CN105567686B

CN105567686B - Application of miR-126-3p in porcine ovarian granular cells

Info

Publication number: CN105567686B
Application number: CN201610034858.9A
Authority: CN
Inventors: 李加琪; 张爱玲; 邓熙; 张哲�; 张豪
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2016-01-19
Filing date: 2016-01-19
Publication date: 2020-03-17
Anticipated expiration: 2036-01-19
Also published as: CN105567686A

Abstract

The invention discloses application of miR-126-3p in porcine ovarian granulosa cells, and belongs to the technical field of cell engineering and genetic engineering. The application of the miR-126-3p in the porcine ovarian granulosa cells is researched by using a cell biology method by taking the miR-126-3p as a research object. The application of miR-126-3p and PIK3R2 in the porcine ovarian granulosa cells and the targeting relationship between miR-126-3p and genes PIK3R2 and TSC1 in the porcine ovarian granulosa cells are proved for the first time; PIK3R2 and TSC1 supplemented with exogenous interference can revert to a cell functional phenotype caused by miR-126-3p, and further shows that PIK3R2 and TSC1 are important functional targets of miR-126-3p in granulosa cells, and miR-126-3p can regulate the development of granulosa cells through PIK3R2 and TSC 1.

Description

Application of miR-126-3p in porcine ovarian granular cells

Technical Field

The invention belongs to the technical field of cell engineering and genetic engineering, and particularly relates to application of miR-126-3p in swine ovarian granulosa cells.

Background

In commercial pork production, the utilization period of the sow is directly related to the economic benefit of a pig farm. Factors influencing the utilization age of the sows mainly comprise breeding obstacles, natural environment, varieties, nutrition, diseases and the like, wherein the breeding obstacles are one of the most main reasons for causing the sows to be prematurely eliminated from the swinery. Ovarian disease is an important cause in sow reproductive disorders.

The ovary is an important gonad of mammals and is an important reproductive organ for follicular development and ovulation. The follicle serves as the basic constitutional unit of the ovary, maintaining the presence, development and atresia of the ovum. Granulosa cells are flat or cuboidal cells surrounding the follicle, and their growth and differentiation play an important regulatory role in the development of the follicle. Only a few follicles in the female mammalian ovary are capable of developing to maturity and ovulating, while the vast majority of follicles undergo atretic degeneration at various stages of development, a significant and direct cause of apoptosis in granulosa cells. Apoptosis of granulosa cells is an important mechanism for initiating follicular atresia.

MicroRNAs (miRNAs) are endogenous non-coding RNAs with a regulating function, and are found in eukaryotes, and the size of the RNAs is about 20-25 nucleotides. It degrades target mRNA or inhibits normal translation of mRNA mainly by complete or incomplete pairing with the 3' untranslated region (UTR) of target gene mRNA, and further regulates expression of the gene after transcription. In recent years, the regulation of miRNA on mammalian ovarian development has also received attention from researchers, and many studies have shown that miRNA is widely involved in various biological processes such as oogenesis, follicular development, atresia, and luteal formation, degeneration. MiRNA is also involved in the regulation of several ovarian diseases, mainly ovarian cancer and premature ovarian failure.

In the earlier stage research, through a Solexa high-throughput sequencing technology, miRNA expression profiles of ovarian tissues of sows in different development stages (3-month-old young sows, 3-gestation-age sows and reproductive failure sows) are identified, wherein miR-126-3p is significantly differentially expressed, and is presumed to possibly play an important regulation and control role in granulosa cells.

In the current research, because the relation between miRNA and target gene is many-to-many, and one phenotype of animal changes, it is difficult to control by regulating and controlling one miRNA and its target gene, and it is difficult to carry out individual level verification of living pig basically; in addition, the cost of using pigs for live testing is too high without a high degree of confidence in successful tests.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the application of miR-126-3p in the porcine ovarian granulosa cells.

Changing the expression quantity of the miRNA in the granular cells through a genetic engineering technology, and further influencing the apoptosis of the miRNA on the granular cells; the miRNA target gene is predicted through bioinformatics software, and the function of the target gene is researched, so that the purpose of applying the miRNA in the swine ovary granular cells is achieved.

Another objective of the invention is to provide a target gene of the miR-126-3 p.

Still another object of the present invention is to provide a method for producing a target gene.

The purpose of the invention is realized by the following technical scheme:

the invention provides application of miR-126-3p in porcine ovarian granulosa cells.

The target genes of the miR-126-3p are PIK3R2 and TSC 1.

The target genes PIK3R2 and TSC1 are applied to the porcine ovarian granulosa cells.

After the exogenous interfering target genes PIK3R2 and TSC1 are supplemented in granular cells, the functional phenotype of the cells caused by miR-126-3p can be recovered.

The sequence of the miR-126-3p is 5'-UCGUACCGUGAGUAAUAAUGCG-3';

the verification results of the invention are as follows:

1. preparing miR-126-3p mimics (micic) and inhibitors (inhibitors) and detecting transfection efficiency. miR-126-3p micic and miR-126-3p inhibitor are transfected in granular cells, transfection efficiency is detected through a qRT-PCR method, after miR-126-3p mics with the concentration of 50nM and miR-126-3 pininhibitor with the concentration of 100nM are transfected respectively, compared with a control group NC transfected respectively, transfection efficiency of an experimental group can reach 2000 times of overexpression and inhibit 10 times of overexpression respectively.

2. After the miR-126-3p imic and the miR-126-3p inhibitor which reach the efficiency are transfected, the expression quantity of the marker gene and the change of the apoptosis of the granular cells are detected. The expression of the miR-126-3p mediated apoptosis marker gene is detected by a qRT-PCR means, and the over-expression of miR-126-3p (namely the miR-126-3p mimic is transfected) is found to inhibit the expression of the apoptosis-promoting marker gene Caspase-3 and promote the expression of the apoptosis-inhibiting marker gene BCL 2. And the influence of miR-126-3p on granular cell apoptosis is detected by an Annexin V-FITC method, and the result shows that the overexpression of miR-126-3p inhibits granular cell apoptosis, and the inhibition of miR-126-3p promotes granular cell apoptosis.

3. Bioinformatics software predicts the target gene of pig miR-126-3p, and experiments prove that the target genes are PIK3R2 and TSC 1. TargetScan, MiRanda and RNAhybrid 3 bioinformatics software predicts a miR-126-3p target gene, utilizes KOBAS 2.0 software to analyze the action pathway of the predicted target gene, finds that the target gene targets key PIK3R2 (phosphatase gene) and TSC1 (3-phosphoinositide dependent kinase) genes in a PI3K (phosphatidylinositol 3-kinase) -AKT (serine protein kinase) pathway, finds that miR-126-3p sequence binding sites are contained in PIK3R2 and TSC 13 'UTR, constructs PIK3R2 and TSC1 wild type 3' UTR and 3 'UTR containing seed sequence mutation into a eukaryotic expression vector pmiro, and finds that the wild type 3' upstream reporter genes of PIK3R2 and TSC1 are regulated by miR-126-3p and mutant genes are not regulated by miR-126-3p through GLirO analysis. And verifying that miR-126-3p regulates the expression of PIK3R2 and TSC1 at the transcription level and the translation level by adopting qRT-PCR and Western blotting, finding that miR-126-3p regulates the expression of PIK3R2 at the transcription and translation level, and only regulates the expression of TSC1 at the translation level.

4. Synthesis of 3 pairs of target genes PIK3R2 and TSC1 interference small fragment/control (siRNA-PIK3R2, siRNA-TSC1/siRNA-NC), screening and testing the interference efficiency. Transfecting the gene interference small fragment into a particle cell, and finally screening siRNA-PIK3R2-1 and siRNA-TSC1-3 small fragments with better interference effect by a qRT-PCR method to carry out subsequent experiments.

5. PIK3R2 and TSC1 interference small fragments (siRNA-PIK3R2-1 and siRNA-TSC1-3) transfect granular cells, and research application of target genes PIK3R2 and TSC1 to pig ovarian granular cell apoptosis. Particle cell apoptosis of PIK3R2 and TSC1 after being interfered by small fragments of siRNA-PIK3R2-1 and siRNA-TSC1-3 is detected by an Annexin V-FITC method, and PIK3R2 and TSC1 are found to promote particle cell apoptosis.

6. And the miR-126-3p target genes PIK3R2 and TSC1 function reply verification. Whether the cell function phenotype caused by miR-126-3p can be recovered after the exogenous interfering target genes PIK3R2 and TSC1 are supplemented in the granular cells. The granular cells are co-transfected with miR-126-3p inhibitor/NC and PIK3R2 and TSC1 interference small fragments (siRNA-PIK3R2-1 and siRNA-TSC1-3)/siRNA-NC, the apoptosis condition of the granular cells is detected by an Annexin V-FITC method, and the function of inhibiting the granular cell apoptosis caused by miR-126-3p can be recovered by PIK3R2 and TSC 1.

The application of the miR-126-3p in the porcine ovarian granulosa cells is researched by using a cell biology method by taking the miR-126-3p as a research object. Key points and points to be protected: (1) the application of miR-126-3p in pig ovarian granule cell apoptosis; (2) miR-126-3p is in targeting relation with genes PIK3R2 and TSC1 in the porcine ovarian granulosa cells; (3) the application of the genes PIK3R2 and TSC1 in porcine ovarian granulosa cell apoptosis; (4) whether the cell functional phenotype caused by miR-126-3p can be recovered or not is verified by a target gene recovery experiment, namely after exogenous interfering target genes PIK3R2 and TSC1 are supplemented in the granular cells.

The invention proves the application of miR-126-3p and PIK3R2 in porcine ovarian granulosa cells and the targeting relationship between miR-126-3p and genes PIK3R2 and TSC1 in the porcine ovarian granulosa cells for the first time; the existing invention has only a few reports about target gene function reversion experiments, and the reason is mainly that the change of one phenotype is influenced by multiple aspects, and the relationship between miRNA and target gene is many-to-many, namely one miRNA can target multiple genes, and one gene can also be the target of multiple miRNA, so the reversion experiments of target gene are difficult to obtain the result, however, PIK3R2 and TSC1 supplemented with exogenous interference can revert the cell function phenotype caused by miR-126-3p, further indicating that PIK3R2 and TSC1 are important functional targets of miR-126-3p in granulosa cells, and miR-126-3p can regulate the development of granulosa cells through PIK3R2 and TSC 1.

Compared with the prior art, the invention has the following advantages and effects:

the invention has detailed design and reliable result. In order to prove the application of miR-126-3p in granular cells, the invention not only researches the functions of the miR-126-3p, but also researches the functions of target genes and the target genes in the granular cells; the invention also verifies whether the cell functional phenotype caused by miR-126-3p can be recovered through a target gene recovery experiment, namely after exogenous interfering target genes PIK3R2 and TSC1 are supplemented in the granular cells.

Drawings

FIG. 1 is a graph of results of qRT-PCR detection of miR-126-3p mimic and miR-126-3p inhibitor transfection efficiency.

FIG. 2 is a graph showing the results of qRT-PCR detection of expression levels of miR-126-3 p-mediated apoptosis marker genes BCL2 and Caspase-3; FIGS. 2A and 2B show the expression level of BCL 2; FIGS. 2C and 2D show the expression level of Caspase-3.

FIG. 3 is a graph showing the results of the Annexin V-FITC method for detecting the apoptosis of miR-126-3p regulatory particles.

FIG. 4 is a graph demonstrating that miR-126-3p targets PIK3R 2; wherein, FIG. 4A, FIG. 4B and FIG. 4C respectively show dual fluorescence reporter gene validation, qRT-PCR and Western blotting validation.

FIG. 5 is a graph demonstrating that miR-126-3p targets TSC 1; wherein, FIG. 5A, FIG. 5B and FIG. 5C respectively show dual fluorescence reporter gene validation, qRT-PCR and Western blotting validation.

FIG. 6 is a small fragment interference efficiency of qRT-PCR assay 3 on PIK3R2 and TSC 1; wherein, fig. 6A shows PIK3R2 interference small fragment interference efficiency; fig. 6B shows TSC1 small-segment interference efficiency.

FIG. 7 is PIK3R2 and TSC1 interfering with small fragment regulation of granulosa apoptosis; FIG. 7A is the result of PIK3R2 interference small fragment regulation; fig. 7B shows the TSC1 interference small fragment regulation results.

FIG. 8 is a result of PIK3R2 reverting miR-126-3p to inhibit granulosa cell apoptosis.

FIG. 9 is the result of TSC1 reverting miR-126-3p to inhibit granular cell apoptosis.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The experimental methods in the following examples, which are not specified under specific conditions, are generally performed under conventional conditions.

EXAMPLE 1 culture of ovarian granulosa cells

(1) Collecting ovaries in a slaughterhouse, placing the ovaries in PBS or normal saline (containing 1% double antibody) in a vacuum flask at 37 ℃, and quickly transporting the ovaries back to a laboratory;

(2) cleaning the collected ovaries for 3 times in a sterile culture room by using preheated PBS (containing 1% double antibody), and quickly transferring the ovaries to a superclean bench; the ovarian follicle fluid is absorbed by a 1mL sterile disposable syringe which is inserted into the ovarian follicle with a cavity in a shallow way;

(3) placing the follicular fluid into a 15mL centrifuge tube containing a proper amount of DMEM, and centrifuging at 1000rpm at room temperature for 6 min;

(4) discarding the supernatant, then resuspending and centrifuging by DMEM, and repeatedly cleaning the cells for 2 times; preparing a DMEM complete culture medium: 89% DMEM + 10% FBS + 1% double antibody;

(5) sucking the cell resuspension and complete culture medium and inoculating the cell resuspension and complete culture medium in a 75mL culture bottle; standing at 37 deg.C for 5% CO₂And (5) standing and culturing in an incubator.

The double-resistant is penicillin and streptomycin.

Example 2 inoculation and transfection of ovarian granulosa cells

(1) Growing the granular cells to about 90%, pouring out the culture medium, and washing for 3 times by using preheated PBS containing 1% double antibodies (the double antibodies are penicillin and streptomycin);

(2) adding trypsin for digestion, placing in an incubator for about 3min, observing under a microscope until most cells float, and immediately adding equivalent stop solution to stop digestion;

(3) washing with DMEM for 2 times, and centrifuging at 1000rpm for 5 min;

(4) lightly resuspending the cell sediment with complete culture medium, uniformly distributing the cell sediment into each hole, supplementing the volume with complete culture medium, lightly shaking up, and culturing in an incubator;

(5) observing the granular cell state for about 24h, and preparing to convert when the cell confluence reaches about 80%

Dyeing;

(6) transfection method Invitrogen

3000 kit Instructions

A row; each set was set to 3 replicates;

(7) the transfected well plates were placed at 37 ℃ in 5% CO₂Culturing in an incubator;

(8) and (5) observing the cell state 1-3 days after transfection, and collecting the cells after the cells grow well.

Example 3qRT-PCR

The qRT-PCR detection of the gene and miRNA in the invention respectively adopts SYBR Premix ExTaq Kit and SYBR PrimeScript miRNA RT-PCR Kit of TaKaRa company. The experiment adopts a Ct value comparison method to detect the content of miRNA or gene in a sample, and the specific calculation formula is as follows:

relative gene expression level of 2^{- { (lead) desired gene Ct value in experimental group-lead (reference gene Ct value in experimental group) < - > (lead) desired gene Ct value in control group- < - >}

The internal reference gene is U6 for miRNA detection and GAPDH for gene detection, and the qRT-PCR primers used in the invention are as follows:

qRT-PCR-BCL2 Forward：5′-GAAACCCCTAGTGCCATCAA-3′；

Reverse：5′-GGGACGTCAGGTCACTGAAT-3′；

qRT-PCR-Caspase3 Forward：5′-GACTGTGGGATTGAGACG-3′；

Reverse：5′-ACCCGAGTAAGAATGTGC-3′；

qRT-PCR-PIK3R2 Forward：5′-GGCAAGATCAACCGCACACAAG-3′；

Reverse：5′-CACCACCACAGAGCAGGCAT-3′；

qRT-PCR-TSC1 Forward：5′-GACCCATATCTATGCGGACCC-3′；

Reverse：5′-TGCTGGTACTGAAGCGGTTG-3′；

qRT-PCR-GAPDH Forward：5′-ACGCCTGCCCTGTGTCCCAA-3′；

Reverse：5′-GAAGCACGCCCTCTCGCCTC-3′；

qRT-PCR-U6 Forward：5′-CTCGCTTCGGCAGCACA-3′；

Reverse：5′-AACGCTTCACGAATTTGCGT-3′；

total RNA extraction of cells was performed according to the instructions of TRIzol of Takara, and the specific extraction procedure was as follows:

(1) adding the granular cells into TRIzol directly;

(2) standing at room temperature for 10min to fully lyse cells, centrifuging at 12000g for 5min, discarding the precipitate, and taking the supernatant in a new RNase-free tube;

(3) adding 0.2mL of chloroform (1 mL of TRIzol) and shaking vigorously for 15-30 s, standing at room temperature for 5min, and centrifuging at 4 ℃ and 12000g for 15 min;

(4) absorbing the upper aqueous phase and placing the upper aqueous phase in a new RNase-free EP tube;

(5) adding 0.5mL of isopropanol (per 1mL of TRIzol), gently inverting and mixing, standing at room temperature for 10min, and centrifuging at 4 ℃ at 12000g for 10 min;

(6) discarding the supernatant, placing at room temperature, adding 1mL 75% ethanol-DEPC (per 1mL TRIzol) along the tube wall to wash RNA, centrifuging at 4 ℃ at 12000g for 5min, and discarding the supernatant as much as possible;

(7) vacuum drying for 5-10 min, and taking care to avoid excessive drying of RNA precipitate;

(8) DEPC water was added to dissolve the RNA pellet.

mRNA and miRNA reverse transcription PCR were performed using Thermo Scientific RevertaIdFirst Strand cDNA Synthesis Kit from Thermo company and Reverta Ace qPCR RT Kit from TOYOBO company, respectively.

Example 4 Western Blotting

(1) Extraction of total protein from adherent monolayer cells: the cell culture was decanted and the cells were washed three times with an appropriate amount of pre-cooled PBS to wash out the culture. After discarding the PBS, the flasks were placed on ice. And adding 100-200 mu L of protein lysate and 10 mu L of 100mM PMSF into each well of 6-well plate cells, and lysing the cells for 30 min. The cell lysate was collected and transferred to a 1.5mL centrifuge tube and centrifuged at 14000rpm at 4 ℃ for 5 min. And adding part of the supernatant into the sample buffer, and boiling for 10 min. Slowly recovering to room temperature, centrifuging, and storing at-20 deg.C;

(2) SDS-PAGE electrophoresis: after initial quantification of protein samples by BCA method, 20. mu.g of total protein per group was mixed with 5 Xloading buffer at a ratio of 5:1 and boiled for 5 min. Performing SDS-PAGE electrophoresis until the bromophenol blue just comes out of the gel bottom;

(3) protein transfer: and (3) pretreating the PVDF membrane for 3-5 s by using methanol, and soaking the PVDF membrane in a transfer printing liquid for 30 min. Taking out the gel, and placing the gel on filter paper to form a sandwich structure of a gel transfer printing accumulation layer. This operation must remove the bubbles completely. Constant pressure of 100V for 60-120 min;

(4) immunoblotting: the hybridization membrane was removed, rinsed for 5min in TBST and repeated three times. 5% skimmed milk powder solution was blocked at room temperature for 90min, and rinsed with TBST for 5min, repeated three times. PIK3R2 and TSC1 primary antibodies (PIK3R2 primary antibody: PIK3R2/p85 Beta, available from LSBio Inc.; TSC1 primary antibody: Hamatin, available from biorbyt Inc.) (1:2000) were added and incubated overnight at 4 ℃ and membranes washed with TBST for 5min three times. Adding a second antibody diluent (a second antibody: horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) purchased from Biyuntian corporation (1:10000), incubating at room temperature for 1H, washing the membrane with TBST for 5min, and carrying out three times. The membrane was rinsed with distilled water for 2min, three times. Wiping off the liquid on the PVDF film, adding a chemiluminescent agent, placing on a film, placing in an X-ray cassette, and placing in a dark room. Opening the film box in the darkroom, putting in the film, opening the film box after 5min, and taking out the film. Protein bands in the films were analyzed using Image Plus software.

Example 5 granular cell apoptosis assay

The Annexin V-FITC technology for detecting the Apoptosis refers to the Annexin V-FITC Apoptosis Detection Kit operating instruction of BioVision, and comprises the following specific operating steps:

(1) placing the cell culture plate at room temperature, slightly rinsing cells in the culture plate by using 2mL of PBS solution, and removing the PBS solution;

(2) adding pancreatin without EDTA to digest the cells, and gently resuspending the cells in the medium of step (1) to a density of about 1X 10⁶cell/mL;

(3) 0.5mL of cell suspension was removed from the cell culture plate (approximately 5X 10)⁵Individual cells) were transferred into a clean centrifuge tube and 500 μ L of 1 × Binding Buffer was added;

(4) adding 5 μ L Annexin V-FITC and 5 μ L propidium iodide at room temperature;

(5) reacting at room temperature in dark for 5 min;

(6) analysis was immediately performed using a FACSCalibur flow cytometer (triplicates per group).

Example 6 luciferase reporter Activity assays

The Luciferase reporter gene activity detection refers to a Dual-Luciferase reporter assay System kit of Promega company, and the specific operation steps are as follows:

(1) after transfection for 48h, the old medium was aspirated off, washed twice with PBS, 100. mu.L of GloLysis Buffer was added to each well of cells, shaken slightly at room temperature for 5min, and cell lysates were collected;

(2) after 30. mu.L of cell lysate was added to a 96-well plate, 75. mu.L of Dual-

And (3) uniformly mixing the Luciferase Assay Reagent, and standing for 15-30 min at 20-25 ℃. Detecting a luminous value on a multifunctional microplate reader of Synergy 2 of BioTek company, wherein the luminous value corresponds to the expression level of the firefly luciferase;

(3) then 75. mu.L of Stop was added&

And (3) uniformly mixing the Reagent, and standing for 15-30 min at 20-25 ℃. Detecting a luminescence value corresponding to the level of renilla luciferase expression;

(4) the ratio of the expression amounts of the firefly luciferase and the Renilla luciferase is the relative activity of the firefly luciferase, which is the activity of the corresponding target gene (three repeats).

And (4) analyzing results:

1. preparing miR-126-3p mimics (micic) and inhibitors (inhibitors) and detecting transfection efficiency. miR-126-3p micic and miR-126-3p inhibitor are transfected in granular cells, transfection efficiency is detected through a qRT-PCR method, after miR-126-3p mics with the concentration of 50nM and miR-126-3 pininhibitor with the concentration of 100nM are transfected respectively, compared with a control group NC transfected respectively, transfection efficiency of an experimental group can reach 2000 times of overexpression and inhibit 10 times of overexpression respectively. The results are shown in FIG. 1.

2. After the miR-126-3p imic and the miR-126-3p inhibitor with the efficiency are transfected, the expression quantity of the marker gene and the change of the apoptosis of the granular cells are detected. The expression of the miR-126-3p mediated apoptosis marker gene is detected by a qRT-PCR means, and the over-expression miR-126-3p (namely the miR-126-3p mimic is transfected) is found to inhibit the expression of the apoptosis-promoting marker gene Caspase-3 and promote the expression of the apoptosis-inhibiting marker gene BCL 2; the results are shown in FIG. 2. The influence of miR-126-3p on granular cell apoptosis is detected by an annexin V-FITC method, and the result shows that the overexpression of miR-126-3p inhibits granular cell apoptosis, and the inhibition of miR-126-3p promotes granular cell apoptosis; the results are shown in FIG. 3.

3. Bioinformatics software predicts the target gene of porcine miR-126-3p, and experiments prove that the target genes are PIK3R2 and TSC 1. TargetScan, MiRanda and RNAhybrid 3 bioinformatics software predict miR-126-3p target genes, KOBAS 2.0 software is used, analyzing action pathways of predicted target genes, finding that the target genes target key PIK3R2 (phosphatase gene) and TSC1 (3-phosphoinositide dependent kinase) genes in a PI3K (phosphatidylinositol 3-kinase) -AKT (serine protein kinase) pathway, finding that miR-126-3p sequence binding sites are contained in PIK3R2 and TSC 13 ' UTR, constructing PIK3R2 and TSC1 wild type 3 ' UTR and 3 ' UTR containing seed sequence mutation into a eukaryotic expression vector pmirGLO, through dual-luciferase reporter analysis, it was found that the wild-type (PIK3R2-WT and TSC1-WT) 3' UTR upstream reporter gene of PIK3R2 and TSC1 was regulated by miR-126-3p, whereas the mutant (PIK3R2-MUT and TSC1-MUT) was not regulated by miR-126-3 p. And verifying that miR-126-3p regulates the expression of PIK3R2 and TSC1 at the transcription level and the translation level by adopting qRT-PCR and Western blotting, finding that miR-126-3p regulates the expression of PIK3R2 at the transcription and translation level, and only regulates the expression of TSC1 at the translation level. The results are shown in FIGS. 4 and 5.

4. Synthesis of 3 pairs of target genes PIK3R2 and TSC1 interference small fragment/control (siRNA-PIK3R2, siRNA-TSC1/siRNA-NC), screening and testing the interference efficiency. The results are shown in FIG. 6. Transfecting the gene interference small fragment into a particle cell, and finally screening siRNA-PIK3R2-1 and siRNA-TSC1-3 small fragments with better interference effect by a qRT-PCR method to carry out subsequent experiments.

siRNA-PIK3R2-1：5′-GGAGAAGUUACUUCAGGAA-3′；

siRNA-PIK3R2-2：5′-GGAACAACAAGCUGAUCAA-3′；

siRNA-PIK3R2-3：5′-GGUAUGUAGGCAAGAUCAA-3′；

siRNA-TSC1-1：5′-GCUGGAGAUCUUAGAUUUA-3′；

siRNA-TSC1-2：5′-GCUUCGACUCUCCCUUCUA-3′；

siRNA-TSC1-3：5′-CCACGUGACAGAAAUCUAU-3′；

The small interference fragments are synthesized by Ribo Biotech, Inc., Guangzhou; control siRNA-NC was from Ribo Biotech, Inc., Guangzhou.

5. PIK3R2 and TSC1 interference small fragments (siRNA-PIK3R2-1 and siRNA-TSC1-3) transfect granular cells, and research application of target genes PIK3R2 and TSC1 to pig ovarian granular cell apoptosis. The Annexin V-FITC method is used for detecting the apoptosis of the granular cells after PIK3R2 and TSC1 are interfered by small fragments of siRNA-PIK3R2-1 and siRNA-TSC1-3, and PIK3R2 and TSC1 are found to promote the apoptosis of the granular cells. The results are shown in FIG. 7.

6. And the miR-126-3p target genes PIK3R2 and TSC1 function reply verification. After the exogenous target genes PIK3R2 and TSC1 are supplemented in the granular cells, whether the cell function phenotype caused by miR-126-3p can be recovered or not is judged, the granular cells are co-transfected with miR-126-3p inhibitor/NC, PIK3R2 and TSC1 interference small fragments (siRNA-PIK3R2-1 and siRNA-TSC1-3)/siRNA-NC, and the granular cell apoptosis condition is detected by an Annexin V-FITC method, and the function of inhibiting granular cell apoptosis caused by miR-126-3p can be recovered by PIK3R2 and TSC 1. The results are shown in FIGS. 8 and 9.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

The application of miR-126-3p in the porcine ovarian granulosa cells is characterized in that: in an in vitro environment, the over-expression of miR-126-3p inhibits apoptosis of ovarian granular cells, and the inhibition of miR-126-3p promotes apoptosis of granular cells;

the over-expression miR-126-3p inhibits the expression of a apoptosis-promoting marker gene Caspase-3 and promotes the expression of an apoptosis-inhibiting marker gene BCL 2;

the sequence of the miR-126-3p is 5'-UCGUACCGUGAGUAAUAAUGCG-3'.
2. The use of miR-126-3p in porcine ovarian granulosa cells of claim 1, wherein: the target genes of the miR-126-3p are PIK3R2 and TSC 1.
3. The use of miR-126-3p in porcine ovarian granulosa cells of claim 2, wherein: the expression of the miR-126-3p is reduced under the transcription and translation level, the expression of PIK3R2 is reduced under the translation level, and the expression of TSC1 is reduced under the translation level.
4. The use of miR-126-3p in porcine ovarian granulosa cells according to claim 3, wherein: the functional phenotype for promoting the apoptosis of the granular cells caused by the inhibition of miR-126-3p can be recovered after the granular cells are supplemented with siRNA-PIK3R2-1 or siRNA-TSC 1-3;

siRNA-PIK3R2-1：5′-GGAGAAGUUACUUCAGGAA-3′；

siRNA-TSC1-3：5′-CCACGUGACAGAAAUCUAU-3′。