Application of combination of FGA and Caspase3 in regulation and control of ovarian granular cells

Abstract

The invention discloses application of FGA combined Caspase3 in regulation and control of ovarian granular cells, and belongs to the technical field of cell engineering and gene engineering. According to the invention, FGA is taken as a research object, the application of FGA in ovarian granular cells is researched by adopting a molecular and cell biology method, and regulation of FGA gene on growth and development of ovarian follicles and influence on mouse initial estrus are verified from multiple levels and multiple angles. The method can provide new reference for genetic improvement of reproductive performance of sows, and has good application value.

Description

Technical Field

[0001] The invention belongs to the technical field of cell engineering and gene engineering, and particularly relates to application of FGA combined with Caspase3 in regulating ovarian granulosa cells. Background Art

[0002] In female mammals, the growth and development of follicles determines their reproductive potential, and the mission of each follicle is controlled by endocrine and paracrine factors. The total number of ovarian follicles is determined early in life, and with age, continuous consumption to exhaustion leads to reproductive aging. Follicle development goes through the primordial, primary and secondary follicle stages, and then acquires the antral cavity (tertiary follicles and mature follicles). After the antral stage, follicle development has two fates. Most follicles undergo atresia and degeneration, while a few of them reach the preovulatory stage under the stimulation of cyclic Gn that occurs after puberty. Compared with animal breeds with low fertility, animal breeds with high fertility often have more mature follicles and higher ovulation rates in their ovaries. However, the vast majority of follicles (more than 99%) do not reach the preovulatory stage, but stop growing during development and undergo a degeneration process called atresia. A large number of follicles with atresia will greatly reduce the reproductive performance of mammals. In short, the growth and development of sow ovarian follicles is a key factor affecting their reproductive performance, which can directly affect the economic benefits of the farm. However, the specific mechanism affecting follicle development needs to be further studied to provide a reference for improving the current relatively low reproductive efficiency and utilization rate.

[0003] The main cause of follicular atresia is the apoptosis of granulosa cells (GCs). In actual production, female mammals that are eliminated due to failure to estrus on time or no estrus directly affect the utilization rate and economic benefits of the farm. The main reasons are that on the one hand, the ovarian follicles are immature and cannot secrete enough hormones to promote estrus; on the other hand, excessive apoptosis of granulosa cells in ovarian follicles leads to follicular atresia or excessive development activation, which accelerates the consumption of primordial follicles and leads to premature ovarian failure.

[0004] Fibrinogen alpha chain (FGA) is a cell adhesion molecule that is usually located in cells and has a potential paracrine mechanism for surrounding cells. It can activate the FAK / AKT signaling pathway, promote cell proliferation, migration and invasion, and inhibit cell apoptosis. A large number of studies have shown that FGA can promote angiogenesis by activating the VEGF-VEGFR2-FAK signaling pathway in endothelial cells. In addition to being a key protein in coagulation-related mechanisms such as angiogenesis, FGA also affects cell proliferation, adhesion, migration and invasion in human immortalized ectopic endometrial epithelial cell line (hEM15A cell line), human liver cancer cell line (HepG2 cell line), and gastric cancer cell line (GC-27 cell line) mainly by activating the FAK-AKT-MMP2 / BCL signaling pathway, and inhibits cell apoptosis. However, there are still no reports on the relationship between the FGA gene and follicle growth and development. Summary of the invention

[0005] To solve the related problems, the primary purpose of the present invention is to provide applications of FGA gene-related biological materials.

[0006] Another object of the present invention is to provide an application of FGA combined with Caspase3 in regulating ovarian granulosa cells.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0008] Application of FGA gene-related biomaterials, wherein the biomaterials are any one or more of the following substances A, B, C, D, E, F, G:

[0009] A. Full-length genomic DNA of FGA gene or its homologous nucleic acid molecule;

[0010] B. FGA gene CDS region sequence or its homologous nucleic acid molecule;

[0011] C. The protein encoded by the FGA gene or its homologous amino acid;

[0012] D. A substance that can increase the expression level and / or activity of substance A or B;

[0013] E. Substances that can reduce the expression and / or activity of substance A or B;

[0014] F. A substance that can increase the expression level and / or activity of substance C;

[0015] G. Substances that can reduce the expression level and / or activity of substance C;

[0016] The application is any one or more of the following applications Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ:

[0017] Ⅰ. Application in regulating follicle development / preparing products for regulating follicle development;

[0018] II. Application in regulating granulocyte proliferation / preparing products for regulating granulocyte proliferation;

[0019] III. Application in regulating granulocyte apoptosis / preparing products for regulating granulocyte apoptosis;

[0020] IV. Application in regulating estrogen secretion / preparing products for regulating estrogen secretion;

[0021] Ⅴ. Application in regulating puberty initiation / preparing products for regulating puberty initiation.

[0022] Furthermore, application I includes any one or more of the following applications 1) and 2):

[0023] 1) Use of substance A, B, C, D or F in promoting follicle development / preparing products for promoting follicle development;

[0024] 2) Use of substance E or G in inhibiting follicle development / preparing products for inhibiting follicle development.

[0025] Furthermore, application II includes any one or more of the following applications 1) and 2):

[0026] 1) Use of substance A, B, C, D or F in promoting granulocyte proliferation / preparing products promoting granulocyte proliferation;

[0027] 2) Use of substance E or G in inhibiting granulocyte proliferation / preparing products for inhibiting granulocyte proliferation.

[0028] Furthermore, application III includes any one or more of the following applications 1) and 2):

[0029] 1) Use of substance A, B, C, D or F in inhibiting granulocyte apoptosis / preparing a product for inhibiting granulocyte apoptosis;

[0030] 2) Application of substance E or G in promoting granulosa cell apoptosis / preparing products promoting granulosa cell apoptosis.

[0031] Furthermore, application IV includes any one or more of the following applications 1) and 2):

[0032] 1) Use of substance A, B, C, D or F in promoting estrogen secretion / preparing products promoting estrogen secretion;

[0033] 2) Use of substance E or G in inhibiting estrogen secretion / preparing products for inhibiting estrogen secretion.

[0034] Furthermore, application V includes any one or more of the following applications 1) and 2):

[0035] 1) Use of substance A, B, C, D or F in promoting puberty / preparing products promoting puberty;

[0036] 2) Use of substance E or G in inhibiting puberty onset / preparing products for inhibiting puberty onset.

[0037] Furthermore, in application IV, the estrogen includes estradiol E2.

[0038] Furthermore, the application is an application in an in vitro environment.

[0039] Furthermore, the application is application in mammals.

[0040] Furthermore, the application is application in humans, pigs or mice.

[0041] Furthermore, when the application is in humans:

[0042] The nucleotide sequence of the full-length genomic DNA described in A is shown in the sequence of NCBI Gene ID: 2243 (NCBI Reference Sequence: NC_000004.12).

[0043] The nucleotide sequence of the CDS region sequence described in B is shown in the sequence of NCBI CCDS: CCDS3787.1.

[0044] The amino acid sequence of the protein described in C is shown in the sequence of NCBI NP_000499.1.

[0045] Furthermore, when the application is in pigs or mice:

[0046] The nucleotide sequence of the full-length genomic DNA described in A is shown in the sequence of NCBI Gene ID: 14161 (NCBI Reference Sequence: NM_010196.4).

[0047] The nucleotide sequence of the CDS region sequence described in B is shown in the sequence of GenBank: BC005467.1.

[0048] The amino acid sequence of the protein described in C is shown in the sequence of GenBank: AAH05467.1.

[0049] Furthermore, the homologous nucleic acid molecule described in A refers to a nucleic acid molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the full-length genomic DNA.

[0050] Furthermore, the homologous nucleic acid molecule described in B refers to a nucleic acid molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the CDS region sequence.

[0051] Furthermore, the homologous amino acid described in C refers to an amino acid that has at least 60%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the amino acid sequence of the protein.

[0052] Furthermore, the substance D or F is selected from: an overexpression recombinant vector containing a corresponding nucleic acid molecule.

[0053] Furthermore, the vector used in the overexpression recombinant vector is pcDNA3.1.

[0054] Furthermore, the substance E or G is selected from: siRNA, shRNA, sgRNA for knocking out / knockdown of gene expression or a knockout recombinant vector containing the same.

[0055] When the application is in humans, the targeting sequence corresponding to the siRNA is: 5′-GCTGCAGGATGAAAGGGTT-3′.

[0056] When the application is in humans, the targeting sequence corresponding to the shRNA is: 5′-GGAACAACAAGGATTCTAA-3′.

[0057] FGA combines with Caspase3 to regulate the application of ovarian granulosa cells. In vitro, the FGA gene combines with the apoptosis-related gene Caspase3 to negatively regulate the apoptosis of ovarian granulosa cells.

[0058] Furthermore, increasing the expression of exogenous FGA gene decreased the expression levels of Caspase3 mRNA and protein, inhibiting the apoptosis of ovarian granulosa cells; inhibiting the expression of FGA gene increased the expression levels of Caspase3 mRNA and protein, promoting the apoptosis of ovarian granulosa cells.

[0059] Furthermore, the increase in exogenous FGA gene expression is achieved by gene overexpression technology, and the gene overexpression vector used is prepared by the following method: (1) extracting RNA from ovarian granulosa cells, reverse transcribing it into cDNA, and performing PCR amplification using cDNA as a template to obtain the target fragment; (2) connecting the target fragment to the pcDNA3.1 vector cut with restriction endonucleases BamHI and NHeI to obtain the gene overexpression vector; the primers used for PCR amplification are as follows:

[0060] FGA F: 5′-CGCGGATCCCTTTCAGCTGGAGGTGCTCCTCA-3′

[0061] FGAR: 5′-CTAGCTAGCAAATGCAAGGGGCCATGGGAA-3′.

[0062] Furthermore, the inhibition of FGA gene expression is achieved through siRNA or shRNA.

[0063] When the application is in humans, the targeting sequence corresponding to the siRNA is: 5′-GCTGCAGGATGAAAGGGTT-3′.

[0064] When the application is in pigs or mice, the targeting sequence corresponding to the shRNA is: 5′-GGAACAACAAGGATTCTAA-3′.

[0065] The verification results of the present invention are as follows:

[0066] 1. FGA promotes ovarian granulosa cell proliferation and cell cycle progression

[0067] The present invention transfers the constructed FGA overexpression vector and FGA small interfering fragment into GCs, and EdU detection finds that FGA can promote the proliferation ability of GCs. In order to further understand the expression of genes in pathways related to GCs proliferation, qRT-PCR and WB results show that compared with the control group, FGA can significantly promote the mRNA expression levels of proliferation-related genes such as STAR, PCNA, IKBA, SP1 in GCs, and the expression of proliferation marker proliferating cell nuclear antigen PCNA at protein and mRNA levels is significantly increased, which confirms the results of EdU proliferation experiment. Subsequently, the cell cycle process was detected by flow cytometry, and the results showed that interference with FGA caused a significant decrease in the S phase of the GCs cell cycle, which is the stage of DNA replication, and the downward trend in the G2 / M phase was also significant. However, after overexpressing FGA, the S phase of the GCs cell cycle also had a downward trend, but compared with the inhibition level of the FGA interference group, it has rebounded, and the downward trend in the G2 / M phase is no longer significant, which can be explained that interference with FGA can inhibit the cell cycle process of GCs. The results of qRT-PCR and WB detection of FGA on cell cycle progression-related pathways showed that FGA could promote the cell cycle progression in GCs by upregulating the mRNA expression levels of MYC, CCND1, CDK7, CCNE2, and CDK4 and upregulating the protein expression levels of CCND1 and CCNE2. In summary, FGA can promote the cell cycle progression of GCs by promoting the expression levels of PCNA, CCND1, and CCNE2, thereby upregulating the proliferation rate of GCs.

[0068] 2. Effects of FGA on follicular development in sows and age at puberty in mice

[0069] The present invention first collects pig ovaries and then divides the follicles into large follicles (>7mm) and small follicles (<3mm) to collect GCs respectively. The differential expression of FGA mRNA and protein in pig large and small follicles is detected by qRT-PCR and WB. It is found that the expression of FGA gradually increases with the development of follicles, so it is speculated that the FGA gene is involved in the development of pig follicles. Subsequently, pig follicles are infecting in vitro with overexpression or knockdown FGA lentivirus, and the effect of FGA on follicular development is detected at the tissue level. It is found that overexpression of FGA can delay the disappearance of blood vessels on the surface of follicles and delay the turbidity of follicular fluid, and inhibit the apoptosis level of follicular GCs, while the development of follicles and the apoptosis level of follicular GCs after knockdown of FGA are opposite to the expression results after overexpression of FGA. After that, the animal model C57BL / 6J female mice were treated with the same concentration of lentivirus for 3 times (1 time / week). After 42 days of age, the ovaries of both sides of the mice were collected, and one of the ovaries was sliced. Through HE staining and Tunel experiment detection, it was found that overexpression of FGA in the ovaries of mice could significantly increase the number of antral follicles and corpora lutea in the ovaries, and also inhibit the apoptosis level of ovarian GCs. The results of the knockdown FGA group were opposite to those of the overexpression FGA group. The above results show that FGA can inhibit the apoptosis of ovarian GCs and promote the development of ovarian follicles.

[0070] Furthermore, the present invention found that compared with the control group, overexpression of FGA can advance the estrus age of mice. In addition, FGA can significantly promote the concentration of E2 in mouse serum. Subsequently, the overexpression or knockdown of FGA lentivirus on the apoptosis, proliferation and cycle progression-related pathways of ovarian follicle GCs was studied and found that FGA inhibited the apoptosis of pig follicle GCs cells by inhibiting the mRNA and protein expression levels of pro-apoptotic genes P53, BIM and Caspase9, and activated the expression of PCNA, STAR, CCNE1 gene mRNA and protein, thereby promoting the proliferation and cell cycle progression of pig follicle GCs; the same method was used to detect the effect of FGA on the apoptosis, proliferation and cycle progression-related pathways of mouse ovarian follicles. The results showed that FGA could inhibit the mRNA and protein expression of cysteine ​​protease family Caspase3, Caspase9 genes and pro-apoptotic gene Bim to inhibit the apoptosis of mouse ovarian follicle GCs. In addition, FGA could also regulate the cell proliferation and cell cycle progression of mouse ovarian follicle GCs by affecting the expression of Cdk4 and Pcna. In summary, FGA can promote the growth and development of ovarian follicles by affecting and regulating the apoptosis, proliferation and secretion of E2 of ovarian GCs, thereby advancing the age of puberty in mice.

[0071] Compared with the prior art, the present invention has the following advantages and effects:

[0072] The present invention uses FGA as the research object, uses molecular and cell biology methods to study its application in ovarian granulosa cells, and verifies from multiple levels and angles that the FGA gene regulates the growth and development of ovarian follicles and affects the onset of puberty in mice. The present invention can provide a new reference for the genetic improvement of sow reproductive performance and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 The figures show the research results of the effect of overexpression or interference of FGA on cell apoptosis; (a) the effect of overexpression of FGA on the apoptosis phenotype of GCs; (b) the effect of interference of FGA on the apoptosis phenotype of GCs; (c) the effect of overexpression or interference of FGA on the mRNA expression level of GCs apoptosis-related genes; (d) the effect of overexpression or interference of FGA on the protein expression level of GCs apoptosis-related genes.

[0074] Figure 2 The figures show the research results of the effect of overexpression or interference of FGA on cell proliferation; (a) the effect of overexpression of FGA on GCs proliferation; (b) the effect of interference of FGA on GCs proliferation; (c) the effect of overexpression or interference of FGA on the mRNA expression level of GCs proliferation-related genes; (d) the effect of overexpression or interference of FGA on the protein expression level of GCs proliferation-related genes.

[0075] Figure 3 The figures show the effects of overexpression or interference of FGA on the cell cycle process; (a) the effect of overexpression of FGA on the GCs cycle process; (b) the effect of interference of FGA on the GCs cycle process; (c) the effect of overexpression or interference of FGA on the mRNA expression level of genes related to the cell cycle process; (d) the effect of overexpression or interference of FGA on the protein expression level of genes related to the cell cycle process.

[0076] Figure 4 This is a graph showing the results of a CoIP experiment to detect the binding level of FGA with GCs apoptosis-related gene proteins.

[0077] Figure 5 The figure shows the results of the nuclear-cytoplasmic separation experiment to detect the expression level of Caspase3 in the nucleus and cytoplasm of GCs after overexpression or interference of FGA; wherein, (a) the mRNA expression level of Caspase3 after overexpression or interference of FGA; (b) the protein expression level of Caspase3 after overexpression or interference of FGA.

[0078] Figure 6 The graph shows the relative expression level of FGA in the development of pig ovarian follicles; (a) the mRNA expression level of FGA in pig follicles; (b) the protein expression level of FGA in pig follicles.

[0079] Figure 7 The results of the efficiency detection of FGA overexpression or knockdown lentivirus infecting pig follicles are shown in the figure; wherein, (a) the efficiency detection of FGA overexpression or knockdown lentivirus mRNA level; (b) the efficiency detection of FGA overexpression or knockdown lentivirus protein level.

[0080] Figure 8 These are photos of porcine follicles on days 1, 3, and 5 after being infected with lentivirus overexpressing or knocking down FGA.

[0081] Fig. 9 This figure shows the research results on the effect of lentiviral infection with overexpression or knockdown of FGA on the apoptosis of GCs in follicles.

[0082] Fig.10 Figure 3 shows the effect of FGA overexpression or knockdown on the genes related to the apoptosis proliferation cycle pathway in pig follicles. (a) The effect of FGA overexpression or knockdown on the mRNA expression level of follicular apoptosis-related genes; (b) The effect of FGA overexpression or knockdown on the protein expression level of follicular apoptosis-related genes; (c) The effect of FGA overexpression or knockdown on the mRNA expression level of follicular proliferation cycle-related genes; (d) The effect of FGA overexpression or knockdown on the protein expression level of follicular proliferation cycle-related genes.

[0083] Fig.11 Figure 2 shows the research results of the effect of overexpression or knockdown of FGA on the onset of puberty in mice; (a) the efficiency detection of FGA mRNA expression level in mouse ovaries by overexpression or knockdown of FGA lentivirus; (b) the efficiency detection of FGA protein expression level in mouse ovaries by overexpression or knockdown of FGA lentivirus; (c) the effect of overexpression or knockdown of FGA on the estrus age of mice.

[0084] Fig.12 The figures show the research results of the growth and development of ovarian follicles in mice after infection with lentivirus overexpressing or knocking down FGA. (a) The ovarian sections of mice overexpressing FGA were stained and photographed, CL is corpus luteum, black arrows are antral follicles, and yellow arrows are preantral follicles; (b) The ovarian sections of mice knocking down FGA were stained and photographed, CL is corpus luteum, black arrows are antral follicles, and yellow arrows are preantral follicles.

[0085] Fig.13 The results of the study on the efficiency of FGA overexpression or knockdown lentivirus infection of pig follicles are shown in Figure 1. (a) The effect of FGA overexpression on the apoptosis of GCs in mouse ovarian follicles; (b) The effect of FGA knockdown on the apoptosis of GCs in mouse ovarian follicles.

[0086] Fig.14The figures show the effects of lentiviral infection with overexpression or knockdown of FGA on the concentration of E2 in mouse serum; (a) the concentration level of E2 in the serum of mice with overexpression of FGA; (b) the concentration level of E2 in the serum of mice with knockdown of FGA.

[0087] Fig.15 Figure 3 shows the effect of overexpression or knockdown of FGA lentivirus-infected mouse ovaries on genes in apoptosis, proliferation and cycle-related pathways; (a) the effect of overexpression or knockdown of FGA on the mRNA expression of key genes related to ovarian apoptosis; (b) the effect of overexpression or knockdown of FGA on the protein expression of genes related to ovarian apoptosis; (c) the effect of overexpression or knockdown of FGA on the mRNA expression of genes related to the ovarian proliferation cycle; (d) the effect of overexpression or knockdown of FGA on the protein expression of genes related to the ovarian proliferation cycle. DETAILED DESCRIPTION

[0088] The present invention is further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions. Unless otherwise stated, the reagents and raw materials used in the present invention can be obtained commercially.

[0089] The present invention applies statistical methods to analyze the results of three independent experiments in each embodiment, calculates the "mean ± standard deviation", and uses one-way analysis of variance to perform significant difference analysis (in the figure, "*" indicates P < 0.05, and "**" indicates P < 0.01).

[0090] The following examples use the human ovarian granulosa cell KGN cell line purchased from Wuhan Punosai Life Science Technology Co., Ltd. as a cell model. The ovarian tissue of a grown-up dual-price reserve sow from a slaughterhouse in Guangzhou was selected for in vitro isolation and culture. 21-day-old C57BL / 6J black female mice purchased from the Experimental Animal Center of Southern Medical University in Guangdong Province were selected as the in vivo experimental model. Overexpression and knockdown FGA lentivirus purchased from Guangzhou Dongze Biotechnology Co., Ltd. was used for in vitro culture of pig ovarian follicles and in vivo experiments in mice. Among them, the overexpression lentivirus is constructed by inserting the CDS region sequence (GenBank: BC005467.1) of the mouse FGA gene (NM_010196.4) into the pLVX-C-FLAG-mCMV-ZsGreen-IRES-Puro plasmid through the SpeI+NotI restriction site, and then introduced into 293T cells to produce a high-titer lentivirus containing the target gene; the knockdown lentivirus used is constructed by inserting the shRNA used to knockdown FGA (target sequence: 5'-GGAACAACAAGGATTCTAA-3') into the pLVX-shRNA2-Puro plasmid through the BamHI+EcoRI restriction site, and then introduced into 293T cells to produce a high-titer lentivirus containing the target gene.

[0091] The pMD-18T vector and T4 DNA Ligase involved in the following examples were purchased from TaKaRa (Japan), and the pcDNA3.1(+) vector was purchased from Promega (USA).

[0092] Example 1: Study on the effect of FGA on the proliferation and cell cycle progression of ovarian granulosa cells

[0093] 1. Construction of FGA gene overexpression vector

[0094] (1) Extract total RNA from KGN cell lines according to the instructions of Shanghai Fijet Biotechnology Total RNA Rapid Extraction Kit and reverse transcribe it into cDNA;

[0095] (2) Specific primers for amplifying the CDS region of FGA (Gene ID: 2243) were designed by NCBI and amplified using the extracted cDNA as a template; the amplified fragment was purified and recovered, connected to the pMD18T vector (purchased from Takara), transformed, screened, and sequenced to extract the common plasmid after correct identification. The specific primer sequences are as follows:

[0096] (2) BioEdit software analysis revealed that the FGA gene CDS region sequence did not have the two restriction endonuclease sites of BamHI and NHeI, while the pcDNA3.1 vector had the two restriction endonuclease sites. The upstream and downstream primers were added with the BamHI and NHeI restriction site sequences, respectively. PCR amplification was performed using the recombinant pMD18T common plasmid of the FGA CDS region as a template; the fragment was purified, recovered, double-digested, connected to the pcDNA3.1 vector, transformed, screened, and sequenced to extract the endotoxin-free plasmid (the endotoxin-free plasmid small-scale extraction kit was purchased from Magen, USA), and named pcDNA3.1-FGA.

[0097] The primer sequences are as follows:

[0098] FGA F: 5′- CGC GGATCCCTTTCAGCTGGAGGTGCTCCTCA-3′

[0099] FGA R: 5′- CTA GCTAGCAAATGCAAGGGGCCATGGGAA-3′.

[0100] 2. Transfer the constructed FGA overexpression vector and FGA small interfering fragment into GCs

[0101] After the construction of the FGA overexpression vector was successfully verified by double enzyme digestion, GCs were inoculated in 24-well cell culture plates to verify the overexpression efficiency of pcDNA3.1-FGA and the interference efficiency of si-FGA. With pcDNA3.1 as the control group of pcDNA3.1-FGA, 100ng, 200ng and 500ng of pcDNA3.1 and pcDNA3.1-FGA vector plasmids were transfected, three replicates in each group, and RNA was extracted after 24 hours. The results showed that the mRNA expression of FGA increased significantly with the increase of transfection concentration (P < 0.001). Since the overexpression vectors at different concentrations had good transfection efficiency, 100ng of pcDNA3.1-FGA was selected for subsequent experiments. At the same time, with si-NC as the control of si-FGA (5′-GCTGCAGGATGAAAGGGTT-3′), 50nM and 100nM of si-NC and si-FGA were transfected into GCs, three replicates in each group. The results showed that the interference efficiency of si-FGA was the best at 50 nM (P < 0.001). Therefore, pcDNA3.1-FGA with a transfection concentration of 100 ng and si-FGA with a concentration of 50 nM were selected for subsequent experiments.

[0102] Cell transfection (6-well plate): observe the cell confluence under an optical microscope. The best transfection effect is achieved when the confluence reaches 70-80%. Aspirate the culture medium in the culture plate, add PBS (containing 1% double antibody) along the wall to wash the cells, repeat the washing once and add 1800 μL of incomplete culture medium. Take a 1.5 or 2 mL sterile centrifuge tube, prepare the transfection mixture according to the transfection concentration mentioned above and refer to the transfection instructions of Lipofectamine3000, and incubate at room temperature for 10-15 minutes. Add 200 μL of transfection mixture to each well of the 6-well plate, shake gently with the "cross" method, and return to the incubator for culture. After 24-48 hours of transfection, collect the cells for subsequent experiments. (2) TSA drug treatment: Observe the cell confluence under an optical microscope. The treatment effect is best when the confluence reaches 70-80%. Refer to the TSA drug instructions, weigh the TSA drug, dissolve it with an appropriate amount of DMSO solvent and dilute it to 0.5μM; discard the culture medium in the culture plate, add PBS (containing 1% double antibody) along the wall to wash the cells, repeat the wash once, add incomplete culture medium, and add DMSO or TSA to each well respectively. DMSO is the control group. Repeat three wells for each group; gently shake evenly using the "cross" method, return to the cell culture incubator for culture, and collect the cells for subsequent experiments 24 hours after transfection.

[0103] 3. Cell Apoptosis Detection

[0104] The present invention adopts Annexin V-FITC / PI cell apoptosis detection kit to detect the apoptosis rate of GCs cells.

[0105] 4. Cell Proliferation Assay

[0106] GCs proliferation detection was performed according to the instructions of Cell-Light EdU Apollo567 In Virto Kit of RiboBio. After GCs were transfected with plasmids or treated with small interfering RNA, they were labeled with EdU, fixed and stained, and immediately examined under a microscope or stored at 4°C in the dark.

[0107] 5. Cell cycle distribution detection

[0108] The present invention uses flow cytometry to detect cell cycle progression.

[0109] 6. Western Blot detection of protein expression of target gene

[0110] Total protein extraction, protein quantification, protein denaturation, SDS-PAGE electrophoresis, membrane transfer, blocking, antibody incubation, color development and gray value analysis.

[0111] 7. Chromatin Immunoprecipitation

[0112] ChIP detection was performed according to the instructions of MIKX Chromatin Immunoprecipitation (ChIP) Kit.

[0113] 8. Chromatin Accessibility

[0114] Refer to Epigen TEK EpiQuik TM The assay was performed according to the Chromatin Accessibility Assay Kit instruction manual, including preparation of buffers and solutions after GCs were transfected with NC and HDAC2; treatment of GCs; cell lysis and chromatin extraction; chromatin digestion; DNA purification, sample qRT-PCR and data analysis.

[0115] 9. Co-immunoprecipitation

[0116] Refer to the instructions of ACErProtein A / G Magnetic IP / Co-IP Kit.

[0117] 10. Nuclear and Cytoplasmic Separation

[0118] Nuclear-cytoplasmic separation Extraction of nuclear and cytoplasmic RNA: Inoculate an appropriate amount of GCs in a culture plate, transfect the plasmid or small interfering fragment for 24 hours, wash the GCs with PBS (containing 1% double antibody) and repeat once; add 200 μL of pre-cooled Lysis Buffer J to the culture plate, shake and lyse on ice for 5 minutes; transfer the lysate to a new 2 mL RNase-free tube and centrifuge at 16000 rpm for 10 minutes; transfer the supernatant (cytoplasmic RNA) to a new 2 mL RNase-free tube, and the remaining precipitate is the nuclear RNA; add 200 μL of Buffer SK to the supernatant and vortex for 10 seconds; add 200 μL of anhydrous ethanol and vortex for 10 seconds, transfer the mixed solution to the collection column, centrifuge at 6000 rpm for 1 minute, discard the filtrate, and retain the adsorption column; add 400 μL of Buffer SK to the nuclear RNA and vortex for 10 seconds; the steps are the same as before, and the adsorption column is retained; add 400 μL of Wash Solution, centrifuge at 16000rpm for 1min and discard the filtrate, repeat twice, discard the filtrate, centrifuge at 16000rpm for 1min and discard the filtrate and dry thoroughly; add 50μL ElutionBuffer E to the sponge in the center of the adsorption column, centrifuge at 2000rpm for 2min, centrifuge at 14000rpm for 1min, collect the nuclear and cytoplasmic RNA samples and store at -80℃.

[0119] Nuclear and cytoplasmic separation: extract the protein of the nucleus and cytoplasm: inoculate an appropriate amount of GCs in the culture plate, transfect the plasmid or small interfering fragment for 24 hours, wash the GCs with PBS (containing 1% double antibody) and repeat once; collect the cell pellet with 0.25% trypsin into a 1.5mL centrifuge tube, wash the collected cells twice with PBS, add 200μL pre-cooled CERⅠ, vortex mix for 15s, incubate on ice for 10min; add 11μL pre-cooled CERⅡ, vortex mix for 5s, incubate on ice for 1min, vortex for 5s, centrifuge at 16000rpm for 5min; immediately transfer the supernatant (cytoplasmic protein) to a new 2mL RNase-free tube and store at -80℃; add 100μL pre-cooled NER to the pellet, vortex for 15s, let it stand on ice for 40min, vortex for 15s every 10min, centrifuge at 16000rpm for 10min, and immediately transfer the supernatant (nuclear protein) to a new 2mL Store in RNase-free tubes at -80°C.

[0120] result:

[0121] (1) FGA inhibits granulosa cell apoptosis

[0122] like Figure 1 As shown in the figure, flow cytometry results showed that compared with the control group, pcDNA3.1-FGA significantly inhibited the early apoptosis of GCs (P<0.01); on the contrary, si-FGA promoted the early apoptosis of GCs. The effect of FGA on the expression of key genes related to apoptosis was further detected. qRT-PCR results showed that pcDNA3.1-FGA significantly inhibited the mRNA expression of apoptosis-related genes such as Bax, Caspase3, Caspase7, Caspase9, CREB1, P53 and BID (P<0.001), and promoted the mRNA level of BAD; while si-FGA significantly promoted the mRNA expression level of apoptosis-related genes such as Bax, Caspase7, Caspase9, CREB1 and BAD (P<0.001). WB results showed that pcDNA3.1-FGA could significantly inhibit the protein expression levels of Caspase3 and Caspase9 (P<0.01), while si-FGA significantly increased the protein expression of Caspase3 (P<0.05) and Caspase9 (P<0.001). The above results indicate that FGA can inhibit the apoptosis of GCs by affecting the mRNA and protein levels of apoptosis-related genes such as Caspase3 and Caspase9.

[0123] (2) FGA promotes granulosa cell proliferation

[0124] like Figure 2As shown in the figure, the EdU method was used to detect the proliferation rate of GCs. Compared with the control group, pcDNA3.1-FGA significantly promoted the proliferation rate of GCs (P<0.001); the corresponding si-FGA significantly reduced the proliferation rate of GCs. The results of qRT-PCR showed that overexpression of FGA could significantly promote the mRNA expression of cell proliferation-related genes such as STAR (P<0.01), PCNA (P<0.01), IKBA (P<0.05), SP1 (P<0.05) and MCL (P<0.01), and inhibit the mRNA expression level of GSK3B (P<0.001). Interference with FGA significantly inhibited the mRNA expression of cell proliferation-related genes such as GSK3B, STAR, SP1 and MCL (P<0.001). WB results showed that pcDNA3.1-FGA significantly promoted the expression of GSK3B (P<0.05) and PCNA (P<0.001) proteins, while si-FGA significantly reduced the expression levels of GSK3B and PCNA proteins (P<0.01). The above results indicate that FGA can promote the proliferation of GCs by promoting the mRNA and protein expression levels of proliferation-related genes such as PCNA.

[0125] (3) FGA accelerates the granulosa cell cycle

[0126] like Figure 3 As shown in the figure, flow cytometry results showed that compared with the control group, overexpression of FGA significantly increased the number of GCs entering G0 / G1 (P<0.05), reduced the number of GCs entering S phase (P<0.01), and had no significant difference in the number entering G2 / M phase. Interference with FGA significantly inhibited the number of GCs entering S phase and G2 / M phase (P<0.001), indicating that interference with FGA blocked GCs in G0 / G1 phase and delayed cell cycle progression. In the detection of the mRNA expression levels of genes related to cell cycle progression affected by overexpression or interference with FGA, it was found that pcDNA3.1-FGA significantly promoted the mRNA expression levels of cycle-related genes such as MYC (P<0.01), CCND1 (P<0.001) and CCNE2 (P<0.01), and inhibited the mRNA expression levels of CDK2 and CCNE1 (P<0.001). si-FGA significantly inhibited the mRNA expression of CCND1, CCNE2 and CDK7 (P<0.001). WB results showed that overexpression of FGA could significantly increase the protein expression of CCND1 (P<0.01) and CCNE2 (P<0.001), while interference with FGA significantly reduced the protein expression of CCND1 (P<0.05), and there was no significant difference in the expression of CCNE2 protein. The above results indicate that FGA can promote the cycle process of GCs by regulating the expression of mRNA and protein of cycle-related genes such as CCND1 and CCNE2.

[0127] (4) Interaction between FGA and Caspase3 affects granulocyte apoptosis

[0128] like Figure 4 As shown, FGA antibody was used to immunoprecipitate with ovarian GCs protein, and the protein complex was collected for denaturation. Finally, WB was used to detect the binding of the denatured protein complex with the key gene antibody related to apoptosis. Combining the negative control group IgG with the positive control group Input group, it was found that FGA protein was bound to Caspase3 protein. In order to further explore whether FGA and Caspase3 proteins are mutually bound, CoIP reverse verification experiment was used to immunoprecipitate ovarian GCs protein with Caspase3 and BIM antibodies. After the protein complex was denatured, WB was used to detect the expression of FGA protein. The results showed that compared with the IgG group, Caspses3 antibody could pull down Caspase3 protein and FGA protein. The above results show that FGA can bind to Caspase3 protein and regulate GCs apoptosis.

[0129] (5) Distribution of Caspase3 in granulosa cells after FGA binds to Caspase3

[0130] like Figure 5 As shown in the figure, the results of nuclear-cytoplasmic separation detection showed that the mRNA expression level of Caspase3 in the nucleus and cytoplasm of GCs was significantly decreased after overexpression of FGA (P<0.001), while interference with FGA significantly promoted the mRNA expression level of Caspase3 in the nucleus and cytoplasm of GCs (P<0.001). The protein level of Caspase3 in the nucleus and cytoplasm of GCs was significantly inhibited after overexpression of FGA (P<0.01), and interference with FGA significantly promoted the protein expression level of Caspase3 in the nucleus and cytoplasm of GCs (P<0.01). In summary, the results showed that the combination of FGA and apoptosis-related gene Caspase3 can significantly inhibit the expression of Caspase3 protein and mRNA in the nucleus and cytoplasm of GCs, thereby inhibiting the apoptosis of GCs.

[0131] Example 2: Study on the effect of FGA on the growth of pig follicles

[0132] The present invention uses healthy commercial sow ovaries as experimental materials and divides the follicles into large follicles (>7mm) and small follicles (<3mm) according to their sizes, extracts RNA and protein from large and small follicles respectively, and detects the expression pattern of FGA in large and small follicles. Figure 6As shown in the figure, qRT-PCR results showed that the mRNA expression of FGA increased significantly with the growth and development of pig follicles (P < 0.001). WB results showed that the expression of FGA protein in large follicles increased significantly compared with small follicles (P < 0.05). The above results indicate that FGA may play a role in promoting the growth and development of follicles.

[0133] Ovaries of healthy commercial sows were taken and separated into single follicles of the same size in the laboratory. The excess connective tissue around the follicles was separated as much as possible. The follicles were infected with lentivirus LV-FGA and sh-FGA respectively. Each group had more than 3 replicates. The follicles were cultured in vitro in a follicle incubator for 5 days. After taking photos and observing on the 1st, 3rd and 5th days, RNA and protein were extracted. The results are as follows Figure 7 As shown in the figure, qRT-PCR results showed that LV-FGA significantly promoted the expression of FGA mRNA (P < 0.001), while sh-FGA significantly inhibited the expression of FGA mRNA (P < 0.001). The expression of FGA protein was similar to the mRNA expression level after LV-FGA and sh-FGA treatment (P < 0.05). The above results show that the overexpression or knockdown of FGA lentivirus has a good effect on interfering with follicles and can be used for subsequent experiments.

[0134] 1. Isolation, culture and lentiviral infection of porcine follicles

[0135] The collected ovarian tissue was stored in pre-cooled PBS (containing 1% double antibody) and quickly brought back, and then washed with PBS (containing 1% double antibody) several times until the PBS liquid was clear and blood-free, soaked in 75% alcohol for 30s, and then washed with PBS (containing 1% double antibody) and sealed and sent to the cell room. 3-5mm follicles were separated with a scalpel under the clean bench, and the excess connective tissue outside the follicles was carefully removed as much as possible. PBS (containing 1% double antibody) was washed twice, and then washed with DMEM / F12 follicle culture medium (containing 1% double antibody) was washed twice. One follicle per well was placed in a 24-well plate containing follicle culture medium, incubated in a 38.5℃ 5% CO2 incubator for 24h, washed with PBS (containing 1% double antibody) twice, replaced with new follicle culture medium, and lentivirus (titer of 10 7 ) follicles were treated and photographed on the 1st, 3rd and 5th days after infection with lentivirus

[0136] 2. Other Experiments Reference Example 1

[0137] result:

[0138] (1) FGA promotes the development of follicular GCs

[0139] After 5 days of in vitro culture of pig follicles infected with lentivirus, Figure 8As shown in the figure, compared with the control group, overexpression of FGA can delay the turbidity of follicular fluid and the disappearance of blood vessels on the surface of follicles. After knocking down FGA, the follicular fluid began to become turbid on the 3rd day, and the blood vessels on the surface of follicles disappeared on the 5th day. The above results indicate that FGA can promote the growth and development of follicles.

[0140] (2) FGA inhibits apoptosis of follicular GCs

[0141] After 5 days of in vitro culture, pig follicles infected with lentivirus were collected and fixed with formaldehyde, and the effect of FGA on the apoptosis of GCs in follicles was studied by Tunel. Fig. 9 As shown in the figure, compared with the control group, LV-FGA inhibited the apoptosis of GCs in follicles, while sh-FGA promoted the apoptosis of GCs in follicles. The above results indicate that FGA can inhibit the apoptosis of GCs in follicles.

[0142] (3) FGA affects the expression of key genes in the follicle function pathway

[0143] In order to study the expression regulation of FGA in follicular development, the present invention detects the effect of FGA on genes related to apoptosis, proliferation and cycle progression in follicles. Fig.10As shown in the figure, qRT-PCR results showed that LV-FGA significantly inhibited the mRNA expression levels of BID (P<0.001), Caspase3 (P<0.001), Caspase8 (P<0.001) and BAD (P<0.01) in follicles, and significantly promoted the mRNA expression of apoptosis-related genes such as Bcl2 (P<0.001), P53 (P<0.001) and BAK (P<0.01). sh-FGA significantly increased the mRNA expression levels of apoptosis-related genes such as BIM, Caspase8, Caspase9, Bcl2, P53 and BAD (P<0.001). WB results showed that overexpression of FGA could significantly reduce the protein expression levels of Caspase9 and P53, and there was no significant difference in the protein expression level of BIM. After knocking down FGA, the protein expression levels of apoptosis-related genes were detected, and the results showed that sh-FGA significantly promoted the protein expression of BIM, Caspase9 and P53. The same method was used to detect the effect of FGA on proliferation and cycle progression-related genes in follicles. Overexpression of FGA significantly promoted the mRNA expression of key proliferation cycle-related genes CDK1, CDK4, CCND1, PCNA, MCL1, CCNB1 and CCND2 (P < 0.001), while there was no significant difference in the mRNA expression of RRM1. Knockdown of FGA significantly inhibited the mRNA expression levels of proliferation cycle-related genes CDK2 (P < 0.05), CDK4 (P < 0.01), CCNE1 (P < 0.05), PCNA (P < 0.05), MCL1 (P < 0.01), CCNB2 (P < 0.01), CCND2 (P < 0.01), CCNE2 (P < 0.001) and RRM1 (P < 0.001). WB results showed that LV-FGA significantly increased the protein expression of proliferation cycle-related genes PCNA, STAR and CCNE1, while sh-FGA had the opposite result, significantly reducing the protein expression levels of genes such as PCNA, STAR and CCNE1. The above results indicate that FGA can regulate the growth and development of follicles by regulating the expression levels of mRNA and protein of genes such as Caspase9, P53, PCNA, STAR, CCNE1, etc.

[0144] Example 3: Study on the effect of FGA on mouse ovarian development

[0145] The present invention uses C57BL / 6J female mice as an experimental animal model to study the effect of FGA on the growth and development of ovarian follicles. Fig.11As shown in the figure, compared with the control group, the overexpression of FGA lentiviral vector significantly promoted the mRNA expression level of FGA in the mouse ovary (P < 0.001), while knocking down FGA had the opposite result and could significantly inhibit the protein expression level of FGA in the mouse ovary (P < 0.001). WB results showed that LV-FGA could significantly promote the protein expression level of FGA in the mouse ovary (P < 0.001), while sh-FGA significantly inhibited the protein expression level of FGA in the mouse ovary (P < 0.01). The above results indicate that the overexpression or knockdown of FGA lentiviral vector can play a regulatory role in the constructed mouse experimental model and can be used in subsequent experiments.

[0146] 1. Mouse husbandry and lentivirus treatment

[0147] Twenty-six 21-day-old C57BL / 6J female mice were selected and randomly divided into four groups with toe clipping and marking: LV-NC group (5 mice), LV-FGA group (8 mice), sh-NC group (5 mice) and sh-FGA group (8 mice). After one week of adaptive breeding at the Experimental Animal Center of South China Agricultural University, the mice were injected with mouse lentiviral vectors by intraperitoneal injection once a week for three consecutive weeks. The mice were weighed every day and the estrus of the mice was observed and recorded.

[0148] The estrus status of mice was observed every day to explore the effect of FGA on the age of mice at first estrus.

[0149] 3. HE staining

[0150] The present invention adopts Hematoxylin-eosin staining (HE) to detect the development of mouse ovarian follicles. The specific experimental steps are carried out with reference to the official website of Wuhan Saiweier Biotechnology Co., Ltd.

[0151] 4. Tunel detection of ovarian follicle apoptosis

[0152] The present invention uses paraffin section fluorescence Tunel to detect ovarian follicle apoptosis, and the specific experimental steps are carried out according to the official website of Wuhan Saiweier Biotechnology Co., Ltd.

[0153] 5.ELISA to detect estrogen secretion

[0154] Mouse serum estrogen detection was performed with reference to the estradiol enzyme-linked immunosorbent assay kit. The specific steps are as follows: (1) Add 100 μL of the sample to be tested to the antibody-coated reaction well (and make a blank well at the same time) and incubate at 37°C for 2 h; (2) Aspirate the supernatant, add 300 μL of washing solution to each well, soak for 2 min, and then aspirate the supernatant; (3) Add 100 μL of biotinylated antibody working solution to each well and incubate at 37°C for 1 h; (4) Same as (2); (5) Add 100 μL of enzyme conjugate working solution to each well and incubate at 37°C for 30 min; (6) Same as (2); (7) Add 100 μL of TMB substrate solution to each well and incubate at 37°C for 30 min in the dark; (8) Add 100 μL of 2 M sulfuric acid, and the color changes from blue to yellow; (9) Detect the OD value at 450 mm using an ELISA reader within 10 min and calculate the result.

[0155] 6. Other experiments refer to Example 1 or Example 2

[0156] result:

[0157] (1) FGA promotes the onset of puberty in mice

[0158] The results are as follows Fig.11 As shown in the figure, the estrus age of mice in the FGA overexpression lentiviral vector group was earlier than that of mice in the control group (P < 0.05). The estrus age of mice in the FGA knockdown lentiviral vector group was later than that of mice in the control group on average (P < 0.05). The above results indicate that FGA can promote the age of first estrus in mice.

[0159] (2) FGA promotes mouse follicle development

[0160] Hematoxylin and eosin staining was used to detect the growth and development of mouse ovarian follicles. Fig.12 As shown in the figure, compared with the control group, the number of preantral follicles in the ovaries of mice in the overexpression FGA group was significantly reduced, and the number of corpora lutea and antral follicles was significantly increased. The number of preantral follicles in the ovaries of mice in the knockdown FGA group was significantly increased compared with that in the control group. The above results indicate that FGA can promote the growth and development of ovarian follicles in mice.

[0161] (3) FGA inhibits mouse ovarian apoptosis

[0162] The Tunel slice experiment was used to detect the apoptosis level of GCs in mouse ovarian follicles. Fig.13 As shown in the figure, compared with the control group, the apoptosis rate of GCs in the ovarian follicles of mice in the overexpression FGA group was significantly reduced. The results after knocking down FGA were the opposite. The apoptosis level of GCs in the ovarian follicles of mice in the knockdown FGA group was significantly higher than that in the control group. The above results indicate that FGA can inhibit the apoptosis level of GCs in the ovarian follicles of mice.

[0163] (4) FGA promotes E2 secretion in mouse serum

[0164] The blood of mice was collected and centrifuged to obtain serum. The concentration of E2 in the serum of mice was detected by ELISA. Fig.14 As shown in the figure, the E2 concentration level in the serum of mice overexpressing FGA was significantly higher than that in the control group (P < 0.01). Compared with the control group, the E2 concentration level in the serum of mice knocked down FGA was significantly reduced (P < 0.01). The above results indicate that FGA can significantly promote the secretion of estrogen in mice.

[0165] (5) FGA affects the expression of key genes in the mouse ovarian function pathway

[0166] The present invention uses qRT-PCR and WB to detect the effect of FGA on the mRNA and protein expression levels of key genes in the apoptosis and proliferation cycle-related pathways of mouse ovarian GCs. Fig.15As shown in the results, overexpression of FGA significantly inhibited the mRNA expression of apoptosis-related genes such as Creb1, Caspase3, Caspase7, Bax, P53 and Bcl2 (P<0.001). Knockdown of FGA significantly increased the mRNA expression levels of Creb1 (P<0.01), Caspase3 (P<0.01), Caspase7 (P<0.01), Bax (P<0.001), P53 (P<0.01), Bcl2 (P<0.001) and Bim (P<0.001), while there was no significant difference in Caspase8 and Caspase9. Compared with the control group, overexpression of FGA significantly reduced the protein expression levels of Caspase3 (P<0.01), Bim (P<0.01), and Caspase9 (P<0.05). The protein expression levels of Caspase3 (P<0.001), Bim (P<0.001), and Caspase9 (P<0.01) in the knockdown FGA group were significantly higher than those in the control group. The same method was used to detect the effect of FGA on the proliferation cycle-related pathways of the mouse ovary. Compared with the control group, the LV-FGA group could significantly promote the mRNA expression levels of proliferation cycle-related genes such as Star (P<0.05), Mcl1 (P<0.001), Pcna (P<0.05), P65 (P<0.01), Cdk4 (P<0.02), and Ccne1 (P<0.05), and significantly inhibited the expression of Sp1 gene mRNA (P<0.001), and there was no significant difference in the mRNA expression of Ccnb2. The mRNA expression levels of Star (P<0.01), Mcl1 (P<0.001), Sp1 (P<0.001), Pcna (P<0.01), P65 (P<0.001), Cdk4 (P<0.001) and Ccnb2 (P<0.001) in the sh-FGA group were significantly higher than those in the control group. The protein level results showed that overexpression of FGA could significantly increase the protein expression level of the proliferation cycle-related gene P65 (P<0.001), and the protein levels of Cdk4 and Pcna were not significantly different from those in the control group. The results of knocking down FGA were the opposite. sh-FGA significantly inhibited the protein expression of P65, Cdk4 and Pcna (P<0.05). The above results indicate that FGA can inhibit the apoptosis of mouse ovarian GCs by inhibiting the mRNA and protein expression of Caspase3, Bim and Caspase9; and promote the proliferation rate and GCs cycle process of mouse ovarian GCs by regulating the expression of P65, Cdk4 and Pcna.

[0167] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Application of FGA gene-related biomaterials, characterized by: The biological material is any one or more of the following substances A, B, C, D, E, F, G: A. Full-length genomic DNA of FGA gene or its homologous nucleic acid molecule; B. FGA gene CDS region sequence or its homologous nucleic acid molecule; C. The protein encoded by the FGA gene or its homologous amino acid; D. A substance that can increase the expression level and / or activity of substance A or B; E. Substances that can reduce the expression and / or activity of substance A or B; F. A substance that can increase the expression level and / or activity of substance C; G. Substances that can reduce the expression level and / or activity of substance C; The application is any one or more of the following applications Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ: Ⅰ. Application in regulating follicle development / preparing products for regulating follicle development; II. Application in regulating granulocyte proliferation / preparing products for regulating granulocyte proliferation; III. Application in regulating granulocyte apoptosis / preparing products for regulating granulocyte apoptosis; IV. Application in regulating estrogen secretion / preparing products for regulating estrogen secretion; Ⅴ. Application in regulating puberty initiation / preparing products for regulating puberty initiation.

2. The use of the FGA gene-related biomaterial according to claim 1, characterized in that: Application I includes any one or more of the following applications 1) and 2): 1) Use of substance A, B, C, D or F in promoting follicle development / preparing products for promoting follicle development; 2) Use of substance E or G in inhibiting follicular development / preparing products for inhibiting follicular development; Application II includes any one or more of the following applications 1) and 2): 1) Use of substance A, B, C, D or F in promoting granulocyte proliferation / preparing products promoting granulocyte proliferation; 2) Use of substance E or G in inhibiting granulocyte proliferation / preparing a product for inhibiting granulocyte proliferation; Application III includes any one or more of the following applications 1) and 2): 1) Use of substance A, B, C, D or F in inhibiting granulocyte apoptosis / preparing a product for inhibiting granulocyte apoptosis; 2) Use of substance E or G in promoting granulosa cell apoptosis / preparing products promoting granulosa cell apoptosis; Application IV includes any one or more of the following applications 1) and 2): 1) Use of substance A, B, C, D or F in promoting estrogen secretion / preparing products promoting estrogen secretion; 2) Use of substance E or G in inhibiting estrogen secretion / preparing products for inhibiting estrogen secretion; Application V includes any one or more of the following applications 1) and 2): 1) Use of substance A, B, C, D or F in promoting puberty / preparing products promoting puberty; 2) Use of substance E or G in inhibiting puberty onset / preparing products for inhibiting puberty onset.

3. The use of the FGA gene-related biomaterial according to claim 2, characterized in that: In application IV, the estrogen includes estradiol E2.

4. The use of the FGA gene-related biomaterial according to any one of claims 1 to 3, characterized in that: The application is application in mammals; The application is an application in an in vitro environment.

5. The use of the FGA gene-related biomaterial according to claim 4, characterized in that: The application is application in humans, pigs or mice.

6. The use of the FGA gene-related biological material according to claim 5, characterized in that: When the use is in humans: The nucleotide sequence of the full-length genomic DNA described in A is shown in the sequence of NCBI Gene ID: 2243; The nucleotide sequence of the CDS region sequence described in B is shown in the sequence of NCBI CCDS: CCDS3787.1; The amino acid sequence of the protein described in C is shown in the sequence of NCBI NP_000499.1; When the application is in pigs or mice: The nucleotide sequence of the full-length genomic DNA described in A is shown in the sequence of NCBI Gene ID: 14161; The nucleotide sequence of the CDS region sequence described in B is shown in the sequence of GenBank: BC005467.1; The amino acid sequence of the protein described in C is shown in the sequence of GenBank: AAH05467.

1.

7. The use of the FGA gene-related biomaterial according to any one of claims 1 to 3, characterized in that: Substance D or F is selected from: an overexpression recombinant vector containing a corresponding nucleic acid molecule; The vector used in the overexpression recombinant vector is pcDNA3.1; Substance E or G is selected from: siRNA, shRNA, sgRNA or knockout recombinant vectors containing the same for knocking out / knockdown of gene expression; When the application is for use in humans, the targeting sequence corresponding to the siRNA is: 5′-GCTGCAGGATGAAAGGGTT-3′; When the application is in pigs or mice, the targeting sequence corresponding to the shRNA is: 5′-GGAACAACAAGGATTCTAA-3′.

8. Application of FGA combined with Caspase3 in regulating ovarian granulosa cells, characterized in that: In vitro, the FGA gene binds to the apoptosis-related gene Caspase3 and negatively regulates the apoptosis of ovarian granulosa cells.

9. The use of FGA combined with Caspase3 in regulating ovarian granulosa cells according to claim 8, characterized in that: Increasing the expression of exogenous FGA gene decreased the expression levels of Caspase3 mRNA and protein, inhibiting the apoptosis of ovarian granulosa cells; inhibiting the expression of FGA gene increased the expression levels of Caspase3 mRNA and protein, promoting the apoptosis of ovarian granulosa cells.

10. The use of FGA combined with Caspase3 in regulating ovarian granulosa cells according to claim 9, characterized in that: The increased expression of exogenous FGA gene is achieved by gene overexpression technology. The gene overexpression vector is prepared by the following method: (1) extracting RNA from ovarian granulosa cells, reverse transcribing it into cDNA, and performing PCR amplification using cDNA as a template to obtain the target fragment; (2) connecting the target fragment to the pcDNA3.1 vector digested with restriction endonucleases BamHI and NHeI to obtain the gene overexpression vector; the primers used for PCR amplification are as follows: FGA F: 5′-CGCGGATCCCTTTCAGCTGGAGGTGCTCCTCA-3′ FGA R: 5′-CTAGCTAGCAAATGCAAGGGGCCATGGGAA-3′; The inhibition of FGA gene expression is achieved by siRNA or shRNA.

Images