Application of miR-424-3p as a target in the preparation of products promoting the proliferation of goat ovarian granulosa cells

Abstract

The present invention provides the use of miR-424-3p as a target in the preparation of a product that promotes the proliferation of goat ovarian granulosa cells, belonging to the technical field of ovarian granulosa cell proliferation. The present invention proposes for the first time that overexpression of miR-424-3p can significantly inhibit the proliferation of goat ovarian granulosa cells and promote apoptosis of goat ovarian granulosa cells, while inhibiting the expression of miR-424-3p promotes the proliferation of goat ovarian granulosa cells and inhibits apoptosis of goat ovarian granulosa cells. The present invention also proposes for the first time that inhibiting the expression of miR-424-3p can promote the transition of goat ovarian granulosa cells from the G1 phase to the S phase.

Description

Technical Field

[0001] The present invention belongs to the technical field of ovarian granulosa cell proliferation, and in particular relates to the application of miR-424-3p as a target in the preparation of a product for promoting the proliferation of goat ovarian granulosa cells. Background Art

[0002] With economic growth and rising living standards, the demand for mutton has increased annually, significantly boosting the sheep farming industry. However, the market is currently in short supply. Low goat fertility is a major constraint on the development of the sheep farming industry. Improving goat reproductive performance can significantly boost the industry's development. Follicular development is a key factor influencing animal reproductive performance. Comprehensive understanding of the mechanisms of follicular development is crucial for improving animal reproductive performance. The follicle is composed of granulosa cells, oocytes, and theca cells. Granulosa cells play a crucial role in follicular development, transmitting signals and exchanging substances with the oocyte through gap junctions. They also secrete steroid hormones and cytokines to influence the growth of surrounding follicles. Granulosa cell proliferation and apoptosis directly influence follicular development. Extensive granulosa cell apoptosis can lead to follicular atresia. Furthermore, granulosa cell proliferation is inhibited in the later stages of follicular development.

[0003] MicroRNA (miRNA) is a short, non-coding RNA that regulates target gene expression by binding to the 3′ UTR region of a target gene through complete or incomplete pairing of its seed sequence, affecting the stability or translation of the target gene mRNA. MiRNA is essential for female reproduction, participating in the regulation of granulosa cell proliferation and apoptosis, the synthesis and secretion of follicular hormones, and oocyte maturation. However, no studies have yet investigated the relationship between miR-424-3p and the proliferation or apoptosis of granulosa cells in goat ovaries. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide the use of miR-424-3p as a target in the preparation of a product for promoting the proliferation and / or inhibiting the apoptosis of goat ovarian granulosa cells.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of miR-424-3p as a target in preparing a product for promoting proliferation and / or inhibiting apoptosis of goat ovary granulosa cells.

[0007] Preferably, the target gene of miR-424-3p is PIP5K1C.

[0008] Preferably, the effect is exerted by inhibiting the expression of miR-424-3p.

[0009] The present invention also provides a method for studying the function of genes related to goat ovarian granulosa cell proliferation, or screening drugs for promoting goat ovarian granulosa cell proliferation or inhibiting goat ovarian granulosa cell apoptosis, or analyzing gene therapy drugs for goat ovarian granulosa cell apoptosis, the method comprising knocking out the miR-424-3p gene or reducing the expression of the miR-424-3p gene.

[0010] Preferably, the method of reducing the expression of the miR-424-3p gene includes using a miR-424-3p inhibitor, and the nucleotide sequence of the miR-424-3p inhibitor is shown in SEQ ID NO.1.

[0011] The present invention also provides the use of a miR-424-3p inhibitor in preparing a product for promoting proliferation and / or inhibiting apoptosis of goat ovarian granulosa cells. The nucleotide sequence of the miR-424-3p inhibitor is shown in SEQ ID NO.1.

[0012] Preferably, the types of products include medicines.

[0013] The present invention also provides a drug capable of promoting proliferation of goat ovary granulosa cells and / or inhibiting apoptosis of goat ovary granulosa cells, wherein the drug can inhibit the expression of miR-424-3p gene.

[0014] Preferably, the dosage form of the drug includes oral solution, injection, tablet, pill, dispersion, capsule, dripping pill, granule, suspension or emulsion.

[0015] Beneficial effects of the present invention:

[0016] The present invention proposes for the first time that overexpression of miR-424-3p can significantly inhibit the proliferation and promote apoptosis of goat ovarian granulosa cells, while inhibition of miR-424-3p expression promotes the proliferation and inhibits apoptosis of goat ovarian granulosa cells. The present invention also proposes for the first time that inhibition of miR-424-3p expression can promote the transition of goat ovarian granulosa cells from the G1 phase to the S phase. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 5 is the qRT-PCR expression detection result of the miR-424-3p gene, among which A is the expression detection result of miR-424-3p in large and small follicles of Leizhou goats; B is the expression detection result of miR-424-3p in goat granulosa cells after transfection of miR-424-3p mimics; C is the expression detection result of miR-424-3p in goat granulosa cells after transfection of miR-424-3p inhibitor.

[0018] Figure 2 Figure 3 shows the effects of CCK8 and EDU on the proliferation of goat granulosa cells detected by miR-424-3p. A shows the effects of CCK8 on the proliferation of goat granulosa cells detected by miR-424-3p mimics and inhibitor. B shows the effects of EDU on the proliferation of goat granulosa cells detected by miR-424-3p mimics and inhibitor.

[0019] Figure 3 Figure 3: Flow cytometry analysis of the effect of miR-424-3p on apoptosis of goat follicular granulosa cells. A shows the effect of overexpression of miR-424-3p mimics on apoptosis of goat follicular granulosa cells; B shows the effect of overexpression of miR-424-3pin inhibitor on apoptosis of goat follicular granulosa cells.

[0020] Figure 4 Figure 3: Flow cytometry analysis of the effect of miR-424-3p on the cell cycle progression of goat granulosa cells. A shows the effect of overexpression of miR-424-3p mimics on the cell cycle progression of goat granulosa cells; B shows the effect of overexpression of miR-424-3p inhibitor on the cell cycle progression of goat granulosa cells.

[0021] Figure 5 Figure 3 shows the effect of overexpression of miR-424-3p on the expression of proliferation marker genes in goat granulosa cells. Figure A shows the expression of proliferation-related genes in goat granulosa cells after overexpression of miR-424-3p mimics by qRT-PCR; Figure 3B shows the expression of PCNA protein, CDK6 protein, and CCND1 protein in goat granulosa cells after overexpression of miR-424-3p mimics by Western Blot.

[0022] Figure 6 Figure 3 shows the effect of overexpression of miR-424-3p on the expression of apoptosis marker genes in goat granulosa cells. A shows the expression results of apoptosis-related genes in goat granulosa cells after overexpression of miR-424-3p mimics by qRT-PCR; B and C show the expression results of BAX protein and BCL2 protein in goat granulosa cells after overexpression of miR-424-3p mimics by Western Blot.

[0023] Figure 7Figure 5 is the screening results of miR-424-3p target genes, where A is the binding pattern of miR-424-3p to the 3′UTR of the goat PIP5K1C gene predicted by RNAhybrid; B is the qRT-PCR detection of the expression of the PIP5K1C gene in goat granulosa cells after overexpressing miR-424-3p mimics; C is the Western Blot detection of the expression of PIP5K1C protein in goat granulosa cells after overexpressing miR-424-3p mimics; D is the dual luciferase detection of the targeting relationship between miR-424-3p and the predicted binding sites of the 3′UTR of the goat PIP5K1C gene. DETAILED DESCRIPTION

[0024] The present invention provides the use of miR-424-3p as a target in preparing a product for promoting proliferation and / or inhibiting apoptosis of goat ovary granulosa cells.

[0025] In the present invention, the target gene of miR-424-3p is preferably the phosphatidylinositol 4-phosphate 5-kinase 1C type (PIP5K1C) gene. PIP5K1C is an important protein that produces phosphatidylinositol PIP2 in cells. MiR-424-3p can inhibit the expression of PIP5K1C by targeting the 3'UTR region of PIP5K1C. In the present invention, when miR-424-3p is used as a target, it is preferred to promote the proliferation of goat ovarian granulosa cells and / or inhibit the apoptosis of goat ovarian granulosa cells by inhibiting the expression of miR-424-3p. The present invention is not particularly limited to the specific method of inhibiting the expression of miR-424-3p. In the present invention, the types of products preferably include drugs.

[0026] The present invention also provides a method for studying the function of genes related to goat ovarian granulosa cell proliferation, or screening drugs for promoting goat ovarian granulosa cell proliferation or inhibiting goat ovarian granulosa cell apoptosis, or analyzing gene therapy drugs for goat ovarian granulosa cell apoptosis, the method comprising knocking out the miR-424-3p gene or reducing the expression of the miR-424-3p gene.

[0027] In the present invention, the method of reducing miR-424-3p gene expression preferably includes using a miR-424-3p inhibitor, and the nucleotide sequence of the miR-424-3p inhibitor is preferably "5'-AUAGCAGCGCCUCACGUUUUG-3' (SEQ ID NO. 1). More preferably, each base of the inhibitor is modified with 2'-O-Methyl (methyl group) (referring to adding a methyl group to the 2'-OH group of the RNA ribose sugar ring).

[0028] The present invention also provides the use of a miR-424-3p inhibitor in the preparation of a product for promoting proliferation and / or inhibiting apoptosis of goat ovarian granulosa cells. The nucleotide sequence of the miR-424-3p inhibitor is preferably "5'-AUAGCAGCGCCUCACGUUUUG-3' (SEQ ID NO. 1). In the present invention, the type of the product preferably includes a drug.

[0029] The present invention also provides a drug capable of promoting proliferation of goat ovary granulosa cells and / or inhibiting apoptosis of goat ovary granulosa cells, wherein the drug can inhibit the expression of miR-424-3p gene.

[0030] In the present invention, the dosage form of the drug preferably includes oral solution, injection, tablet, pill, dispersion, capsule, dripping pill, granule, suspension or emulsion. In the present invention, the drug preferably also includes pharmaceutical excipients. The present invention does not particularly limit the specific type of pharmaceutical excipients, which depends on the specific drug dosage form.

[0031] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] In the following examples, unless otherwise specified, all methods are conventional.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] The primer sequences used in the qRT-PCR experiments are summarized in Table 1.

[0035] Table 1 qRT-PCR primer sequences

[0036]

[0037]

[0038] All data in the following examples are expressed as mean ± standard deviation (SD), with at least three independent replicates. Differences between two or more groups were analyzed using t-test and one-way analysis of variance, respectively. All analyses were performed in GraphPad Prism 9. For "*" in the charts, "*" represents P < 0.05, "**" represents P < 0.01, and "***" represents P < 0.001.

[0039] Example 1

[0040] Isolation of goat follicles

[0041] After slaughtering the Leizhou goats, the ovaries are removed and placed in a culture dish, washed with PBS, and then the follicles are initially cut off from the ovaries with scissors. Then, the ovarian base tissue to which the follicles are adhered is separated as cleanly as possible under a stereomicroscope. The separated follicles are divided into large and small follicles according to their diameter. Follicles with a diameter of less than 3 mm are defined as small follicles, and follicles with a diameter of more than 6 mm and surrounded by rich blood vessels are defined as large follicles. The separated follicles are immediately placed in liquid nitrogen for preservation.

[0042] The expression of miR-424-3p in large and small follicles was verified by qRT-PCR. The specific method is as follows: Cell RNA extraction and reverse transcription: Cell RNA extraction was carried out according to the instructions of Takara's RNAiso Plus reagent. The specific steps are as follows: (1) Cell collection. 48 hours after cell transfection, the cells were digested and centrifuged using 0.25% trypsin; (2) Cell lysis. 1 ml of RNAiso Plus reagent was added to each cell sample, vortexed for 15 seconds, and allowed to stand at room temperature for 5 minutes; (3) Total RNA extraction. 200 μL of chloroform was added, the centrifuge tube was tightly capped, vortexed for 15 seconds, and allowed to stand at room temperature for 5 minutes; centrifuged at 4°C, 12000g for 15 minutes, and then the centrifuge tube was carefully removed. At this time, the solution was divided into three layers. The upper aqueous phase was carefully pipetted into a new centrifuge tube, and then an equal volume of ice-cold isopropanol was added. The tube was gently inverted to mix and allowed to stand at room temperature for 10 minutes; then centrifuged at 4°C, 12000g for 10 minutes; (4) RNA precipitation and washing. Carefully discard the supernatant, add 1 mL of 75% ethanol, invert the tube to wash the RNA precipitate and the tube wall, then centrifuge at 7500g for 5 minutes at 4°C, repeat the operation twice; (5) RNA dissolution. Discard the supernatant, carefully remove the remaining liquid with a pipette, finally open the centrifuge tube cap, and dry the precipitate at room temperature for a few minutes. After the precipitate is dry, add an appropriate amount of RNase-free water to dissolve the precipitate.

[0043] Reverse transcription of miRNA: Use the miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) from Novizan Biotechnology to reverse transcribe miRNA. The steps are as follows: (1) Removal of genomic DNA. Take 1 μg of total RNA, add 2 μl of 5×gDNA Wiper Mix, add an appropriate amount of sterile enzyme-free water to make the system volume reach 10 μl, and incubate at 42°C for 2 minutes. (2) cDNA synthesis. Add 2 μl of 10×RT Mix, 2 μl of HiScript II Enzyme Mix, 1 μl of reverse transcription primer (2 μM) and 5 μl of sterile enzyme-free water to the above reaction system to prepare the reaction system. Gently pipette to mix. After mixing, reverse transcription is performed according to the reaction temperature: 25°C, 5 minutes; 50°C, 15 minutes; 80°C, 5 minutes. The reverse transcription primer sequence was 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGCGCG-3′ (SEQ ID NO. 22). All operations were performed on ice.

[0044] Reverse transcription of mRNA

[0045] Perform mRNA reverse transcription according to the instructions for the Evo M-MLV Reverse Transcription Premix Kit from Acryl. Prepare the reaction mixture and incubate at 37°C for 5 minutes and then at 85°C for 5 seconds. After completion of the reaction, dilute the mixture to 100 μL and store at -20°C.

[0046] qRT-PCR was performed using cDNA as a template according to the instructions of Novizan ChamQ SYBR qPCR Master Mix (Low ROX Premixed). The reaction system was as follows: 10 μL of 2X ChamQ SYBR qPCR Master Mix (Low ROX Premixed), 0.4 μL of each of the upstream and downstream primers (10 μM), 2 μL of cDNA, and 7.2 μL of ddH2O. The reaction program was: 95°C for 30 seconds; 95°C for 10 seconds, 60°C for 30 seconds, 40 cycles; 95°C for 15 seconds, 60°C for 60 seconds, and 95°C for 15 seconds. GAPDH was used as an internal reference, and the results were analyzed using 2 -△△CT The primer sequences are shown in Table 1.

[0047] The results showed that the expression level of miR-424-3p in large follicles of Leizhou goats was significantly higher than that in small follicles, which was consistent with the sequencing results ( Figure 1 A).

[0048] Example 2

[0049] Isolation and identification of granulosa cells

[0050] To investigate the effect of miR-424-3p on the proliferation and apoptosis of goat follicular granulosa cells, miR-424-3p mimics and inhibitors, as well as corresponding negative controls, were transfected into goat follicular granulosa cells, and the expression of miR-424-3p was detected by qRT-PCR (the qRT-PCR detection method was the same as in Example 1, and the primer sequences are shown in Table 1). The specific method is as follows:

[0051] Isolation and identification of goat follicular granulosa cells

[0052] After slaughtering the goat, the ovarian tissue was located and removed using autoclaved scissors and forceps. The ovaries were then immersed in 75% alcohol for approximately 10 seconds, followed by another immersion in saline containing a 2% double-antibody solution for approximately 10 seconds. After immersion, the ovaries were removed and placed in saline containing a 2% double-antibody solution at 37°C. The ovaries were then transported to the laboratory within 2 hours. Once back in the laboratory, granulosa cells were isolated in a biosafety cabinet that had been sterilized with ultraviolet light for 30 minutes. First, the ovaries were removed from physiological saline and soaked in 75% alcohol for 20 seconds. The ovaries were then rinsed three times with PBS containing 2% double antibody. After the rinsing, the ovaries were placed in 37°C DMEM culture medium containing 2% double antibody; a 0.8 ml syringe was used to puncture the follicles to release the granulosa cells in the follicles, and then 0.5 ml of DMEM was drawn up with a syringe to blow the punctured follicles three times. All follicles were operated on in sequence; the DMEM culture medium containing follicular fluid was filtered using a 70-micron cell sieve. After the filtration, the filtrate was transferred to a 15 ml centrifuge tube, allowed to stand at room temperature for 15 minutes, and then centrifuged at 1000 r / min for 10 minutes. After the centrifugation, the supernatant was discarded, and the cells were resuspended in 3 ml of DMEM containing 2% double antibody, centrifuged again and the supernatant was discarded; then 10 ml of complete culture medium (formula: 90% DMEM / F12 + 10% FBS + 1% double antibody + 1% 100x ITS-A) cells were resuspended and inoculated into a cell culture dish; finally, the cells were cultured in an incubator at 5% carbon dioxide, saturated humidity, and 37°C. After 24 hours of culture, unattached cells were discarded and replaced with new complete culture medium. The culture medium was then replaced every 48 hours until the cells reached approximately 80% growth, at which time the cells were passaged and cryopreserved.

[0053] During cell passage, an appropriate amount of cells were seeded into a 24-well cell culture dish containing a cell slide. After 36 hours of cell culture, the FSHR gene was used as a molecular marker for ovarian granulosa cells for immunofluorescence detection to identify whether the cultured cells were granulosa cells. The immunofluorescence identification steps for granulosa cells are as follows: discard the culture medium in the culture plate and then add PBS to soak the cells 3 times, each for 3 minutes; then fix the cell slide with 4% paraformaldehyde for 10 minutes, and after fixation, soak the cell slide with PBS 3 times for 3 minutes each; then add 0.5% Triton to the well. X-100 solution was allowed to stand at room temperature for 30 minutes for cell permeabilization. After permeabilization, the cell slides were washed 3 times with PBS for 3 minutes each time. At room temperature, the cell slides were blocked with 5% BSA for 1 hour. After blocking, the BSA solution was discarded, and 5% BSA-diluted FSHR immunofluorescent antibody (dilution ratio, 1:200) was added thereto and incubated at 4°C overnight. The antibody was discarded and the cell slides were washed 3 times with PBS for 5 minutes each time. The cells were incubated in a dark incubator with immunofluorescent secondary antibody at 37°C for 30 minutes. After incubation, the cell slides were washed 3 times with PBS for 5 minutes each time. Finally, the cell slides were removed from the culture plate and placed on a glass slide, and sealed with anti-fluorescence quenching sealing solution containing DAPI. After sealing, the fluorescence was observed under an inverted fluorescence microscope.

[0054] Synthesis of miRNA mimics and inhibitors

[0055] Log in to the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) to download the goat miR-424-3p sequence, and synthesize miR-424-3p mimics and inhibitors using a chemical synthesis method based on the miR-424-3p sequence. The mimics used in the present invention were synthesized by Ruibo Biotechnology Co., Ltd., and the inhibitor was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequence of the goat miR-424-3p mimic is: "5′-CAAAACGTGAGGCGCTGCTAT-3′ (SEQ ID NO.23)", and the sequence of the goat miR-424-3p inhibitor is "5′-AUAGCAGCGCCUCACGUUUUG-3′ (SEQ ID NO.1), and each base is modified with 2'-O-Methyl".

[0056] Cell passaging and transfection

[0057] Select cells that are at the end of the logarithmic growth phase and have good cell morphology for passaging. Observe the granular cells under a microscope and pass them when the cell density reaches 80%-90%. When passaging, first discard the old culture medium in the culture medium, then use PBS to wash. After washing, add an appropriate amount of 0.25% trypsin digestion solution preheated at 37°C to the culture dish to cover the cells. Digest in a 37°C incubator for 2 minutes, then immediately add complete culture medium to terminate digestion, blow off the cells, mix them, and transfer them to a 15mL sterile centrifuge tube. Centrifuge at 1000rpm for 5 minutes. After centrifugation, discard the supernatant, then add 1 ml of complete culture medium to resuspend the cells, and take an appropriate amount of cell suspension to inoculate into a new culture dish.

[0058] After about 24 hours of cell plating, observe the cells under a microscope and perform cell transfection when the cells grow to about 60%. TM Lipofectamine TM The procedure was performed according to the instructions of the 3000 transfection kit. In short, (1) when the cells in the 6-well plate have grown to about 60%, the culture medium was discarded, and the cells were rinsed with PBS. The PBS was discarded, and 1.75 ml of complete culture medium was added to each cell culture well, and then the well was placed in the incubator for transfection. (2) Transfection solution A was prepared. 125 ml of Opti-MEM was added to a 1.5 ml centrifuge tube. TM Medium and 5 μl of Lipofectamine TM 3000 Reagent, gently pipette to mix. (3) Prepare transfection solution B. Add 125 ml of Opti-MEM to a 1.5 ml centrifuge tube. TM Medium and 10 μL of 20 pmol / μL mimic or inhibitor, gently pipetting to mix. (4) Add transfection solution B to solution A, let it stand at room temperature for 15 minutes, and then add it dropwise to the culture wells. After transfection, continue to culture the cells in a cell culture incubator with 5% CO2, saturated humidity, and 37°C.

[0059] The results showed that overexpression of mimicshe and inhibitor significantly increased and decreased the expression of miR-424-3p in granulosa cells, respectively. Figure 1 B and Figure 1 C).

[0060] Example 3

[0061] Effects of miR-424-3p on proliferation and apoptosis of goat ovarian granulosa cells

[0062] The CCK8 cell proliferation detection kit was used to detect the effects of miR-424-3p mimics and inhibitors on the proliferation of goat ovarian granulosa cells. The specific method of the CCK8 cell proliferation detection kit was as follows: granulosa cells in good growth condition were inoculated into a 96-well plate, wherein only complete medium was added to the outer wells of the 96-well plate without adding cells to prevent the volatilization of the central liquid from affecting the experimental results. After the cells were plated, they were placed in a cell culture incubator for static culture. After the cells grew to about 50%, cell transfection was performed. Then, 10 μL of CCK-8 reagent was added at 12 hours, 24 hours, 48 ​​hours, and 72 hours respectively. After 1 hour, the absorbance value at a wavelength of 450 nm was measured using a microplate reader. The remaining steps were the same as in Example 2.

[0063] The results showed that compared with the control group, overexpression of miR-424-3p could significantly inhibit the proliferation of granulosa cells, while inhibition of miR-424-3p expression promoted the proliferation of granulosa cells ( Figure 2 A).

[0064] Cell EdU proliferation assay

[0065] The cells were seeded in a 96-well plate, and 48 hours after transfection, EDU cell proliferation detection was performed according to the instructions of the Cell-Light EdU Apollo 567 In Vitro Kit of Ruibo Biotechnology. In short, the configured EDU solution was added to the cell culture medium and incubated for 2 hours, the culture medium was removed, and then the cells were fixed with a cell fixative for 30 minutes, permeabilized with a permeabilization solution for 10 minutes, and washed with PBS. Apollo staining was then performed, and after washing with PBS, Hoechst dye was used to stain the cell nucleus DNA. The cell staining was observed under an inverted fluorescence microscope. The remaining steps were the same as in Example 2.

[0066] The results of the EDU experiment also showed that overexpression of miR-424-3p could significantly inhibit the proliferation of granulosa cells, while inhibition of miR-424-3p expression promoted the proliferation of goat follicle cells ( Figure 2 B).

[0067] Flow cytometry was used to assess apoptosis in goat follicular granulosa cells. 48 hours after cell transfection, apoptosis was assessed using a Beckman Coulter CYTOFLEX flow cytometer. Cells were treated according to the instructions for the Annexin V-FITC / PI Apoptosis Detection Kit (Elabscience). The procedure was as follows: 48 hours after transfection, the culture medium was discarded, the cells were washed once with PBS, and then digested with 0.25% EDTA-free trypsin. The cells were harvested by centrifugation at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were then resuspended in ice-cold PBS at 4°C and centrifuged twice at 1000 rpm for 5 minutes at 4°C. 100 μL of 1× Binding Buffer was added to each sample and gently mixed using a pipette. 5 μL of FITC and PI reagents were then added each, protected from light, and incubated at room temperature for 15 minutes. 400 μL of 1× Binding Buffer was added and mixed before loading. The on-machine test was completed within one hour, the flow cytometer speed was selected to be slow, and all data were analyzed using FlowJo V10 software.

[0068] The results showed that compared with the control group, the apoptosis rate of goat follicle granulosa cells in the miR-424-3p mimics transfection group increased significantly, while the apoptosis rate of cells in the inhibitor transfection group decreased significantly ( Figure 3 A and Figure 3 B) These results indicate that overexpression of miR-424-3p can inhibit the proliferation and promote apoptosis of goat granulosa cells.

[0069] Example 4

[0070] Effects of miR-424-3p on cell cycle progression of goat ovarian granulosa cells

[0071] Flow cytometry was used to detect the effect of overexpression of miR-424-3p on the cell cycle progression of granulosa cells. The cell cycle detection steps were as follows: 48 hours after cell transfection, cell cycle detection was performed using a Beckman Coulter CYTOFLEX flow cytometer. Cell treatment was performed according to the instructions of the Cell Cycle Assay Kit (Red Fluorescence) from Elabscience. The operation steps were as follows: (1) Absolute ethanol was placed in a -20°C refrigerator overnight. (2) Digestion and collection of cells, centrifugation at 300×g for 5 minutes, and the supernatant was discarded. (3) Add 1 mL of PBS for washing, centrifugation at 300×g for 5 minutes, and the supernatant was discarded. (4) Add 0.3 mL of PBS to resuspend the cells. (4) Slowly add 1.2 mL of -20°C absolute ethanol to the centrifuge tube on a vortex shaker, mix thoroughly, and place in a -20°C refrigerator overnight. (5) Centrifugation at 300×g for 5 minutes, discard the supernatant, add 1 mL of PBS to resuspend the cells, and place at room temperature for 15 minutes. (6) Centrifuge at 300 × g for 5 min, discard the supernatant, add 100 μL of RNase A Reagent and fully suspend the cells, and incubate at 37°C in a water bath for 30 min. (6) Add 400 μL of PI Reagent (50 μg / mL) and mix thoroughly. Incubate at 2-8°C in the dark for 30 min. (7) After the incubation, immediately test on the instrument, using a PE channel at a slow flow rate. The remaining steps are the same as in Example 2.

[0072] The results showed that compared with the control group, the ratio of G1 phase cells in the miR-424-3p mimics group was significantly increased, while the ratio of S phase and G2 phase cells was significantly decreased. This indicates that overexpression of miR-424-3p can hinder the progression of the cell cycle of granulosa cells ( Figure 4 A). After transfection with miR-424-3p inhibitor, the G1 phase ratio of the inhibitor group decreased significantly, the S phase ratio increased significantly, but the effect on the G2 phase ratio was not significant. This indicates that inhibiting the function of miR-424-3p can promote the transition of follicular granulosa cells from G1 phase to S phase ( Figure 4 B).

[0073] Example 5

[0074] Effect of overexpression of miR-424-3p on the expression of proliferation marker genes in goat granulosa cells

[0075] MiR-424-3p was transfected into granulosa cells (the specific method is the same as in Example 2), and the mRNA and protein expression levels of cell proliferation marker genes PCNA, CCND1, CDK6 and MKI67 were detected using qRT-PCR and Western Blot techniques. The qRT-PCR method is the same as in Example 1, and the primer sequences are shown in Table 1. The Western Blot method is as follows: Cell protein extraction: Wash the cells three times with PBS, aspirate the PBS, add 200 μl of RIPA lysis buffer to each well of the 6-well plate, lyse on ice for 5-10 minutes, scrape the cells with a cell scraper and transfer them to a 1.5 ml centrifuge tube, centrifuge in a centrifuge at 4°C, 12000 rpm for 10 minutes, gently remove the centrifuge tube and place it on an ice box, and use a pipette to aspirate an appropriate amount of supernatant. Protein concentration determination: Use the Novezan BCA protein concentration determination kit to determine the protein concentration. In short, (1) Preparation of BCA working solution. According to the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCAreagent A to 1 volume of BCAreagent B (50:1) and mix thoroughly; (2) Draw the standard curve. Prepare the protein concentration standard solution according to the instructions; (3) Prepare the sample. Take an appropriate amount of the above protein supernatant and dilute it to 20 μL, and add 200 μL of BCA working solution; (4) Vortex and mix, incubate at 37℃ for 30 minutes, and then measure the absorbance at A562 nm using an enzyme reader, with the absorbance value without BSA as the blank control. (5) Draw the standard curve with protein content (μg) as the horizontal axis and absorbance as the vertical axis, and calculate the protein content of the sample. Protein denaturation: Take an appropriate amount of 9.1 lysate supernatant, add 5X protein loading buffer, boil at 100℃ for 10 minutes. SDS-PAGE electrophoresis: Take 20 μg of denatured total protein from each group for electrophoresis, and the electrophoresis conditions are 110V for 90 minutes. Transfer: Activate the PVDF membrane by soaking it in methanol for at least 10 minutes beforehand. After electrophoresis, assemble the membrane into a transfer "sandwich" configuration (filter paper-gel block-PVDF membrane-filter paper), taking care to remove all bubbles. Transfer the membrane at 90V for 90 minutes. For immunoblotting: After transfer, rinse the PVDF membrane three times with 1X TBST for 5 minutes each. Block the membrane with 5% BSA at room temperature for 2 hours, then rinse the membrane three times with 1X TBST for 5 minutes each. Incubate the membrane with the corresponding primary antibody overnight at 4°C, then rinse the membrane five times with 1X TBST for 5 minutes each. Incubate the membrane with HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies at room temperature for 1 hour, then rinse the membrane five times with 1X TBST for 5 minutes each. Finally, treat the membrane with ECL solution, visualize protein bands using a TANON imaging system, and analyze protein bands using ImageJ software.

[0076] The results showed that overexpression of miR-424-3p could significantly reduce the mRNA levels of PCNA and MKI67 genes in granulosa cells compared with the control group, but did not affect the mRNA levels of CDK6 and CCND1 ( Figure 5 A). Protein results showed that overexpression of miR-424-3p could significantly reduce the expression of PCNA, CDK6 and CCND1 proteins ( Figure 5 B. Figure 5 C and Figure 5 D).

[0077] Example 6

[0078] Effect of overexpression of miR-424-3p on the expression of apoptosis marker genes in goat follicular granulosa cells

[0079] miR-424-3p was transfected into granulosa cells, and the mRNA and protein expression levels of apoptosis marker genes BAX, BCL2, and CASP3 were detected using qRT-PCR and Western Blot techniques. The specific methods were the same as in Example 5, and the primer sequences are shown in Table 1.

[0080] The results showed that overexpression of miR-424-3p could significantly inhibit the expression of BCL2 mRNA and protein, and significantly increase the expression of BAX protein, but had no significant effect on the expression of BAX and CASP3 gene mRNA. Figure 6 A- Figure 6 C).

[0081] Example 7

[0082] Screening of miR-424-3p target genes

[0083] Sequence alignment revealed that the goat miR-424-3p sequence was highly similar to the miR-424 sequence of mammals such as humans, pigs, and horses. Using RNAhybrid and miRWalk online software to predict miR-424-3p target genes, it was found that the seed sequence of miR-424-3p could bind to the 3′UTR region of PIP5K1C ( Figure 7 A). Then, miR-424-3p mimics were transfected into granulosa cells (the specific method was the same as in Example 2), and the changes in the mRNA level of PIP5K1C were detected by qRT-PCR (the specific method was the same as in Example 1, and the sequence was shown in Table 1). The results showed that compared with the control group, the expression of the PIP5K1C gene in the miR-424-3p overexpression group was significantly downregulated ( Figure 7 B).

[0084] Then, a dual-luciferase assay was used to verify that miR-424-3p could bind to the predicted binding site in the 3′UTR of PIP5K1C and inhibit protein translation. The specific method was as follows: Dual-luciferase reporter gene assay: Log in to the NCBI website to download the predicted 3′UTR sequence of PIP5K1C (Gene ID: 102174559), and then use RNAhybrid online software (bibiserv.cebitec.uni-bielefeld.de / rnahybrid?id=rnahybrid_view_submission) to predict the binding site of miR-424-3p to PIP5K1C. The binding site of the 3′ UTR was identified. Primers were then designed using the Novizan online primer design tool for fragments approximately 200 bp upstream and downstream of the predicted binding site. High-fidelity enzymes were used to amplify the fragments approximately 200 bp upstream and downstream of the predicted binding site of the target gene using goat DNA as a template. The amplified product was purified and ligated into the pmirGLO vector using seamless cloning. After transformation, screening, and sequencing, the plasmid was extracted using an endotoxin-free plasmid miniprep kit. The extracted plasmid was named PIP5K1C-WT. A PIP5K1C-MUT mutant binding site was synthesized using whole gene synthesis. The mutant vector was constructed and extracted according to the above method, and the extracted plasmid was named PIP5K1C-MUT. The primer sequences are: upstream primer 5′-TGTTTAAACGAGCTCGCTAGCCCCCGCCCTGGCTCCCCG-3′ (SEQ ID NO. 24); downstream primer 5′-CAGGTCGACTCTAGACTCGAGGCTATCCCAGCCACCACTGTG-3′ (SEQ ID NO. 25). Wild-type (WT) and mutant (MUT) dual-luciferase reporter vectors were co-transfected with miR-424-3p mimics and mimics NC into 293T cells. Forty-eight hours later, dual-luciferase activity was detected according to the instructions of the Dual Luciferase Reporter Assay Kit from Novizan Biotechnology.

[0085] The results showed that ( Figure 7 D), miR-424-3p mimics can bind to the PIP5K1C 3′UTR region and inhibit the activity of the firefly luciferase gene. Finally, using Western Blot (the specific method is the same as Example 5), it was detected that miR-424-3p can inhibit the expression of PIP5K1C protein in granulosa cells. It was found that compared with the control group, overexpression of miR-424-3p can significantly inhibit the expression of PIP5K1C protein ( Figure 7C) These results indicate that miR-424-3p can target the 3′UTR region of PIP5K1C and inhibit the expression of PIP5K1C.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a miR-424-3p inhibitor in the preparation of a product for promoting proliferation or inhibiting apoptosis of goat ovarian granulosa cells, characterized in that: The nucleotide sequence of the miR-424-3p inhibitor is shown in SEQ ID NO. 1, and the goat is a Leizhou goat.

2. The use according to claim 1, characterized in that The types of products described include pharmaceuticals.

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