Application of LncRNA IFA in regulating the expression of ACTG1 in porcine ovarian granulosa cells

Abstract

The present invention discloses the use of lncRNA IFA to regulate the expression of ACTG1 in porcine ovarian granulosa cells. The present invention predicts a sequence complementary to ACTG1 mRNA within the lncRNA IFA sequence and detects the binding of lncRNA IFA to ACTG1 mRNA through pull-down and experimental testing. Quantitative polymerase chain reaction (qPCR) is used to detect the expression levels of lncRNA IFA and ACTG1 in granulosa cells of follicles at different developmental stages. The expression level of ACTG1 is then tested after overexpression and interference with lncRNA IFA, revealing that lncRNA IFA can promote ACTG1 expression. Furthermore, CCK8, EdU, and flow cytometry analysis revealed that ACTG1 can promote proliferation and inhibit apoptosis of ovarian granulosa cells, enhance cell activity, and accelerate cell cycle progression in ovarian granulosa cells after overexpression and interference with ACTG1. This provides valuable information for research into the expression and regulation mechanisms of ACTG1.

Description

Technical Field

[0001] The present invention belongs to the technical fields of cell engineering and genetic engineering, and particularly relates to the application of lncRNA IFA in regulating the expression of ACTG1 in pig ovarian granulosa cells. Background Art

[0002] LncRNAs are typically endogenous cellular RNA molecules consisting of linked sequences exceeding 200 nucleotides. They have a structure similar to mRNA but lack protein-coding capabilities. LncRNA IFA (Inhibitor of Follicular Atresia, tentatively named) is a lncRNA identified through RNA-seq of porcine ovarian granulosa cells. LncRNA IFA can inhibit apoptosis, promote proliferation and cell cycle progression, and enhance cellular activity in granulosa cells.

[0003] In eukaryotic cells, actin has multiple important biological functions. In addition to providing a structural framework to maintain cell shape and polarity, it also participates in regulating cell migration, proliferation, and apoptosis. ACTG1 (actin gamma 1), a type of actin, is generally expressed at high levels in cells and plays a role in maintaining the cytoskeleton, morphology, and motility. Loss of ASAP3 destabilizes ACTG1, thereby inhibiting cancer cell migration. ACTG1 regulates the cell cycle in vitro by downregulating cyclins and cyclin-dependent kinases, promoting cell proliferation, and inhibiting apoptosis by regulating mitochondrial function. Overexpression of ACTG1 increases apoptosis in a 6-OHDA-induced Parkinson's disease cell model. RRAD inhibits the proliferation of hepatocellular carcinoma (HCC) cells by downregulating ACTG1.

[0004] A large number of studies generally believe that the development of follicles, the ovulation process and the proliferation and differentiation of granulosa cells are closely related, and excessive apoptosis of granulosa cells can cause follicular atresia.

[0005] Furthermore, studies have shown that lncRNAs can directly bind to mRNA, thereby affecting RNA homeostasis and regulating target gene expression, thereby exerting their biological effects. For example, lncRNA-PXN-AS1 can enhance PXN RNA homeostasis by targeting PXN mRNA, thereby increasing PXN expression and promoting the occurrence of liver cancer. Summary of the Invention

[0006] To solve the related problems, the primary purpose of the present invention is to provide a method for regulating the expression of ACTG1 in porcine ovarian granulosa cells by lncRNA IFA.

[0007] Another object of the present invention is to provide the use of ACTG1 in porcine ovarian granulosa cells.

[0008] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0009] A method for regulating the expression of ACTG1 in porcine ovarian granulosa cells using lncRNA IFA. In vitro, lncRNA IFA positively regulates the expression of ACTG1. By regulating the expression level of lncRNA IFA, the expression of ACTG1 in porcine ovarian granulosa cells can be regulated. The nucleotide sequence of the lncRNA IFA is shown in SEQ ID NO: 1.

[0010] Furthermore, when the expression level of lncRNA IFA increased, the expression level of ACTG1 increased; when the expression level of lncRNA IFA decreased, the expression level of ACTG1 decreased.

[0011] Furthermore, the expression level of lncRNA IFA was increased by gene overexpression technology, and the gene overexpression vector used was prepared by the following method:

[0012] (1) RNA was extracted from pig ovarian granulosa cells, reverse transcribed into cDNA, and PCR amplified using the cDNA as a template to obtain the target fragment;

[0013] (2) The target fragment was ligated to the pcDNA3.1 vector digested with restriction endonucleases BamH I and Xba I to obtain a recombinant vector.

[0014] The primers used for PCR amplification in step (1) are as follows:

[0015] lncRNA IFA F: 5'-GGCGATCCTGGGTACATGG-3';

[0016] lncRNA IFA R: 5'-GAGACCGCGTCCACTCCGCC-3'.

[0017] Furthermore, the expression level of lncRNA IFA was decreased by RNA interference technology.

[0018] The targeting sequence of the siRNA used was: 5'-TGACCTGGGCGACGTAGCA-3' (SEQ ID NO. 6).

[0019] The application of ACTG1 in pig ovarian granulosa cells: in vitro environment, the ACTG1 positively regulates any one or more functional phenotypes of pig ovarian granulosa cell proliferation, cell apoptosis, cell activity, and cell cycle progression.

[0020] Furthermore, increasing exogenous ACTG1 promotes cell proliferation, inhibits cell apoptosis, increases cell activity and / or accelerates cell cycle progression; inhibiting ACTG1 expression inhibits cell proliferation, promotes cell apoptosis, reduces cell activity and / or blocks cell cycle progression.

[0021] Furthermore, the increase of exogenous ACTG1 is achieved by gene overexpression technology, and the gene overexpression vector used is prepared by the following method:

[0022] (1) RNA was extracted from pig ovarian granulosa cells, reverse transcribed into cDNA, and PCR amplified using the cDNA as a template to obtain the target fragment;

[0023] (2) The target fragment was ligated to the pcDNA3.1 vector digested with restriction endonucleases BamH I and Xba I to obtain a recombinant vector.

[0024] The primers used for PCR amplification in step (1) are as follows:

[0025] ACTG1 F: 5'-GCTCCGCTTAAATAGGGGCG-3';

[0026] ACTG1 R: 5'-GTACGCATCTGCTCGCAGTC-3'

[0027] Furthermore, inhibition of ACTG1 expression was achieved by RNA interference technology.

[0028] The targeting sequence of the siRNA used was: 5'-CCGACTACCTCATGAAGAT-3' (SEQ ID NO. 7).

[0029] Use of ACTG1 and / or its related biomaterials in the preparation of a drug, wherein the drug is any one or more combinations of the following drugs:

[0030] Ⅰ. Drugs that promote the proliferation of porcine ovarian granulosa cells;

[0031] II. Drugs that inhibit apoptosis of porcine ovarian granulosa cells;

[0032] III. Drugs that enhance the activity of porcine ovarian granulosa cells;

[0033] IV. Drugs that accelerate the cell cycle progression of porcine ovarian granulosa cells;

[0034] Ⅴ. Drugs that promote follicle development.

[0035] Furthermore, the ACTG1-related biomaterial is any one or more combinations of the following biomaterials:

[0036] 1) DNA molecules encoding ACTG1;

[0037] 2) an expression cassette containing the DNA molecule described in 1);

[0038] 3) a recombinant vector containing the DNA molecule described in 1), or a recombinant vector containing the expression cassette described in 2);

[0039] 4) A recombinant cell containing the DNA molecule described in 1), or a recombinant cell containing the expression cassette described in 2), or a recombinant cell containing the recombinant vector described in 3).

[0040] LncRNA IFA (Inhibitor of Follicular Atresia, tentative name) is a lncRNA obtained by RNA-seq of pig ovarian granulosa cells, and its nucleotide sequence is shown in SEQ ID NO: 1.

[0041] In the early stage of this invention, genetic engineering and cell engineering technologies were used to study the effects of lncRNA IFA on ovarian granulosa cell proliferation, apoptosis, cell activity, and cell cycle. It was confirmed that lncRNA IFA can promote the proliferation of ovarian granulosa cells, inhibit apoptosis, enhance the cell activity of ovarian granulosa cells, and accelerate the cell cycle process of ovarian granulosa cells. Specifically, EdU (5-Ethynyl-2'-deoxyuridine) experiments demonstrated that, in vitro, overexpression of lncRNA IFA promoted the proliferation of ovarian granulosa cells (P<0.01), while interference with lncRNA IFA inhibited the proliferation of ovarian granulosa cells (P<0.05). CCK8 (Cell Counting Kit-8) experiments demonstrated that, in vitro, overexpression of lncRNA IFA significantly enhanced the cell viability of ovarian granulosa cells (P<0.01), while interference with lncRNA IFA significantly inhibited the cell viability of ovarian granulosa cells (P<0.01). Flow cytometry (FCM) experiments demonstrated that, in vitro, overexpression of lncRNA IFA significantly inhibited the apoptosis of ovarian granulosa cells (P<0.01) and accelerated the cell cycle progression of ovarian granulosa cells (P<0.05). Interference with lncRNA IFA significantly decreased the apoptosis rate of ovarian granulosa cells (P<0.001) and significantly inhibited the cell cycle progression (P<0.01).

[0042] The present invention found through sequence alignment that ACTG1 and lncRNA IFA have a large number of complementary sequences.

[0043] The verification results of the present invention are as follows:

[0044] 1. The present invention predicts that the lncRNA IFA sequence and the ACTG1 mRNA sequence have a large number of complementary regions by bioinformatics means ( Figure 1 (a), and there is also a possibility that lncRNA IFA binds to ACTG1 protein ( Figure 1 (b)

[0045] 2. The present invention detected through pull-down experiments that lncRNA IFA directly binds to ACTG1 mRNA ( Figure 1 c); but no binding to ACTG1 protein ( Figure 1 (d)

[0046] 3. The present invention detected through qPCR and WB experiments that the expression trends of lncRNA IFA and ACTG1 were the same in granulosa cells of follicles at different developmental levels ( Figure 2 (a)

[0047] 4. The present invention detected through qPCR experiments that after overexpression / interference of lncRNA IFA, the RNA expression of ACTG1 was upregulated / downregulated ( Figure 2 (b)

[0048] 5. The present invention detected through WB experiments that after overexpression / interference of lncRNA IFA, the protein expression of ACTG1 was upregulated / downregulated ( Figure 2 (c)

[0049] 6. The present invention detected by CCK8 and EdU that after overexpression of ACTG1, the activity and proliferation level of granulosa cells increased ( Figure 3 Middle a); After interfering with ACTG1, the activity and proliferation of granulosa cells decreased ( Figure 3 (b)

[0050] 7. The present invention detected by flow cytometry that after overexpression of ACTG1, the cell cycle process of granulosa cells was accelerated and the apoptosis level was reduced ( Figure 4 After ACTG1 interference, the cell cycle progression of granulosa cells was blocked and the apoptosis level was increased ( Figure 4 (b)

[0051] This study used lncRNA IFA and ACTG1 as research objects and adopted molecular cell biology methods to first verify the direct binding of lncRNA IFA to ACTG1 mRNA; then detected the regulatory effect of lncRNA IFA on ACTG1 expression, proving that lncRNA IFA promotes the expression of ACTG1 and further regulates the activity, proliferation, cycle and apoptosis of granulosa cells.

[0052] The present invention has the following advantages and effects compared to the prior art:

[0053] This study used bioinformatics and molecular biology experiments to predict and verify the sequence within the lncRNA IFA that is complementary to the ACTG1 mRNA. The study also examined ACTG1 expression after overexpression or interference with the lncRNA IFA. Furthermore, the activity, proliferation, cycle, and apoptosis of granulosa cells were examined after overexpression or interference with ACTG1. The results of this study demonstrate that lncRNA IFA binds to ACTG1 mRNA, thereby promoting ACTG1 expression and further regulating the activity, proliferation, cycle, and apoptosis of granulosa cells. This study provides valuable insights into the regulatory mechanisms of ACTG1 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Figure 1 is a diagram showing the prediction and verification results of lncRNA IFA binding to ACTG1 mRNA and ACTG1 protein; wherein, a is the prediction result of lncRNA IFA and ACTG1 mRNA complementary sequence, b is the prediction result of lncRNA IFA binding to ACTG1 protein, c is the pull-down experiment result of lncRNA IFA binding to ACTG1 mRNA, and d is the pull-down experiment result of lncRNA IFA binding to ACTG1 protein;

[0055] Figure 2 Figure 2 is a test result diagram of the correlation between lncRNA IFA and ACTG1 expression; a is the expression level of lncRNA IFA and ACTG1 in granulosa cells at different developmental levels of follicles, b is the effect of overexpression / interference of lncRNA IFA on ACTG1 RNA expression, and c is the effect of overexpression / interference of lncRNA IFA on ACTG1 protein expression;

[0056] Figure 3 Figure 1 is a graph showing the activity and proliferation of follicular granulosa cells after overexpression / interference of ACTG1; a is the activity and proliferation level test result of cells after overexpression of ACTG1, and b is the activity and proliferation level test result of cells after interference of ACTG1;

[0057] Figure 4 Figure 1 is a graph showing the detection results of cell cycle and apoptosis of granulosa cells after overexpression / interference of ACTG1; a is the detection result of cell cycle progression and apoptosis level of cells after overexpression of ACTG1, and b is the detection result of cell cycle progression and apoptosis level of cells after interference of ACTG1. DETAILED DESCRIPTION

[0058] The present invention will be described in further detail below in conjunction with Examples, but embodiments of the present invention are not limited thereto. It should be understood that the examples described in this specification are merely for explaining the present invention and are not intended to limit the present invention. The parameters, ratios, etc. of the examples can be selected according to local conditions and have no substantial effect on the results. In the examples, unless otherwise specified, all are conventional reagents and method steps in the art. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0059] Example 1: Construction of overexpression vectors for lncRNA IFA and ACTG1

[0060] BioEdit software analysis revealed that the lncRNA IFA sequence lacked restriction endonuclease sites for BamH I and Xba I, whereas the pcDNA3.1 vector (purchased from Invitrogen, catalog number V79020) contained these sites. Full-length primers for lncRNA IFA were designed using the NCBI primer blast online tool, and the BamH I and Xba I restriction endonuclease sites were added to the upstream and downstream primer sequences (lncRNA IFA F: 5'-GGCGATGCCTGGGTACATGG-3' (SEQ ID NO. 2); lncRNA IFAR: 5'-GAGACCGCGTCCACTCCGCC-3' (SEQ ID NO. 3)). RNA was extracted from the porcine ovarian granulosa cell line and reverse transcribed to obtain a cDNA library. The target fragment was then amplified by PCR using the cDNA as a template. After purification, recovery, double enzyme digestion, ligation to the pcDNA3.1 vector, transformation, screening, and sequencing, the endotoxin-free plasmid was extracted (the endotoxin-free plasmid mini-preparation kit was purchased from Magen). The obtained recombinant vector was named pcDNA3.1-lncRNA IFA.

[0061] The process for constructing the ACTG1 overexpression vector is the same as that for lncRNA IFA, and the upstream and downstream primer sequences are:

[0062] ACTG1 F: 5'-GCTCCGCTTAAATAGGGGCG-3' (SEQ ID NO. 4); ACTG1 R: 5'-GTACGCATCTGCTCGCAGTC-3' (SEQ ID NO. 5); BamH I and Xba I were selected as the restriction enzyme cleavage sites.

[0063] Sequence of lncRNA IFA (SEQ ID NO. 1):

[0064] .

[0065] Example 2: Culture of ovarian granulosa cells

[0066] (1) Pig ovarian tissue collected from the slaughterhouse was placed in PBS or normal saline containing 1% (w / w) double-antibody, placed on ice and quickly brought back to the laboratory;

[0067] (2) The collected ovaries were washed three times with PBS or physiological saline (containing 1% (w / w) double-antibody) in a sterile culture room, and then quickly transferred to a clean bench. A 1 mL sterile disposable syringe was inserted shallowly into the antral follicles of the ovary to aspirate the follicular fluid;

[0068] (3) The aspirated follicular fluid was placed in a centrifuge tube containing an appropriate amount of DMEM medium and centrifuged at 800 rpm for 5 min at room temperature;

[0069] (4) Discard the supernatant, resuspend the cells in DMEM medium, centrifuge, and wash the cells twice; prepare complete DMEM medium: 89% (w / w) high-glucose DMEM medium + 10% (w / w) FBS + 1% (w / w) double antibody;

[0070] (5) Resuspend the cells in complete culture medium and inoculate them into a 75 mL culture flask; place the flask in a 37°C, 5% CO2 incubator for static culture.

[0071] The dual resistance is penicillin and streptomycin.

[0072] Example 3: Pull down experiment

[0073] Pull down experiment refers to Thermo's Pierce TM Follow the instructions of the Magnetic RNA-Protein Pull-Down Kit. The specific steps are as follows:

[0074] (1) Cell lysate can be prepared using standard lysis buffer to ensure that the cell lysate protein concentration is greater than 2 mg / mL.

[0075] (2) Binding of streptavidin magnetic beads to biotin-labeled probes:

[0076] Use 25-100 pmol of biotin probe per 20-50 μL of magnetic beads, using 50 pmol of probe as an example. Add 50 μL of streptavidin magnetic beads to a 1.5 mL sterile centrifuge tube; place the centrifuge tube on a magnetic rack, collect the beads on the side of the tube, and discard the supernatant; wash with an equal volume of 20 mM Tris (pH 7.5) and resuspend the beads; repeat the wash once, place the centrifuge tube on a magnetic rack, collect the beads on the side of the tube, remove and discard the supernatant; add an equal amount of 1× RNA Capture Buffer and resuspend the beads; add 50 pmol of biotin-labeled probe to the beads and gently mix with a pipette; incubate on a shaker at room temperature for 15-30 minutes.

[0077] (3) Binding of RNA-binding protein to biotin probe:

[0078] Place the centrifuge tube on a magnetic rack, collect the beads on the side of the centrifuge tube and discard the supernatant; wash with an equal volume of 20mM Tris and resuspend the magnetic beads; repeat the wash and place on a magnetic rack, collect the beads on the side of the centrifuge tube, remove and discard the supernatant; dilute 10× Protein-RNA Binding Buffer to 1× with ultrapure water; add 100μL of 1× Protein-RNA Binding Buffer to the magnetic beads and mix; place the centrifuge tube on a magnetic rack, collect the beads on the side of the centrifuge tube and discard the supernatant; add 100μL of RNA-Protein Binding Reaction to the centrifuge tube and mix by pipetting or gentle vortexing; incubate on a shaker at 4°C for 30-60 minutes.

[0079] (4) Cleaning and elution of Probe-Binding Protein Complexes:

[0080] Place the centrifuge tube on a magnetic rack, collect the beads on the side of the tube, remove and discard the supernatant; wash twice with an equal amount of 1× Wash buffer; place the centrifuge tube on a magnetic rack, collect the beads on the side of the tube, remove and discard the supernatant; add 50μL of Elution Buffer, mix well, and incubate on a shaker at 37℃ for 15-30 minutes; place the centrifuge tube on a magnetic rack and aspirate the supernatant for downstream analysis.

[0081] Example 4: Inoculation and transfection of ovarian granulosa cells

[0082] (1) When the confluence of porcine ovarian granulosa cells reaches about 70-90%, discard the culture medium and wash the cells three times with preheated PBS;

[0083] (2) Add 0.25% trypsin for digestion and place in an incubator for about 3 minutes. Observe under a microscope until most cells float up, and immediately add an equal amount of stop solution (DMEM complete medium) to terminate the digestion.

[0084] (3) Wash twice with PBS and centrifuge at 800 rpm for 5 min;

[0085] (4) Gently resuspend the cell pellet in complete medium, evenly distribute it to each well, replenish the volume with complete medium, shake gently, and culture in an incubator;

[0086] (5) After about 24 hours, observe the status of the porcine ovarian granulosa cells and prepare for transfection when the cell confluence reaches about 70-90%;

[0087] (6) The transfection method was according to Invitrogen The experiment was performed according to the instructions of the 3000 kit; 3 replicates were set for each group;

[0088] (7) The transfected cells were placed in a 37°C, 5% CO2 incubator for further culture;

[0089] (8) Collect cells 24h or 48h after transfection according to the experimental purpose.

[0090] Example 5: RNA extraction and reverse transcription

[0091] Total RNA extraction from cells was performed according to the Takara TRIzol operating instructions. The specific steps are as follows:

[0092] (1) After the pig ovarian tissue was ground with liquid nitrogen, 1 mL of TRIzol was added for every 50-100 mg of tissue and the mixture was pipetted several times. When total RNA was extracted from adherent pig ovarian granulosa cells, 1 mL of TRIzol was added for every 10 cm 2 Add 1 mL of TRIzol directly to the bottom area of ​​the cell culture plate;

[0093] (2) Place on ice for 10 min to fully lyse the tissue / cells, centrifuge at 12,000 rpm for 5 min, discard the precipitate, and aspirate the supernatant into a new 1.5 mL RNase-free tube;

[0094] (3) Add 200 μL of chloroform (per 1 mL of TRIzol) and shake vigorously for 15–30 s, place on ice for 15 min, and centrifuge at 12,000 rpm at 4°C for 15 min.

[0095] (4) Aspirate the upper aqueous phase and place it in a new 1.5 mL RNase-free EP tube;

[0096] (5) Add 500 μL of isopropanol (per 1 mL of TRIzol), gently invert the tube to mix, and place on ice for 10 min. Centrifuge at 12,000 rpm at 4°C for 10 min.

[0097] (6) Discard the supernatant and place at room temperature. Add 1 mL of 75% ethanol-DEPC water along the tube wall to wash the RNA. Centrifuge at 12,000 rpm at 4°C for 5 min and discard the supernatant.

[0098] (7) Vacuum dry for 5–10 min, taking care to avoid over-drying the RNA precipitate;

[0099] (8) Add DEPC water to dissolve the RNA precipitate.

[0100] mRNA reverse transcription was performed with reference to TaKaRa's PrimeScript™ RT Master Mix (Perfect RealTime) cDNA reverse transcription kit.

[0101] Example 6: Western blotting

[0102] (1) Protein sample preparation:

[0103] Remove the cell culture medium, wash once with PBS, and add 150-250 μL of RIPA lysis buffer to each well of a 6-well plate (increase or decrease the amount of lysis buffer for other wells accordingly). Let the plate rest for 10-20 minutes to allow the cells to fully lyse. Centrifuge at 10,000-14,000 rpm for 3-5 minutes and collect the supernatant. For tissue samples, grind and add 150-250 μL of RIPA lysis buffer per 20 mg of sample.

[0104] (2) Protein quantification:

[0105] The protein sample concentration was detected by the BCA method. Standards and protein samples of different concentrations were added to a 96-well plate. After adding BCA working solution, the plates were incubated at 37°C for 30 minutes. The absorbance was detected at a wavelength of 570 nm on the ELISA plate. A standard curve was drawn to obtain a regression equation. The average OD value of the samples was substituted into the regression equation to calculate the sample concentration.

[0106] (3) SDS-PAGE electrophoresis:

[0107] After protein denaturation, add the sample, cover the electrophoresis tank, connect the electrophoresis instrument, turn on the electrophoresis instrument, and run the electrophoresis at a voltage of 140V for 30 to 50 minutes (the specific time depends on the length of the stacking gel). Stop the electrophoresis when the bromophenol blue indicator (blue line) in the sample migrates to 1 to 2 cm from the leading edge.

[0108] (4) Transfer:

[0109] Using eBlot TM L1 fast wet transfer instrument is used for membrane transfer. The specific time depends on the molecular weight of the target protein.

[0110] (5) Closure:

[0111] Prepare 5% skimmed milk powder as the blocking solution, and wash it with TBST after the transfer. Place the membrane in an incubation box, pour in an appropriate amount of blocking solution to cover the PVDF membrane, and place it on a decolorization shaker at 40 rpm for 1 to 2 hours.

[0112] (6) Primary antibody incubation:

[0113] Refer to the antibody instructions for dilution ratio, dilute with TBST, incubate at room temperature for 1-2 hours or at 4°C overnight; use GAPDH or α-Tubulin as the internal reference protein; after incubation, rinse three times with TBST on a shaker, each time for about 5 minutes.

[0114] (7) Secondary antibody incubation:

[0115] Prepare secondary antibodies of the corresponding species according to the species of the primary antibody, dilute them with TBST according to the dilution factor in the instructions, mix them on a vortex and set aside; after the primary antibody incubation is completed and the membrane is washed, add the diluted secondary antibody and incubate at room temperature for 1-2 hours; after the incubation is completed, rinse the membrane with TBST three times and then expose it.

[0116] Example 7: Ovarian granulosa cell proliferation detection

[0117] Granulocyte proliferation was detected using the EdU method. The procedure was performed according to the instructions of the Cell-Light EdU Apollo 567 Invitro Kit (using a 48-well cell culture plate as an example):

[0118] (1) Add 150 μL of 50 μM EdU culture medium to each well of the cell culture plate, incubate in a cell culture incubator for 2 h, discard the culture medium, and wash the cells twice with PBS;

[0119] (2) Add 150 μL / well of cell fixative (PBS containing 80% acetone) and incubate at room temperature for 30 min. Discard the fixative. Wash the cells twice with PBS.

[0120] (3) Add 150 μL / well of permeabilization agent (PBS containing 0.5% Triton X) to permeabilize the cells for 3 min and wash the cells three times with PBS;

[0121] (4) Add 150 μL / well of 1× Apollo staining solution, incubate at room temperature for 30 min in the dark, and discard the staining solution. Wash on a shaker with PBS for 6 times, 5 min each time.

[0122] (5) Add 150 μL / well DAPI staining solution, incubate at room temperature in the dark for 30 min, discard the staining reaction solution, and add 150 μL / well PBS to wash three times;

[0123] (6) After staining, take photos using a fluorescence microscope.

[0124] Example 8: Detection of ovarian granulosa cell apoptosis

[0125] Granulocyte apoptosis detection was performed according to the instructions of the Annexin V-FITC / PI double staining apoptosis detection kit:

[0126] (1) Place the cell culture plate at room temperature, gently rinse the cells in the culture plate with PBS, and discard the PBS;

[0127] (2) Add trypsin to digest the cells and place them in an incubator for about 3 minutes. Observe under a microscope until most cells float up, and immediately add an equal amount of stop solution (complete culture medium) to terminate the digestion.

[0128] (3) Collect the cells by centrifugation at 1000 × g for 5 min, discard the supernatant, and wash the cells twice with pre-cooled PBS; adjust the number of cells per tube to 0.2–1.0 × 10 6 Add 400 μL of 1× Binding Buffer to resuspend the cells;

[0129] (4) Add 5 μL of FITC-Annexin V to each sample tube and react at room temperature in the dark for 15 min;

[0130] (5) Then add 10 μL of PI, mix gently, and react at 4°C in the dark for 5 min;

[0131] (6) After the reaction is completed, flow cytometry is used for analysis.

[0132] Example 9: Detection of ovarian granulosa cell activity

[0133] Granulocyte activity detection was performed according to the instructions of the Cell Counting Kit-8 from Biosharp (using a 96-well cell culture plate as an example):

[0134] (1) Aspirate the culture medium in the cell culture plate, rinse the cells with PBS, and discard the PBS.

[0135] (2) Dilute the CCK8 solution with complete culture medium to a final concentration of 10%;

[0136] (3) Add 100 μL of 10% CCK8 solution to each well and place the culture plate in an incubator for 1–4 h.

[0137] (4) Aspirate the CCK8 solution after incubation and measure the absorbance at 450 nm using a microplate reader.

[0138] Example 10: Ovarian granulosa cell cycle detection

[0139] Granulocyte activity assay was performed according to the instructions of Keygi's PI / RNase Staining Buffer:

[0140] (1) Place the cell culture plate at room temperature, gently rinse the cells in the culture plate with PBS, and discard the PBS;

[0141] (2) Add trypsin to digest the cells and place them in an incubator for about 3 minutes. Observe under a microscope until most cells float up. Immediately add an equal amount of stop solution (complete culture medium) to terminate the digestion.

[0142] (3) Collect the cells by centrifugation at 1000 × g for 5 min, discard the supernatant, and wash the cells twice with pre-chilled PBS;

[0143] (4) Add 0.5 mL of PI / RNase Staining Buffer to each tube of cell sample, slowly and thoroughly resuspend the cell pellet, and incubate at 37°C in the dark for 30 min;

[0144] (5) After the reaction is completed, flow cytometry is used for analysis.

[0145] Result Analysis

[0146] 1. The NCBI BLAST online tool predicted that there were a large number of reverse complementary sequences between the lncRNA IFA sequence and the mRNA sequence of ACTG1 (Gene ID: 397653) ( Figure 1 (a); At the same time, the RPIseq website predicted that lncRNA IFA may also bind to the ACTG1 protein ( Figure 1 (b)

[0147] 2. Pull-down assay showed that the expression of ACTG1 in the ChRIP probe group was significantly higher than that in the LacZ group (P<0.01) ( Figure 1 c); while the expression of ACTG1 protein was detected in both ChRIP probe group and LacZ group ( Figure 1 (d) The above experimental results indicate that lncRNA IFA directly binds to ACTG1 mRNA but not to ACTG1 protein.

[0148] The above-mentioned ChIRP probe was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.; the control group LacZ was from Guangzhou Ruibo Biotechnology Co., Ltd. (LacZ is the company's general negative control product).

[0149] 3. According to the diameter of the follicles, the follicles were divided into three categories: small (diameter ≤ 3mm), medium (3mm < diameter < 5mm), and large (diameter ≥ 3mm). Granulosa cells were extracted and RNA was reverse transcribed. qPCR and WB detected that the expression levels of lncRNA IFA and ACTG1 in the granulosa cells of small, medium, and large follicles showed a gradual increase ( Figure 2 (a)

[0150] 4. Granulosa cells were transfected with the overexpression vector of lncRNA IFA and siRNA (5'-TGACCTGGGCGACGTAGCA-3'), and RNA and protein were extracted 24 hours after transfection. qPCR detection showed that the expression of ACTG1 RNA increased significantly after overexpression of lncRNA IFA (P<0.05), while the expression of ACTG1 decreased significantly after interference with lncRNA IFA (P<0.05) ( Figure 2b); WB detected that the expression of ACTG1 protein increased significantly after overexpression of lncRNA IFA (P<0.05), while the expression of ACTG1 decreased significantly after interference with lncRNA IFA (P<0.05) ( Figure 2 (c) The above experimental results show that lncRNA IFA can promote the expression of ACTG1.

[0151] 5. The overexpression vector of ACTG1 and siRNA (5'-CCGACTACCTCATGAAGAT-3') were transfected into granulosa cells respectively. The activity of granulosa cells was detected 12h, 24h, 36h and 48h after transfection. The results showed that the activity of granulosa cells transfected with pcDNA3.1-ACTG1 was significantly increased compared with the control group (P<0.01) ( Figure 3 (a), while the activity of granulosa cells transfected with si-ACTG1 was significantly decreased (P<0.05) ( Figure 3 (B) This study confirmed that lncRNA IFA promotes the activity of ovarian granulosa cells.

[0152] 6. The overexpression vector and siRNA of ACTG1 were transfected into granulosa cells respectively. The proliferation rate of granulosa cells was detected 24 hours later. The results showed that the proliferation rate of granulosa cells transfected with pcDNA3.1-ACTG1 was significantly increased compared with the control group (P<0.05) ( Figure 3 a); while the proliferation rate of granulosa cells transfected with si-ACTG1 was significantly decreased (P<0.05) ( Figure 3 Middle b). This indicates that ACTG1 can promote the proliferation of ovarian granulosa cells.

[0153] 7. The overexpression vector and siRNA of ACTG1 were transfected into granulosa cells respectively. After 48 hours, the cells were collected and the apoptosis rate and cell cycle progression of ovarian granulosa cells were detected by flow cytometry. The results showed that compared with the control group, the apoptosis rate of granulosa cells transfected with pcDNA3.1-ACTG1 was significantly reduced (P<0.01), and the S phase cell rate was significantly increased (P<0.01) ( Figure 4 a); however, the apoptosis rate of granulosa cells transfected with si-ACTG1 increased significantly (P<0.05), and the rate of cells in the S phase decreased ( Figure 4 Middle b). This indicates that ACTG1 can inhibit the apoptosis of ovarian granulosa cells and accelerate the cell cycle process.

[0154] The above siRNA was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.; the control group NC was from Guangzhou Ruibo Biotechnology Co., Ltd. (NC is the company's general negative control product).

[0155] 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 should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Overexpression ACTG1 The invention is used for promoting the proliferation of pig ovarian granulosa cells, inhibiting the apoptosis of pig ovarian granulosa cells and / or improving the activity of pig ovarian granulosa cells in vitro, characterized in that: The ACTG1 The nucleotide sequence is shown in the sequence with accession number XM_003357928.4 at NCBI.

2. The use according to claim 1, characterized in that: The gene overexpression vector used in the overexpression is prepared by the following method: (1) RNA was extracted from pig ovarian granulosa cells, reverse transcribed into cDNA, and PCR amplified using cDNA as a template to obtain the target fragment; (2) The target fragment was ligated to the pcDNA3.1 vector digested with restriction endonucleases BamH I and Xba I to obtain a recombinant vector.

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