p were predicted. The predicted target genes of miR-103-3p were preliminarily verified by qPCR, and it was found that FBXW7 had an opposite expression pattern to miR-103-3p. Therefore, FBXW7 was preliminarily used as the target mRNA of miR-103-3p ( Fig.13 ).

[0097] Based on the predicted target site, the target site and its preceding and following sequences were constructed into a dual luciferase reporter gene vector (pmirGLO), and the wild-type dual luciferase reporter gene (pmirGLO-FBXW7-WT) and mutant dual luciferase reporter gene (pmirGLO-FBXW7-MT) were constructed, in which the sequence of the binding site was mutated from "ATGCTGC" to "TACGACG". The results of the dual luciferase reporter assay in DF-1 cells in vitro showed that after co-transfection of the dual luciferase reporter gene and miR-103-3p mimics, the firefly luciferase activity in the pmirGLO-FBXW7-MT plasmid decreased, but there was no change in the cells co-transfected with the mutant reporter gene, indicating that FBXW7 is a target gene of miR-103-3p ( Fig.14 ).

[0098] Three siRNA sequences (S1, S2, and S3 in Table 5) were designed. After chicken primary granulosa cells were transfected with siRNA of FBXW7 (si-FBXW7) in vitro, the expression level of FBXW7 was detected by qPCR. The results showed that after transfection of siRNA of FBXW7 in granulosa cells, the expression level of FBXW7 could be significantly reduced compared with other transfection groups s3-FBXW7. The sequence of S3 was: S: GGGUGAAUUUAUCCGAAAUTT, AS: AUUUCFFAUAAAUUCACCCTT ( Fig.15 ).

[0099] Table 5 3 siRNA sequences

[0100]

[0101] In the sequence listing, U is changed to W.

[0102] qPCR was used to detect the expression of proliferation-related genes CCND1, CCND2, CDK2 and PCNA. The results showed that FBXW7 could reduce the expression of CCND1, CCND2, CDK2 and PCNA ( Fig.16 Western Blot results showed that FBXW7 could reduce the abundance of proliferation-related protein PCNA ( Fig.16 The results of EdU staining showed that FBXW7 could reduce the number of EdU-stained cells, and the results of flow cytometry analysis showed that FBXW7 could reduce the number of granulosa cells in the S and G2 phases and increase the number of granulosa cells in the G0 and G1 phases ( Fig.17 ).

[0103] In summary, FBXW7 can inhibit the proliferation of chicken primary granulosa cells, while inhibiting the expression of FBXW7 can promote the proliferation of chicken primary granulosa cells.

[0104] qPCR verified the mRNA levels of apoptosis-related genes Caspase3, Caspase8, Caspase9 and BCL-2. The results showed that FBXW7 could increase the mRNA levels of Caspase3, Caspase8 and Caspase9, and reduce the expression of BCL-2 ( Fig.18 Western Blot results showed that FBXW7 could significantly increase the protein expression levels of Caspase3 and Caspase9 ( Fig.18 Figures B and C in the middle). Flow cytometry cell cycle detection results showed that FBXW7 can increase the apoptosis rate of granulosa cells ( Fig.18 (D in the middle).

[0105] In summary, FBXW7 promotes the apoptosis of granulosa cells, and inhibiting the expression of FBXW7 can promote the development of chicken primary granulosa cells.

[0106] qPCR verified the mRNA levels of steroid hormone synthesis-related genes CYP11A1, CYP19A1, and STAR. The results showed that FBXW7 could inhibit the mRNA levels of CYP11A1, CYP19A1, and STAR ( Fig.19 Figure A in the middle). ELISA detection of reproductive hormones E2 and PROG levels revealed that FBXW7 could reduce the secretion levels of reproductive hormones E2 and PROG in granulosa cells ( Fig.19 Western Blot results showed that FBXW7 could significantly reduce the protein expression of STAR and CYP19A1 ( Fig.19 (D and E in the middle).

[0107] In summary, FBXW7 can inhibit the secretion and synthesis of steroid hormones in chicken primary granulosa cells, while inhibiting the expression of FBXW7 can promote the secretion and synthesis of steroid hormones in chicken primary granulosa cells.

[0108] 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 a reagent for enhancing or promoting miR-103-3p expression in the preparation of a reagent, composition or medicine for promoting the proliferation and development of chicken granulosa cells or promoting follicle development, wherein the reagent for enhancing or promoting miR-103-3p expression has a sequence as shown in SEQ ID. 12.

Images