Method for regulating and controlling SLIT2 transcription and application of method in ovarian granular cells

Abstract

The invention discloses a method for regulating and controlling SLIT2 transcription and application of the method in ovarian granular cells, and belongs to the technical field of cell engineering and gene engineering. According to the invention, SLIT2e, SLIT2 gene promoter region histone acetylation and an SLIT2 gene are taken as research objects, and the SLIT2 gene promoter histone acetylation is verified through TSA or HDAC-siRNA to promote the expression of the SLIT2 gene promoter histone acetylation. The invention explores the mechanism that the enhancer regulates the gene promoter region histone acetylation and chromatin opening degree so as to regulate the transcription expression of the SLIT2 gene. The invention has a certain application value for researching the molecular mechanism of influence of histone acetylation of the SLIT2 gene promoter region on ovarian follicle development.

Description

Technical Field

[0001] This invention belongs to the fields of cell engineering and genetic engineering technology, specifically relating to a method for regulating SLIT2 transcription and its application in ovarian granulosa cells. Background Technology

[0002] In female mammals, follicle growth and development determine their reproductive potential. The follicle is the basic structural and functional unit of the ovary. Follicle development progresses through primordial, primary, and secondary follicle stages, before acquiring the antral cavity (tertiary and mature follicles). After the antral phase, follicle development has two fates: most follicles undergo atresia and degeneration, while a minority reach the pre-ovulatory stage under the stimulation of circulating Gn after puberty. During development, granulosa cells initiate their own programmed cell death to regulate follicle atresia and degeneration. When 10% of the granulosa cells in a follicle undergo apoptosis, the follicle is considered to have entered an atretic state. Extensive follicular atresia leads to a significant reduction in reproductive performance in mammals. In conclusion, the growth and development of follicles in the sow's ovary is a key factor affecting its reproductive performance and directly impacts the economic benefits of the farm.

[0003] Slit guide ligand 2 (SLIT2) is a key member of the SLIT / ROBO pathway, which includes three secreted SLIT glycoproteins (SLIT1, SLIT2, and SLIT3) and four transmembrane ROBO receptors (ROBO1, ROBO2, ROBO3, and ROBO4). Studies have shown that the SLIT / ROBO pathway is involved in many processes, including cell proliferation, apoptosis, and follicular development. Expression of the SLIT / ROBO pathway has been confirmed in adult human ovaries, and overexpression of SLIT2 significantly reduces the number of proliferating oocytes in developing ovaries. The expression of SLIT2, SLIT3, and ROBO2 increases during the late luteal phase and is negatively regulated by hCG and cortisol. Inhibiting SLIT-ROBO activity in luteal cells enhances cell migration and reduces apoptosis. Therefore, studying the regulation of SLIT2 may have scientific significance for improving reproductive performance.

[0004] Histone acetylation is an epigenetic regulation process that plays a crucial role in regulating the expression of various genes and maintaining normal cellular function. Epigenetics includes DNA methylation and demethylation, histone modification, and lncRNA. Histone acetylation can remodel chromatin structure, thereby regulating gene expression; this process is mainly accomplished by histone acetyltransferases. Histone deacetylases (HDACs), on the other hand, remove acetyl groups from histones, resulting in a more compact chromatin structure and inhibiting gene transcription.

[0005] Enhancers are cis-regulatory elements in the genome that transcribe eRNAs that influence histone acetylation and chromatin openness, thereby regulating gene expression. Enhancers are typically located in loosely packed chromatin regions far from genes and possess specific chromatin markers, thus linking them to histone modifications. They can act as distal regulatory elements, regulating gene transcription through p300 and related acetyltransferases. Enhancers also exhibit specificity, specifically promoting or inhibiting the expression of target genes while having no effect on other adjacent genes. Summary of the Invention

[0006] To address these issues, the primary objective of this invention is to provide a method for regulating SLIT2 gene transcription.

[0007] Another object of the present invention is to provide an application of the above-described method for regulating SLIT2 gene transcription.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0009] A method for regulating SLIT2 transcription is proposed, which regulates the transcriptional activity of the SLIT2 gene in porcine ovarian granulosa cells by adjusting the histone acetylation level in the promoter region of the SLIT2 gene.

[0010] Furthermore, the histone acetylation in the SLIT2 gene promoter region is the histone H3K4 acetylation in the SLIT2 gene promoter region; the H3K4 acetylated region is the -1919 to -1782 bp region of the SLIT2 gene promoter.

[0011] Furthermore, in the method described, the level of histone acetylation in the promoter region of the SLIT2 gene is increased, and the transcriptional activity of the SLIT2 gene is increased.

[0012] Furthermore, the increased histone acetylation level in the SLIT2 gene promoter region is achieved by inhibiting the expression of histone deacetylation enzyme HDAC using histone deacetylation inhibitors or RNA interference technology. Specifically, the siRNA used in the RNA interference technology to inhibit HDAC is as follows:

[0013] HDAC1-siRNA: 5′-GAATACTTCGGACCAGATT-3′.

[0014] A core promoter functional regulatory region of the SLIT2 gene in porcine ovarian granulosa cells is the region from -1919bp to -1782bp of the SLIT2 gene promoter.

[0015] A functional region of the core enhancer of the SLIT2 gene in porcine ovarian granulosa cells, specifically the 691bp–1085bp region of the SLIT2 gene enhancer.

[0016] The nucleotide sequence of the SLIT2 gene enhancer is shown in SEQ ID NO.1.

[0017] A nucleic acid fragment that regulates the expression of the SLIT2 gene in porcine ovarian granulosa cells is a siRNA of the SLIT2 enhancer region, and the target sequence corresponding to the siRNA is: 5′-GCACAGTAGCTTTCTATCA-3′.

[0018] The application of the aforementioned core promoter functional regulatory regions, core enhancer functional regions, or nucleic acid fragments in regulating SLIT2 gene expression in porcine ovarian granulosa cells.

[0019] The application of the aforementioned core promoter functional regulatory region, core enhancer functional region, or nucleic acid fragment is at least one of the following applications:

[0020] I. Application in promoting the proliferation of porcine ovarian granulosa cells / in the preparation of drugs that promote the proliferation of porcine ovarian granulosa cells;

[0021] II. Application in promoting porcine follicle development / in the preparation of drugs that promote porcine follicle development.

[0022] Furthermore, the core promoter functional regulatory region, the core enhancer functional region, and the nucleic acid fragment described herein play a role in regulating the expression of the SLIT2 gene in porcine ovarian granulosa cells.

[0023] This invention, through database analysis, discovered an active enhancer signal (chr8, 16915620-16917621 bp) near the SLIT2 gene. This enhancer signal increased H3K4ac levels by interfering with histone deacetylases, and decreased SLIT2 mRNA and protein levels by interfering with the SLIT2 enhancer region. Therefore, we propose that SLIT2e regulates the transcriptional level of the SLIT2 gene through histone acetylation.

[0024] The verification results of this invention are as follows:

[0025] 1. The presence of an active enhancer signal (chr8, 16915620-16917621bp) near the SLIT2 gene was predicted using the bioinformatics website Pig-eRNAdb.

[0026] 2. We constructed the pGL3-enhancer vector and demonstrated the transcriptional activity of SLIT2e using a dual-luciferase activity assay. Subsequently, we isolated granulosa cells from small (≤3 mm), medium (3–5 mm), and large (≥5 mm) follicles and used qRT-PCR to detect the expression levels of SLIT2 gene and SLIT2e in follicles at each stage. qRT-PCR results showed that as the follicles developed and enlarged, the mRNA expression levels of both SLIT2 gene and SLIT2e gradually decreased, but the downregulation of SLIT2e was not significant. In conclusion, SLIT2e may promote follicle development by downregulating SLIT2 expression.

[0027] 3. We treated ovarian granulosa cells with TSA or HDAC-siRNA, respectively, and used qRT-PCR, Western Blot, and ChIP to detect the expression levels of the SLIT2 gene and H3K4ac, respectively. qRT-PCR and Western Blot results showed that TSA significantly promoted the mRNA and protein expression levels of the SLIT2 gene, as well as the protein expression level of H3K4ac. Meanwhile, ChIP results showed that HDAC-siRNA significantly increased the enrichment level of H3K4ac in the SLIT2 gene promoter region. Chromatin accessibility experiments showed that si-HDAC significantly increased the chromatin accessibility of the SLIT2 gene promoter region. In conclusion, si-HDAC may promote SLIT2 gene expression by increasing the H3K4ac level and chromatin accessibility in the SLIT2 gene promoter region.

[0028] 4. By transfecting the constructed SLIT2 overexpression vector and the SLIT2 small interfering fragment into GCs, qRT-PCR results showed that pcDNA3.1-SLIT2 significantly increased SLIT2 mRNA levels in a dose-dependent manner; si-SLIT2-2 and si-SLIT2-3 both significantly decreased SLIT2 mRNA levels, with si-SLIT2-3 showing the best interference effect. qRT-PCR and Western blot analysis revealed that SLIT2 overexpression significantly increased the mRNA levels of CASP9 and CASP3 in the apoptosis signaling pathway, while SLIT2 interference showed the opposite effect. In the cell cycle signaling pathway, SLIT2 overexpression significantly decreased PCNA mRNA levels, while SLIT2 interference significantly increased the mRNA levels of PCNA and CDK1. Subsequently, flow cytometry analysis showed that SLIT2 overexpression significantly promoted GC apoptosis; SLIT2 interference significantly reduced the percentage of apoptotic cells. To further investigate the effects of SLIT2 on granulocyte proliferation and apoptosis, EdU assays revealed that overexpression of SLIT2 significantly inhibited granulocyte proliferation, while interference with SLIT2 significantly promoted granulocyte proliferation. In summary, SLIT2 can promote granulocyte apoptosis and inhibit granulocyte proliferation by increasing the expression levels of CASP9 and CASP3 and inhibiting the expression levels of PCNA and CDK1.

[0029] The present invention has the following advantages and effects compared with the prior art:

[0030] (1) The enrichment of H3K4ac in the SLIT2 gene promoter region and chromatin accessibility, as well as the direct or indirect involvement of the SLIT2 gene in follicular atresia and follicular development, are studied in this invention. This study focuses on SLIT2e, histone acetylation in the SLIT2 gene promoter region, and the SLIT2 gene (ID: 397679). Molecular and cell biological techniques were employed to investigate the transcriptional regulation of the SLIT2 gene by SLIT2e regulating SLIT2 promoter region acetylation. This research has significant application value in studying the molecular mechanisms by which histone acetylation in the SLIT2 gene promoter region participates in ovarian follicular atresia and the onset of puberty.

[0031] (2) The technical solution of this invention is well-designed and the results are reliable. To verify the effect of histone acetylation in the promoter region of the SLIT2 gene on the transcriptional regulation of the SLIT2 gene, this invention verifies from multiple levels and perspectives. First, it verifies at the real level by detecting the effect of histone deacetylase on H3K4ac in the SLIT2 promoter region. Then, it detects the effect of SLIT2e on histone acetylation and gene expression in SLIT2. Attached Figure Description

[0032] Figure 1This is a diagram illustrating the effect of the SLIT2 gene enhancer.

[0033] Figure 2 This is a schematic diagram of the enhancer segmentation of the SLIT2 gene.

[0034] Figure 3 This is a transcriptional level diagram detected after segmentation of the SLIT2 gene enhancer; where A is the quantification of the enhancer fragment, and B is the amplification of the segment with the strongest transcriptional activity.

[0035] Figure 4 This is a graph showing the effect of SLIT2 gene expression level and H3K4ac enrichment level in the SLIT2 gene promoter region after transfection with SLIT2e-siRNA; where A is the effect of the interfering fragment SLIT2e-siRNA on the H3K4ac enrichment level in the SLIT2 gene promoter region, and B is the effect of SLIT2e-siRNA on the SLIT2 gene protein and mRNA levels.

[0036] Figure 5 This is a schematic diagram of the SLIT2 gene promoter segmentation.

[0037] Figure 6 This is a graph showing the enrichment level of H3K4ac and chromatin accessibility of different segments of the SLIT2 promoter region after transfection with HDAC-siRNA. Among them, A is the effect of interfering fragment HDAC-siRNA on the H3K4ac level of the SLIT2 promoter region, with HDAC1 showing the most significant effect; B is the effect of interfering fragment HDAC1-siRNA on the H3K4ac level of different regions of the promoter after segmentation; C is the effect of interfering fragment HDAC1-siRNA on the chromatin accessibility of different regions of the promoter after segmentation.

[0038] Figure 7 This is a graph showing the changes in the expression levels of the SLIT2 gene and SLIT2e in different follicles; where A represents the change in the expression level of SLIT2e in different follicles, and B represents the change in the expression level of the SLIT2 gene in different follicles.

[0039] Figure 8 Figure 1 shows the results of a study on the effects of SLIT2 overexpression or interference on cell proliferation and apoptosis. Specifically, a) shows the effect of different quality pcDNA3.1-SLIT2 on SLIT2 mRNA levels; b) shows the effect of different interference fragments on SLIT2 mRNA levels; c) shows the effect of SLIT2 overexpression or interference on the mRNA expression levels of genes related to GC proliferation and apoptosis; d) shows the effect of SLIT2 overexpression or interference on the protein expression levels of genes related to GC apoptosis; e) shows the apoptosis rate of GCs after SLIT2 overexpression and interference detected by flow cytometry; and f) shows the GC proliferation rate after SLIT2 overexpression and interference detected by EdU. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions.

[0041] In this invention, statistical methods are applied to analyze the results of three independent experiments in each embodiment, and the "mean ± standard deviation" is calculated respectively. One-way ANOVA is used to analyze the significance of differences (in the figure, "*" indicates P<0.05, and "**" indicates P<0.01).

[0042] The experimental methods involved in the embodiments of this invention are as follows:

[0043] 1. Culture of porcine ovarian granulosa cells

[0044] (1) Healthy ovaries of commercial sows were collected from Kongwangji Slaughterhouse in Guangzhou, washed twice, placed in PBS containing 1% double antibiotics and autoclaved, and quickly brought back to the laboratory in an ice box.

[0045] (2) Wash the ovaries three times with PBS containing 1% double antibiotics and autoclaved, and transfer the ovaries into the clean bench of the cell culture room (wipe with alcohol swabs and irradiate with UV for 30 min in advance).

[0046] (3) Prepare complete culture medium: 89% DMEM, 10% serum and 1% penicillin antibiotics, mix by inverting the container;

[0047] (4) First, add 5 mL of complete culture medium to a 15 mL centrifuge tube, and carefully draw 2 mL of follicular fluid into the centrifuge tube using a 1 mL disposable sterile syringe; centrifuge at 1000 rpm for 5 min;

[0048] (5) Discard the supernatant, add 5 mL of PBS buffer solution to wash twice; centrifuge at 1000 rpm for 3 min each time, and finally resuspend the cell pellet with 3 mL of complete culture medium;

[0049] (6) Add 12 mL of complete culture medium to a 75 mL culture flask, then add the above resuspension, and gently shake to mix.

[0050] (7) Incubate at 37℃ in a 5% CO2 incubator and observe the growth of granule cells after 48 hours.

[0051] (8) Wash twice with preheated PBS containing 1% penicillin and streptomycin, and then incubate for 24 hours before proceeding with subsequent experiments. The penicillin and streptomycin are penicillin and streptomycin, respectively.

[0052] 2. Plating and transfection of porcine ovarian granulosa cells

[0053] (1) When the granular cells have grown to about 90%, discard the culture medium and wash twice with preheated PBS containing 1% double antibiotics.

[0054] (2) Add 5 mL of trypsin, place in an incubator to digest for about 5 min, observe under a microscope until most cells float up, and immediately add an equal amount of complete culture medium to stop digestion.

[0055] (3) Transfer to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min; discard the supernatant and wash twice with PBS containing 1% penicillin and antibiotics, centrifuging at 1000 rpm for 5 min during the process;

[0056] (4) Gently resuspend the cell pellet in complete culture medium, evenly distribute it into each well, replenish the volume with complete culture medium, gently shake well, and incubate in an incubator.

[0057] (5) After about 24 hours, observe the cell status and perform transfection when the cell confluence reaches about 80%.

[0058] (6) The transfection method is as per Invitrogen's specifications. Follow the instructions in the 3000 kit manual, with 3 replicates per group;

[0059] (7) After transfection, the well plate was placed in a 37°C, 5% CO2 incubator and cultured for 24-72 hours after transfection. The cell status was observed. If the cells grew well, subsequent experiments could be carried out.

[0060] 3. qRT-PCR:

[0061] This invention segments enhancers and detects the segment with the highest transcriptional activity. The qRT-PCR detection of genes and enhancer fragments was performed using the Maxima SYBR Green qPCR Master Mix (2X) kit (Shanghai Yisheng Biotechnology Co., Ltd.). The Ct value method was used to detect the gene content in the samples, and the specific calculation formula is as follows:

[0062] Relative gene expression level = 2 - {<(Ct value of target gene in experimental group) - (Ct value of internal reference gene in experimental group)> - <(Ct value of target gene in control group) - (Ct value of internal reference gene in control group)>}

[0063] GAPDH was used as an internal control for gene detection.

[0064] Total RNA extraction from cells: Total RNA was extracted from cells using an RNA extraction kit (purchased from Magen). Specifically, approximately 1 × 10⁻⁶ ppm was collected. 7One cell sample was placed in a 1.5 mL deenzyme-free centrifuge tube, and 100 μL of PBS buffer was added to resuspend the cells by pipetting. 500 μL of cell lysis buffer (CRL) was added, vortexed for 13 s, and incubated for 1 min. The liquid was transferred to a column, centrifuged, and the liquid was removed. 600 μL of buffer CW was added, centrifuged for 1 min, and the liquid was removed. The column was then centrifuged again for 2 min. The column was placed in a 1.5 mL deenzyme-free centrifuge tube, 60 μL of RNase-free water was added, and the column was incubated for 2 min. The RNA was then washed away by centrifugation and stored at -80°C.

[0065] Using PrimeScript from TaKaRa TM RT Master Mix (Perfect Real Time) cDNA Reverse Transcription Kit for Reverse Transcription of Total RNA.

[0066] 4. Western Blot:

[0067] (1) Extraction and quantification of total protein from ovarian granulosa cells: Discard the culture medium, wash the cells twice with pre-cooled PBS, and add an appropriate amount of lysis buffer containing 0.2% protease inhibitor (5-10×10⁻⁶). 6 Add 500 μL to each cell, mix well, shake at 4°C for 20 min, centrifuge at 13000 rpm at 4°C for 10 min, and take the supernatant into a pre-cooled 1.5 mL sterile centrifuge tube;

[0068] (2) Determining protein sample concentration using the BCA method: ① Prepare BCA working solution (reagent A: reagent B = 50:1) according to the number of samples; ② Take 10 μL of protein standard in a 1.5 mL sterile centrifuge tube and dilute with PBS to a final concentration of 0.5 mg / mL; ③ Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the diluted protein standard to a 96-well plate, and then add 20, 19, 18, 16, 12, 8, 4, and 0 μL of PBS to the corresponding wells; ④ Add 2 μL of protein sample and 18 μL of PBS to the sample wells; add 0.2 mL of BCA working solution to each well and incubate at 37 °C for 30 min; ⑤ Measure the absorbance using a microplate reader (wavelength 570 nm) and calculate the protein concentration.

[0069] (3) SDS-PAGE electrophoresis: Take 20 μg of total protein and 5× loading buffer for each group, mix them at a ratio of 5:1, and boil for 5 min. Perform SDS-PAGE electrophoresis until bromophenol blue just appears at the bottom of the gel;

[0070] (4) Transfer: First, activate the PVDF membrane with methanol for 3 min, then equilibrate it with PVDF membrane equilibration solution for 2 min. Then, stack the sponge, PVDF membrane, gel, and sponge on the positive electrode of the transfer clamp in sequence, cover the negative electrode of the transfer clamp, and insert it into the channel of the rapid transfer instrument for transfer (using the instrument's default parameters).

[0071] (5) Immunoblotting: Remove the PVDF membrane, wash it 3 times with TBST for 10 min each time, and block it with 6% skim milk powder at room temperature for 2.5 h; wash off the excess skim milk powder with TBST, dilute the antibody with TBST (SLIT2: 20217-1-AP, proteintech, 1:1000; GAPDH: 10494-1-AP, proteintech, 1:30000), and incubate overnight at 4℃; Secondary antibody incubation: Remove the membrane, wash it 3 times with TBST for 10 min each time, dilute the secondary antibody with TBST according to the ratio (mouse source: 1:3000, rabbit source: 1:5000), and incubate at room temperature for about 2 h; After the secondary antibody incubation, remove the membrane, wash it 3 times with TBST for 10 min each time, then mix A and B chemiluminescence solutions at a 1:1 ratio, immerse both sides of the membrane in the mixture for 30 s, develop it with a chemiluminescence analyzer, take pictures and save them, and finally analyze the protein bands using Image Plus software.

[0072] 5. Chromatin accessibility testing

[0073] We divided the SLIT2 gene promoter into P1 (-539bp to -436bp), P2 (-805bp to -512bp), P3 (-1629bp to -1382bp), and P4 (-1919bp to -1782bp). The chromatin status of different regions of the SLIT2 promoter was then detected using the EpiQuik Chromatin Accessibility Assay Kit (Epigentek, USA). Chromatin extracted from granulosa cells was digested at 37°C with a mixture of exogenous nucleases (Nse) for 4 minutes, followed by the addition of a reaction stop solution. The enrichment fold (FE) of the gene was calculated by qPCR using the following formula:

[0074] FE = 2^(Nse CT - no Nse CT), where: FE represents the enrichment factor, NseCT (No-Nuclease Mix) represents the number of amplification cycles for the sample treated with Nse, and no Nse CT represents the number of amplification cycles for the control sample.

[0075] 6. ChIP-qPCR

[0076] We segmented the enhancer segments predicted on Pig-eRNAdb and used Pierce... TMThe ChIP Kit (Thermo, Rockford, USA) was used to detect the level of H3K4ac (acetylation of lysine at position 4 of histone H3) in the SLIT2 promoter region.

[0077] Chromatin fragments were incubated with H3K4ac antibody (39382, Active Motif, USA) and IgG antibody (30000-0-AP, Proteintech), and the purified DNA was used for qPCR analysis.

[0078] Example 1:

[0079] (1) Database analysis revealed an active enhancer signal (chr8, 16915620-16917621 bp) near the SLIT2 gene (ID: 397679), the sequence of which is shown below and named SLIT2e. The nucleotide sequence of the enhancer signal is as follows (SEQ ID NO.1):

[0080]

[0081] (2) Vector Construction: Based on the enhancer sequence of SLIT2, the enzyme restriction sites in the region were detected using Primer Premier 5.0 software. Simultaneously, referring to the pGL3-promoter vector map, Mlul and Nhel were selected as restriction sites. Primers were used to amplify the desired target fragment, and the fragment was recovered from the gel. The pGL3-promoter vector was double-digested and then subjected to agarose gel electrophoresis. The target band was excised and recovered from the gel. The target fragment was ligated to the linearized vector for plasmid recombination. The plasmid was then incubated on ice for 30 min, followed by heat shock at 42°C for 2 min, and immediately incubated on ice. The recombinant plasmid was plated overnight, and single colonies were picked and tested the next day. The primers used were:

[0082] SLIT2 enhancer F: 5′-TCTCACAGCAATTCCCAGCTTT-3′;

[0083] SLIT2 enhancer R: 5′-CTTCAGATGCTTGCTGGATCTTT-3′;

[0084] (3) Dual Luciferase Activity: The procedure for this experiment was performed according to the instructions of the Dual Luciferase Reporter Gene Assay Kit from Shanghai Yisheng Company. The specific steps are as follows: The constructed recombinant plasmid and Tk plasmid were co-transfected into a 96-well plate. After culturing for 24 hours, the culture medium was discarded, and the cells were washed twice with PBS solution containing 1% penicillin and antibiotics. 100 μl of cell lysis buffer was added to each well to ensure that the lysis buffer fully covered the cells. The cells were incubated on ice for 5 min. The lysed cells were added to a new 1.5 ml sterile EP tube and centrifuged at 12,000 rpm for 1 min at 4 degrees Celsius. The supernatant was then transferred to a new EP tube. 20 μl of cell lysis buffer was added to the microplate, with 5 replicates per group. Solution A (firefly luciferase reaction working solution) and solution B (Reniform luciferase reaction working solution) were prepared. 100 μl of solution A was added, and the mixture was allowed to stand for 5 min. After mixing, the luminescence value of firefly luciferase was detected. 100 μl of solution B was added. Solution B, let stand for 5 minutes, and then measure the luminescence value of Renida luciferase; the test must be completed within 30 minutes; the data should be analyzed according to the formula: relative activity of firefly luciferase = luminescence value of firefly luciferase / luminescence value of Renida luciferase.

[0085] (4) We will enhance the sub-segments E1 (135bp-248bp), E2 (691-1085bp), E3 (698-885bp), E4 (1192-1321bp) and E5 (1347-1472bp) ( Figure 2 ); qRT-PCR was used to detect transcriptional activity in different regions. The qRT-PCR primers used were:

[0086] qRT-PCR-SLIT2 F: 5′-CTGCGAATGCGAGGAAGGATG-3′;

[0087] qRT-PCR-SLIT2 R: 5′-GCTGTAGGAGAAGGCGTTGATG-3′;

[0088] qRT-PCR-E1 F: 5′-TCTCGGGGCTCTGAAAATGG-3′;

[0089] qRT-PCR-E1 R: 5′-TCTCGGGGCTCTGAAAATGG-3′;

[0090] qRT-PCR-E2 F: 5′-ATGAAATGTCTGCCTCTCGG-3′;

[0091] qRT-PCR-E2 R: 5′-TGAGGCTCAGATTTATCACA-3′;

[0092] qRT-PCR-E3 F: 5′-GTGCCTGACCTTTGACATCG-3′;

[0093] qRT-PCR-E3 R: 5′-AACCCTGTGGGTGAGAGACA-3′;

[0094] qRT-PCR-E4 F: 5′-CATGAAACGTGACAGTGGGC-3′;

[0095] qRT-PCR-E4 R: 5′-TGGTACCCACGGGTCTTACT-3′;

[0096] qRT-PCR-E5 F: 5′-GATGGTTCTGCTCTCCTCGG-3′;

[0097] qRT-PCR-E5 R: 5′-GGAACTCTGCTGTGGCTTTTG-3′;

[0098] qRT-PCR-GAPDH F: 5′-GGACTCATGACCACGGTCCAT-3′;

[0099] qRT-PCR-GAPDH R: 5′-CTCCTGTGGTGGCATTAGCA-3′.

[0100] Depend on Figure 1 The results show that the predicted enhancer has the effect of enhancing gene expression. Figure 3The results showed that the E2 region had the highest transcriptional activity and could be successfully amplified.

[0101] Example 2:

[0102] We treated ovarian granulosa cells with si-SLIT2e and used qRT-PCR, Western Blot, and ChIP-qPCR to detect the expression level of the SLIT2 gene and the enrichment level of H3K4ac in the SLIT2 gene promoter region. si-SLIT2e was synthesized by a company outsourced, and its sequence is as follows:

[0103] si-SLIT2e: 5′-GCACAGTAGCTTTCTATCA-3′.

[0104] qRT-PCR and Western Blot results showed that si-SLIT2e significantly reduced the mRNA and protein expression levels of the SLIT2 gene. Simultaneously, ChIP-qPCR results showed that si-SLIT2e significantly reduced the H3K4ac enrichment level in the P4 region of the SLIT2 gene promoter. Figure 4 In summary, SLIT2e may affect SLIT2 transcription by influencing the H3K4ac level in the SLIT2 gene promoter region.

[0105] Example 3:

[0106] We then transfected histone deacetylase HDAC small interfering RNA (si-HDAC1) into granulosa cells, and used qRT-PCR and Western blot to detect the expression level of the SLIT2 gene. Furthermore, we divided the SLIT2 gene promoter into segments: P1 (-539bp—-436bp), P2 (-805—-512bp), P3 (-1629—-1382bp), and P4 (-1919—-1782bp). Figure 5 Subsequently, the H3K4ac enrichment level and chromatin accessibility of each segment of the SLIT2 promoter region were detected using ChIP-qPCR and a chromatin accessibility assay kit, respectively. The si-HDAC1 sequence is as follows:

[0107] si-HDAC1: 5′-GAATACTTCGGACCAGATT-3′;

[0108] The qRT-PCR primers used in this example are:

[0109] P1 Forward: 5′-CAGCAGCCGAGCGAGAATA-3′;

[0110] P1 Reverse: 5′-GGCTTTGAATCCAGTCCCAC-3′;

[0111] P2 Forward: 5′-CAGATTCCACCGACCCAACT-3′;

[0112] P2 Reverse: 5′-CACTAGTGACAACCCAGGCA-3′;

[0113] P3 Forward: 5′-TGTCGGTAGACGACGAGGTT-3′;

[0114] P3 Reverse: 5′-GAGGCTCAGTAGGAACGGTG-3′;

[0115] P4 Forward: 5′-TCTGCGAGAATAGACGACGC-3′;

[0116] P4 Reverse: 5′-TAGAGCTGACACAGGTACGC-3′.

[0117] qRT-PCR and Western Blot results showed that si-HDAC1 significantly increased the mRNA and protein expression levels of the SLIT2 gene. ChIP-qPCR and chromatin accessibility assays indicated that si-HDAC1 significantly increased the H3K4ac enrichment level and chromatin accessibility in the promoter P4 region. Figure 6 ).

[0118] In summary, si-HDAC1 increases chromatin openness by increasing H3K4ac enrichment in the P4 promoter region of the SLIT2 gene, thereby promoting SLIT2 gene transcription.

[0119] Example 4:

[0120] We selected large follicles (diameter > 5 mm) and small follicles (diameter < 3 mm) for qRT-PCR to detect the changes in the expression levels of SLIT2 gene and SLIT2e in different follicles.

[0121] qRT-PCR results showed that SLIT2e expression was higher in small follicles, and the expression level of the SLIT2 gene in small follicles was significantly higher than that in large follicles. In conclusion, SLIT2e promotes follicle development by regulating SLIT2 expression. Figure 7 ).

[0122] Example 5:

[0123] 1. Constructing an overexpression vector for the SLIT2 gene

[0124] (1) Total RNA was extracted from porcine ovarian granulosa cells according to the instructions of the Shanghai Feijie Biotechnology Total RNA Rapid Extraction Kit and reverse transcribed into cDNA.

[0125] (2) Specific primers for amplifying the CDS region of SLIT2 (Gene ID: 100620577) were designed using NCBI and amplified using the extracted cDNA as a template. The amplified fragment was purified, recovered, ligated into the pMD18T vector (purchased from Takara), transformed, screened, and sequenced to confirm its correctness before extracting ordinary plasmids.

[0126] (3) Analysis using BioEdit software revealed that the CDS region sequence of the SLIT2 gene lacked HindIII and KpnI restriction endonuclease sites, while the pcDNA3.1 vector contained HindIII (A^AGCTT) and KpnI (G^GTACC) restriction sites. HindIII and KpnI restriction site sequences were added to the upstream and downstream primers, respectively. PCR amplification was performed using the recombinant pMD18T plasmid of the SLIT2 CDS region as a template. The fragment was purified, double-digested, ligated into the pcDNA3.1 vector, transformed, screened, and sequenced to confirm its correctness. An endotoxin-free plasmid (an endotoxin-free plasmid small-scale extraction kit was purchased from Magen, USA) was then extracted and named pcDNA3.1-SLIT2. The PCR amplification primer sequences are as follows:

[0127] SLIT2 F:5′-CCAAGCTTCCCACGAGCTAGGAGGCGGC-3′

[0128] SLIT2 R:5′-GGGGTACCTTCTCCAAGGACCAGAGCCG-3′

[0129] 2. Transfer the constructed SLIT2 overexpression vector and SLIT2 small perturbation fragment into GCs.

[0130] After double enzyme digestion to verify the successful construction of the SLIT2 overexpression vector, GCs were seeded in 24-well cell culture plates to verify the overexpression efficiency of pcDNA3.1-SLIT2 and the interference efficiency of si-SLIT2. pcDNA3.1 was used as the control group for pcDNA3.1-SLIT2. 100 ng, 200 ng, and 500 ng of pcDNA3.1 and pcDNA3.1-SLIT2 vector plasmids were transfected, with three replicates per group. RNA was extracted after 24 h. The results showed that the mRNA expression level of SLIT2 significantly increased with increasing transfection concentration (P < 0.001). Since different concentrations of the overexpression vector all showed good transfection efficiency, 500 ng of pcDNA3.1-SLIT2 was selected for subsequent experiments. Meanwhile, si-NC was used as a control for si-SLIT2-1 (5′-GGAAGAAGGTCACCAGTCATT-3′), si-SLIT2-2 (5′-CGGAGAGACTGGATTTGAATG-3′), and si-SLIT2-3 (5′-CGGAAGATTATCGATCAAAAT-3′). si-SLIT2-1, si-SLIT2-2, si-SLIT2-3, and si-NC were transfected into GCs, with three replicates per group. The results showed that si-SLIT2-3 exhibited the best interference efficiency according to qRT-PCR (P < 0.001). Therefore, 500 ng concentrations of pcDNA3.1-SLIT2 and si-SLIT2-3 were selected for subsequent experiments.

[0131] Cell transfection (6-well plate): Observe the cell confluence under an optical microscope. The transfection effect is best when the confluence reaches 70-80%. Discard the culture medium in the culture plate, add PBS (containing 1% penicillin and bismuth subsalicylate) along the wall to wash the cells, repeat the washing once, and then 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 described above, and refer to the transfection instructions of Lipofectamine 3000. Incubate at room temperature for 10-15 min. Add 200 μL of transfection mixture to each well of the 6-well plate, gently shake in a cross shape to mix, and return to the incubator for culture. After 24-48 h of transfection, collect the cells for subsequent experiments.

[0132] 3. Apoptosis detection

[0133] This invention uses the Annexin V-FITC / PI cell apoptosis detection kit to detect the apoptosis rate of GCs cells.

[0134] 4. Cell proliferation detection

[0135] GC proliferation detection was performed according to the instructions of Ribobio's Cell-Light EdU Apollo567 In Virto Kit. GCs were treated with 50 μM EdU for 2 hours at room temperature, followed by treatment with 80% acetone for 30 minutes, 0.5% Triton X-100 for 10 minutes, 1x Apollo reagent for 30 minutes, and Hoechst staining for 30 minutes. They were then examined under a microscope immediately or stored at 4°C protected from light.

[0136] 5. Western blot detection of target gene protein expression

[0137] Protein samples separated by SDS-PAGE gel electrophoresis were transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was then blocked with skim milk powder and incubated overnight at 4°C with the following primary antibodies: SLIT2 (20217-1-AP, Proteintech, 1:1000), H3K4ac (39382, Active Motif, 1:1000), CASP9 (10380-1-AP, Proteintech, 1:1000), CASP8 (13423-1-AP, Proteintech, 1:1000), CASP3 (19677-1-AP, Proteintech, 1:1000), CAT (21260-1-AP, Proteintech, 1:8000), and SOD1 (10269-1-AP, Proteintech, 1:1). The following bands were measured: SOD2 (24127-1-AP, Proteintech, 1:10000), xCT (26864-1-AP, Proteintech, 1:1000), GPX4 (30388-1-AP, Proteintech, 1:1000), GAPDH (10494-1-AP, Proteintech, 1:20000), and Tubulin (11224-1-AP, Proteintech, 1:5000). ImageJ software was used to measure the bands.

[0138] result:

[0139] (1) SLIT2 promotes apoptosis of ovarian granulosa cells and inhibits the proliferation of ovarian granulosa cells.

[0140] like Figure 8As shown, qRT-PCR results indicated that, compared with the control group, pcDNA3.1-SLIT2 significantly increased the mRNA expression levels of apoptosis-related genes Caspase3 (P < 0.01) and Caspase9 (P < 0.01) (P < 0.01), and significantly decreased the mRNA expression level of cell proliferation-related genes PCNA (P < 0.05). Conversely, si-SLIT2 significantly promoted the mRNA levels of cell proliferation-related genes PCNA (P < 0.01) and CDK1 (P < 0.05) (P < 0.05), and inhibited the mRNA expression levels of apoptosis-related genes Caspase3 and Caspase9 (P < 0.05). Western blot results showed that si-SLIT2 significantly inhibited the protein expression level of Caspase9 (P < 0.005). Flow cytometry analysis revealed that pcDNA3.1-SLIT2 significantly promoted early apoptosis of GCs compared to the control group (P < 0.01); conversely, si-SLIT2 inhibited early apoptosis of GCs. EdU assay analysis of GC proliferation showed that pcDNA3.1-SLIT2 significantly inhibited GC proliferation compared to the control group (P < 0.01); while si-SLIT2 significantly increased GC proliferation (P < 0.01).

[0141] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for regulating SLIT2 transcription, characterized in that: The transcriptional activity of the SLIT2 gene in porcine ovarian granulosa cells was regulated by modulating the histone acetylation level in the promoter region of the SLIT2 gene.

2. The method for regulating SLIT2 transcription according to claim 1, characterized in that: The histone acetylation in the promoter region of the SLIT2 gene is the histone H3K4 acetylation in the promoter region of the SLIT2 gene; the H3K4 acetylated region is the -1919 to -1782 bp region of the SLIT2 gene promoter.

3. The method for regulating SLIT2 transcription according to claim 2, characterized in that: In the method described, the level of histone acetylation in the promoter region of the SLIT2 gene is increased, and the transcriptional activity of the SLIT2 gene is increased.

4. The method for regulating SLIT2 transcription according to claim 3, characterized in that: The increased histone acetylation level in the promoter region of the SLIT2 gene is achieved by inhibiting the expression of histone deacetylation enzyme HDAC using histone deacetylation inhibitors or RNA interference technology.

5. The method for regulating SLIT2 transcription according to claim 4, characterized in that: The siRNAs used in RNA interference technology to inhibit HDAC are as follows: HDAC1-siRNA: 5′-GAATACTTCGGACCAGATT-3′.

6. A core promoter functional regulatory region of the SLIT2 gene in porcine ovarian granulosa cells, characterized in that: This refers to the region from -1919bp to -1782bp of the SLIT2 gene promoter.

7. A core enhancer functional region of the SLIT2 gene in porcine ovarian granulosa cells, characterized in that: The region from 691bp to 1085bp is the enhancer region of the SLIT2 gene; the nucleotide sequence of the SLIT2 gene enhancer is shown in SEQ ID NO.

1.

8. A nucleic acid fragment regulating the expression of the SLIT2 gene in porcine ovarian granulosa cells, characterized in that: The siRNA is for the SLIT2 enhancer region, and the siRNA is as follows: 5′-GCACAGTAGCTTTCTATCA-3′.

9. The application of the core promoter functional regulatory region of claim 6, the core enhancer functional region of claim 7, or the nucleic acid fragment of claim 8 in regulating the expression of the SLIT2 gene in porcine ovarian granulosa cells.

10. The application of the core promoter functional regulation region of claim 6, the core enhancer functional region of claim 7, or the nucleic acid fragment of claim 8, characterized in that: For at least one of the following applications: I. Application in promoting the proliferation of porcine ovarian granulosa cells / in the preparation of drugs that promote the proliferation of porcine ovarian granulosa cells; II. Application in promoting porcine follicle development / in the preparation of drugs that promote porcine follicle development.

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