CN114874993A

CN114874993A - Method for regulating and controlling pig ovarian granulosa cell MMP2 gene expression

Info

Publication number: CN114874993A
Application number: CN202210599960.9A
Authority: CN
Inventors: 袁晓龙; 李泳光; 李加琪; 吕媛媛; 方明; 张哲�; 张豪
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-05-30
Filing date: 2022-05-30
Publication date: 2022-08-09
Anticipated expiration: 2042-05-30
Also published as: CN114874993B

Abstract

The invention discloses a method for regulating and controlling the expression of MMP2 gene of a porcine ovary granulosa cell, belonging to the technical field of cell engineering and gene engineering. The invention takes MMP2 gene and transcription factor ESR1 as research objects, constructs an overexpression vector of the transcription factor ESR1 and designs an interference fragment thereof by utilizing technologies of molecular biology, cell biology and the like, and researches the influence of the transcription factor ESR1 on MMP2 gene expression. The technical scheme of the invention is thoroughly designed and has reliable results. The transcription factor ESR1 can be combined with the promoter (-1317-1274 bp) region of the MMP2 gene, and can inhibit the expression of the MMP2 gene. By researching the application of the transcription factor ESR1 to the MMP2 gene transcription expression mechanism, the method has good application value for researching the molecular mechanism that the MMP2 gene influences ovarian follicle atresia, sow initial condition starting and the like.

Description

Method for regulating and controlling pig ovarian granulosa cell MMP2 gene expression

Technical Field

The invention belongs to the technical field of cell engineering and genetic engineering, and particularly relates to a method for regulating and controlling the expression of pig ovarian granulosa cell MMP2 gene.

Background

Gene expression refers to the process by which a gene converts the genetic information contained in the gene into RNA, polypeptides and proteins by transcription and translation. A plurality of regulatory elements exist in a 5' untranslated region of the gene, including an internal ribosome entry site, a 5' UTR secondary structure, a G-tetramer, a 5' cap structure, an upstream initiation codon ATG, a 5' UTR intron and the like, and the 5' UTR plays a certain role in maintaining the stability of mRNA, intranuclear transportation, RNA splicing, cell proliferation and the like in the regulation of gene expression.

Matrix metalloproteinase 2 (MMP 2) is one of important members of MMPs family, and can decompose gelatin, fibrin and type IV collagen in follicular fluid, and affect proliferation and apoptosis of granulosa cells. In atretic follicles, the expression level of MMP2 gene mRNA is significantly higher than in normal follicles.

Disclosure of Invention

In order to solve the related problems, the invention mainly aims to provide a method for regulating and controlling the expression of the MMP2 gene of the porcine ovarian granulosa cells.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for regulating and controlling MMP2 gene expression of a swine ovary granular cell adopts a transcription factor ESR1 as a transcription factor of an MMP2 gene promoter region, and realizes regulation and control of MMP2 gene transcription activity in the swine ovary granular cell by regulating the expression level of the transcription factor ESR 1.

Furthermore, the transcription factor ESR1 inhibits the expression of the transcription factor ESR1 by combining with a promoter-1317-1274 bp region of an MMP2 gene; increasing an exogenous transcription factor ESR1, and reducing the transcriptional activity of MMP2 gene; the expression of a transcription factor ESR1 is inhibited, and the transcriptional activity of an MMP2 gene is increased.

Furthermore, the increase of the exogenous transcription factor ESR1 is realized by a gene overexpression technology, and the adopted gene overexpression vector is prepared by the following method: (1) extracting RNA of the pig ovarian granulosa cells, carrying out reverse transcription on the RNA to form cDNA, and carrying out PCR amplification by taking the cDNA as a template to obtain a target fragment; (2) the objective fragment was ligated to pcDNA3.1 vector digested with restriction enzymes EcoRI and XbaI to obtain a recombinant vector.

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

note: the underlined part is the protecting base and the italicized part is the cleavage site.

Furthermore, the inhibition of the expression of the transcription factor ESR1 is realized by an RNA interference technology, and the adopted siRNA has the following sequence:

siRNA-ESR1-2：5′-GGATTTAAGCCTCCATGAT-3′。

the core promoter function regulating region of the ovarian granulosa cell MMP2 gene is a region of MMP2 gene promoter-1317-1274 bp.

A nucleic acid segment for regulating and controlling the expression of an MMP2 gene of an ovarian granulosa cell is siRNA for inhibiting the expression of a transcription factor ESR1, and the siRNA has the following sequence:

siRNA-ESR1-2：5′-GGATTTAAGCCTCCATGAT-3′。

the application of the core promoter function regulating region or the nucleic acid fragment in regulating and controlling the expression of the MMP2 gene of the ovarian granulosa cell.

In the early stage of the invention, a binding site of an Estrogen receptor 1 (ESR 1) exists in an MMP2 promoter region predicted by a bioinformatics website. ESR1 is one of important members of transcription factor nuclear receptor family, ESR1 is in inactive state under the condition of no hormone, ESR1 becomes homodimer after being combined with estrogen, then the homodimer interacts with a regulatory element combined with a gene promoter to regulate the gene transcription level, and the developmental maturation of the reproductive system of a mammal can be regulated by receiving and transmitting signals emitted by E2 and regulating the expression of key genes of an E2 signal channel. In conclusion, the transcription factor ESR1 may bind to MMP2 promoter region and regulate the expression of MMP2, thereby affecting the function of granulosa cells.

The verification results of the invention are as follows:

1. the potential binding site of the transcription factor ESR1 in the promoter region (-2142/+1, +1 is the transcription initiation site) of the MMP2 gene is predicted by a bioinformatics website, and 6 binding sites of ESR1 exist in the promoter region of the MMP2 gene and are respectively positioned as follows: -2078/-2071bp, -2060/-2053bp, -1317/-1310bp, -1274/-1267bp, -406/-399bp, -392/-385bp (FIG. 1).

2. According to the binding site of a transcription factor ESR1 in a promoter region of an MMP2 gene, recombinant plasmids (P1-P4) of an MMP2 gene promoter deletion fragment are constructed, after the recombinant plasmids are transfected into granular cells, a multifunctional enzyme labeling instrument is used for detecting the relative activity of the MMP2 gene promoter deletion fragment, and the activity of an MMP2 gene promoter deletion fragment carrier P3 is found to be remarkably higher than that of P2(P <0.001), while the activity of a recombinant carrier P4 is remarkably lower than that of P3(P <0.001) (figure 1).

3. Primers were designed to amplify the CDS region of the transcription factor ESR 1: searching the sequence of a transcription factor ESR1(NCBI Gene ID:397435) from NCBI, determining enzyme cutting sites (the enzyme cutting site of ESR1 is EcoRI and XbaI), and designing a specific primer by using an NCBI website; the target fragment is specifically amplified, purified and enzyme-cut by PCR, then is connected to a pcDNA3.1 expression vector, and finally the overexpression vector pcDNA3.1-ESR1 of ESR1 is successfully constructed. The expression level of ESR1 mRNA was detected by qRT-PCR after transfecting overexpression vectors with different concentrations (100, 400 and 800ng) in granular cells, and it was found that the higher the concentration of the overexpression vector transfected with pcDNA3.1-ESR1, the higher the expression level of ESR1 mRNA, and the difference in both were significant. When 400ng of ESR1 overexpression vector is transfected in granular cells and the expression level of ESR1 protein is detected by using Western Blot, the expression level of ESR1 protein is remarkably increased after the ESR1 overexpression vector is transfected compared with a control group (figure 2). The constructed ESR1 overexpression vector is proved to be normally expressed in granular cells, and the transfection concentration of 400ng pcDNA3.1-ESR1 is selected in subsequent research.

4. 3 pairs of small interfering RNA/control (ESR1-siRNA/siRNA-NC) interfering with ESR1 were synthesized, and subsequently different concentrations (30nmol, 50nmol, and 100nmol) of small interfering RNA were transfected into granulosa cells, and their interference efficiency was examined by qRT-PCR. As shown in the figure, the ESR1-siRNA-2 small interfering RNA has the best interference effect, then 50nmol ESR1-siRNA-2 is transfected in granular cells, the expression level of ESR1 protein is detected by Western Blot, and the expression level of ESR1 protein is remarkably reduced after ESR1-siRNA-2 small interfering RNA is transfected compared with a control group (figure 3). Proved that the synthesized small interfering RNA can interfere the expression of ESR1 in granular cells with high efficiency, and finally 50nmol ESR1-siRNA-2 small interfering RNA is selected for subsequent experiments.

siRNA-ESR1-1：5′-GCCTTCTTCAAGAGAAGTA-3′；

siRNA-ESR1-2：5′-GGATTTAAGCCTCCATGAT-3′；

siRNA-ESR1-3：5′-GCATTCCTTGCAAATGTAT-3′。

5. pcDNA3.1-ESR1 or ESR1-siRNA-2 is transfected into granular cells respectively, expression levels of mRNA and protein of MMP2 genes are detected by qRT-PCR and Western Blot respectively, overexpression of ESR1 remarkably reduces the expression level of mRNA and protein of MMP2 genes (P <0.01), and interference of ESR1 remarkably increases the expression level of mRNA and protein of MMP2 genes (P <0.01) (figure 4).

6. To further demonstrate that ESR1 can inhibit MMP2 gene expression, we treated granulosa cells with estrogen (E2) and an E2 antagonist and examined the effect of E2 and E2 antagonists on MMP2 gene mRNA expression levels by qRT-PCR. The results showed that E2 significantly reduced MMP2 gene mRNA expression levels (P <0.05), while E2 antagonists significantly increased MMP2 gene mRNA expression levels (P <0.05), indicating that E2 can inhibit MMP2 gene transcription, while E2 antagonists can promote MMP2 gene transcription (fig. 5).

7. To verify whether the transcription factor ESR1 binds to the promoter region P2-P3(-1594/-718bp) region of the MMP2 gene, the study examined whether the transcription factor ESR1 binds to the promoter region sequence of the MMP2 gene by ChIP assay. The results show that the experimental group (Anti-ESR1) and the positive control (Anti-polyII) lanes of the MMP2 gene promoter (-1317 to-1274 bp) region have obvious single bands, while the negative control (Anti-IgG) lanes have no obvious band, which indicates that the transcription factor ESR1 can be combined with the MMP2 gene promoter (-1317 to-1274 bp) region (FIG. 6).

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

the technical scheme of the invention is thoroughly designed and has reliable results. The MMP2 gene and the transcription factor ESR1 directly or indirectly participate in growth and differentiation, follicular development and primordial situation starting of granulosa cells, and the MMP2 gene and the transcription factor ESR1 are used as research objects in the research, and the influence of the transcription factor ESR1 on the MMP2 gene expression is researched by adopting molecular and cell biology technologies. To demonstrate that the transcription factor ESR1 inhibits the expression of MMP2 gene, the invention was validated from multiple levels, multiple angles, first at the mRNA level and protein level, then further validated by drug treatment, and finally validated for specific binding sites by ChIP. The gene has good application value in researching the expression regulation mechanism of the MMP2 gene in ovarian granulosa cells.

Drawings

FIG. 1 is a graph showing the results of relative activity measurement of the binding site of the transcription factor ESR1 in the promoter region of MMP2 gene and a promoter deletion fragment; wherein, a is the binding site of a transcription factor ESR1 in a promoter region of an MMP2 gene, and b is the relative activity of a promoter deletion fragment of the MMP2 gene;

FIG. 2 is a graph showing the efficiency test of the overexpression vector pcDNA3.1-ESR 1; wherein, a is the efficiency detection of the overexpression vector pcDNA3.1-ESR1 at the mRNA level, and b is the efficiency detection of the overexpression vector pcDNA3.1-ESR1 at the protein level;

FIG. 3 is a graph of the efficiency detection of ESR1 small interfering RNA; wherein, ESR1 small interfering RNA efficiency test at mRNA level, b is ESR1 small interfering RNA efficiency test at protein level;

FIG. 4 is a graph of the results of the effects of over-expression or interference of ESR1 on MMP2 gene expression; wherein, a is the effect of over-expressing ESR1 on MMP2 gene mRNA level, b is the effect of over-expressing ESR1 on MMP2 gene protein level, c is the effect of interfering ESR1 on MMP2 gene mRNA level, and d is the effect of interfering ESR1 on MMP2 gene protein level;

FIG. 5 is a graph of the results of the effects of E2 and E2 antagonists on MMP2 gene expression; wherein a is the effect of E2 on MMP2 gene expression and b is the effect of E2 antagonist on MMP2 gene expression;

FIG. 6 shows the results of ChIP verification of the binding of the transcription factor ESR1 to the promoter region (-1317 to-1274 bp) of MMP2 gene.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The experimental methods in the following examples, which are not specified under specific conditions, are generally performed under conventional conditions.

In the present invention, the results of 3 independent experiments in each example were analyzed by a statistical method, and the "mean ± standard deviation" was calculated, and the analysis of significance of the difference was performed by a one-way variance analysis (in the figure, "+" indicates P <0.05, and "+" indicates P < 0.01).

Example 1: MMP2 gene promoter deletion fragment vector construction and double fluorescence activity analysis

(1) Using NCBI website to design primers, and using extracted DNA of porcine granulosa cells as a template to amplify a promoter region P1(-2142 to +191), P2(-1594 to +191), P3(-718 to +191) and P4(-323 to +191) of MMP2 gene; the amplified fragment was purified, recovered, ligated with pMD18T vector (purchased from Takara), transformed, screened, sequenced and identified as correct, and then the ordinary plasmid was extracted. The BioEdit software analyzes the distribution of enzyme cutting sites in the promoter region of the MMP2 gene, and finally selects two restriction enzymes KpnI and BglII as enzyme cutting sites. The restriction site sequences of KpnI and BglII enzymes are added to the upstream and downstream primers respectively. Then, taking each recombinant pMD18T common plasmid as a template, and carrying out PCR amplification by using each enzyme digestion primer; the fragment is purified and recovered, subjected to double digestion, connected with pGL3-basic vector, transformed, screened, sequenced and identified to be correct, and then endotoxin-free plasmid (endotoxin-free plasmid miniprep kit is purchased from America magenta), wherein pGL3-basic is used as a negative control, and pGL3-control is used as a positive control.

The MMP2 gene promoter deletion fragment primer used in the invention comprises the following components:

(2) Luciferase reporter gene activity assay, referred to the dual luciferase assay kit instructions of Shanghai assist san Francisco Biotech Co., Ltd., comprises the following steps: after 24h of transfection, the medium was removed, washed 2 times with PBS, 80. mu.L of cell lysate was added, the plate was gently rotated to cover the cells completely with lysate, and incubated on ice for 5 min; centrifuging at 4 ℃ and 13000rpm for 1min, and taking the supernatant into a new 1.5mL sterilized centrifuge tube; adding 25 μ L of the cell lysate to a 96-well culture plate, and repeating 6 wells per group; adding 100 μ L firefly luciferase reaction solution, standing for 5min, and detecting the luminescence value of firefly luciferase; adding 100 μ L of renilla luciferase reaction solution, standing for 5min, and detecting the luminescent value of renilla luciferase; and (3) calculating: relative activity of firefly luciferase-the luminescence value of firefly luciferase/the luminescence value of renilla luciferase.

Example 2: construction of an overexpression vector of ESR1

(1) Using NCBI to design a specific primer for amplifying an ESR1(Gene ID:397435) CDS region, and using extracted cDNA of the swine ovarian granulosa cells as a template for amplification; the amplified fragment was purified, recovered, ligated with pMD18T vector (purchased from Takara), transformed, screened, sequenced and identified as correct, and then the ordinary plasmid was extracted.

(2) The CDS region sequence of ESR1 gene was analyzed by BioEdit software to find that there are no restriction enzyme cutting sites of EcoRI and XbaI, while there are EcoRI and XbaI cutting sites in pcDNA3.1 vector. EcoRI and XbaI enzyme cutting site sequences are added to the upstream primer and the downstream primer respectively. PCR amplification is carried out by taking CDS region recombinant pMD18T common plasmid of ESR1 as a template; the fragment is purified, recovered, double digested, connected to pcDNA3.1 vector, transformed, screened, sequenced and identified to be correct, and then endotoxin-free plasmid (endotoxin-free plasmid miniprep kit is purchased from America magenta) is extracted and named as pcDNA3.1-ESR 1.

The ESR1 gene CDS region amplification primer used in the invention comprises the following components:

Example 3 qRT-PCR

The qRT-PCR detection of the gene of the invention employed the Maxima SYBR Green qPCR Master Mix (2X) kit (assist in san Yang Biotech, Inc.). The content of the sample gene is detected by adopting a Ct value comparison method in the experiment, and the specific calculation formula is as follows:

relative gene expression level 2- { (Ct value of target gene of experimental group) -in experimental group (Ct value of reference gene of experimental group) -in control group (Ct value of target gene of control group) -in control group

GAPDH is used as an internal reference for detecting genes, and qRT-PCR primers used by the invention are as follows:

qRT-PCR-MMP2 F：5′-GACAAGTGGTGCGTGTGAAG-3′(SEQ ID NO.11)；

qRT-PCR-MMP2 R：5′-CATGGTGAACAGGGCTTCGT-3′(SEQ ID NO.12)；

qRT-PCR-ESR1 F：5′-GCAAAGAGGGTGCCAGGATT-3′(SEQ ID NO.13)；

qRT-PCR-ESR1 R：5′-AAGCGAGACGATGTAGCCAG-3′(SEQ ID NO.14)；

qRT-PCR-GAPDH F：5′-GGACTCATGACCACGGTCCAT-3′(SEQ ID NO.15)；

qRT-PCR-GAPDH R：5′-TCAGATCCACAACCGACACGT-3′(SEQ ID NO.16)。

extraction of total RNA of cells: total cellular RNA was extracted using an RNA extraction kit (purchased from magenta) as follows: collecting about 1 × 107 cell samples in a 1.5mL enzyme-removing centrifuge tube, adding 100 μ L PBS buffer solution to blow and resuspend cells; adding 500 μ L cell lysate (CRL), vortexing for 13s, and standing for 1 min; transferring the liquid to the column, centrifuging, removing the liquid, adding 600 μ L Buffer CW, centrifuging for 1min, removing the liquid, and then allowing to air-separate for 2 min; the column was placed in a 1.5mL enzyme removal centrifuge tube, 60. mu.L RNase Free Water was added, allowed to stand for 2min, centrifuged to elute RNA, and stored at minus 80 ℃.

PrimeScript from TaKaRa was used ^TM The RT Master Mix (Perfect Real Time) cDNA reverse transcription kit reverse transcribes total RNA.

Example 4: culture and transfection of porcine ovarian granulosa cells

(1) Collecting healthy ovaries of commercial sows in a Kongwang Ji slaughterhouse in Guangzhou city, cleaning for 2 times, placing the ovaries in 1% double-resistant autoclaved PBS, and quickly taking the ovaries back to a laboratory in an ice box;

(2) washing ovary with 1% double-anti-autoclave PBS for 3 times, transferring ovary into superclean bench of cell room (wiping with alcohol cotton in advance and irradiating with ultraviolet for 30 min);

(3) preparing a complete culture medium: 89% DMEM, 10% serum and 1% double antibody are mixed by turning upside down;

(4) firstly, adding 5mL of complete culture medium into a 15mL centrifuge tube, and carefully sucking 2mL of follicular fluid into the centrifuge tube by using a 1mL disposable sterile syringe; centrifuging at 1000rpm for 5 min;

(5) pouring off the supernatant, and adding 5mL of PBS buffer solution for washing twice; centrifuging at 1000rpm for 3min, and finally suspending the cell pellet with 3mL of complete culture medium;

(6) adding 12mL of complete culture medium into a 75mL culture bottle, and then adding the resuspension;

(7) observing with microscope, and standing at 37 deg.C and 5% CO ₂ Culturing in an incubator, observing the growth condition of granular cells after 24 hours, pouring out the culture medium when the granular cells grow to about 90%, and washing for 2 times by using preheated PBS containing 1% double antibody;

(8) adding 5mL of trypsin, placing the mixture in an incubator for digestion for about 5min, observing most cells under a microscope until the cells float, and immediately adding an equivalent complete culture medium to terminate digestion;

(9) transferring to a 15mL centrifuge tube, and centrifuging at 1000rpm for 5 min; discarding the supernatant, washing with 1% double antibody-containing PBS for 2 times, and centrifuging at 1000rpm for 5 min;

(10) 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;

(11) observing the cell state after about 24 hours, and performing transfection when the confluence degree of the cells reaches about 80%;

(12) transfection method Invitrogen

3000 kit instructions, each set 3 replicates;

(13) the transfected well plates were placed at 37 ℃ in 5% CO ₂ Culturing in an incubator, observing the cell state 24-72 h after transfection, and collecting the cells after good growth.

The double-resistant is penicillin and streptomycin.

Example 5: western Blot

(1) Extraction and quantification of total protein of ovarian granulosa cells: discarding the culture medium, washing the cells with pre-cooled PBS for 2 times, adding appropriate amount of lysis buffer (5-10 × 10) containing 0.2% protease inhibitor ⁶ Adding 500 mu L of the supernatant into each cell), uniformly mixing, oscillating for 20min at 4 ℃, centrifuging for 10min at 13000rpm at 4 ℃, and taking the supernatant into a precooled 1.5mL sterile centrifuge tube;

(2) protein sample concentration was determined using BCA method: firstly, preparing a BCA working solution according to the number of samples (reagent A: reagent B: 50: 1); ② putting 10 mu L of protein standard substance into a 1.5mL sterile centrifuge tube, adding PBS to dilute the protein standard substance to a final concentration of 0.5 mg/mL; thirdly, adding the diluted protein standard substance into a 96-well plate according to 0, 1, 2, 4, 8, 12, 16 and 20 mu L, and then sequentially adding 20, 19, 18, 16, 12, 8, 4 and 0 mu LPBS into corresponding wells; adding 2 mu L of protein sample and 18 mu L of LPBS into the sample hole; 0.2mL BCA working solution is added into each well, and the mixture is incubated at 37 ℃ for 30 min; measuring the absorbance by a microplate reader (wavelength of 570 nm), and calculating the concentration of the protein.

(3) SDS-PAGE electrophoresis: 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 bromophenol blue just comes out of the bottom of the gel;

(4) film transfer: activating the PVDF membrane by methanol for 3min, balancing by PVDF membrane balancing solution for 2min, sequentially overlapping sponge, PVDF membrane, gel and sponge on the anode of a membrane rotating clamp, covering the cathode of the membrane rotating clamp, and inserting into a channel of a rapid membrane rotating instrument for membrane rotating (using default parameters of the instrument);

(5) immunoblotting: taking off the PVDF membrane, washing with TBST for 3 times, each time for 10min, and sealing with 6% skimmed milk powder at room temperature for 2.5 h; washing off the excess skim milk powder with TBST, diluting the antibody (MMP 2: 10373-2-AP, proteintech,1: 1000; ESR 1: 04-820-25UL, Merck,1: 2000; GAPDH: 10494-1-AP, proteintech,1:30000) with TBST, incubating overnight at 4 ℃; and (3) secondary antibody incubation: taking out the membrane, washing the membrane with TBST for 3 times, 10 min/time, diluting the secondary antibody with TBST in proportion (1: 3000 for mouse source and 1:5000 for rabbit source), and incubating at room temperature for about 2 h; after the secondary antibody is incubated, the membrane is taken out, the membrane is washed for 3 times and 10 min/time by TBST, then A and B luminous liquids are mixed according to the ratio of 1:1, the front and back sides of the membrane are soaked in the mixed liquid for 30s, a chemiluminescence instrument is used for developing, photographing and storing are carried out, and finally protein bands are analyzed by using Image Plus software.

Example 6: chromatin co-immunoprecipitation (ChIP)

The chromatin co-immunoprecipitation operation process is carried out according to the Pierce Agarose ChIP Kit specification, and the specific steps are as follows:

(1) crosslinking and GCs separation: adding a proper amount of 37 percent formaldehyde into each culture dish containing 8mL of cell culture medium to ensure that the final concentration of the formaldehyde is 1 percent, uniformly mixing, and incubating for 10 min; adding a proper amount of 10X glycine till the final concentration is 1X, uniformly mixing, incubating at room temperature for 5min, completely absorbing and removing the culture medium, and washing the cells for 2 times by using a precooled PBS buffer solution; ③ adding 10 mu L of Halt Cocktail into 1mL of precooled PBS, adding the mixed solution into a culture dish, stripping cells by using a cell scraper, and transferring the cells into a 1.5mL centrifuge tube; centrifuging at 3000rpm for 5min, removing liquid, and storing cell precipitate at-80 deg.C or directly performing the next step;

(2) cracking and digesting: putting frozen cells on ice for thawing, adding 100 mu l of lysine Buffer 1 (prepared on ice in advance), vortexing for 15s, uniformly mixing, incubating on ice for 10min, and centrifuging at 9000rpm for 3 min; discarding the supernatant, adding 100 μ L MNase digest Buffer Working Solution to blow and beat the heavy suspension cell sediment, adding 0.25 μ L Micrococcus nucleic (ChIP Grade) (10U/μ L), vortex and mixing uniformly, water bath at 37 ℃ for 15min, inverting and mixing uniformly every 5 min; ③ adding 10 mul MNase Stop Solution vortex to terminate the reaction, incubating for 5min on ice, centrifuging for 5min at 9000rpm, and removing the supernatant to leave a precipitate; adding 50 mul Lysis Buffer 2 to resuspend the cell sediment, incubating on ice for 15min, vortexing and mixing for 15s every 5min, centrifuging at 9000rpm for 5min, transferring the supernatant to a new 1.5mL sterile centrifuge tube, and then performing immunoprecipitation, or storing the sample in a refrigerator at-80 ℃;

(3) and (3) performing immunoprecipitation: transferring 5 mu l of supernatant to a 1.5mL centrifuge tube, storing the supernatant at-20 ℃ as an Input control group, adding 450 mu l of 1 XPIP Dilution Buffer into the residual supernatant, uniformly mixing, and averagely transferring the mixture to 3 columns; adding 10 mu L of Anti-RNA Polymerase II Antibody, 1-2 mu L of normal rabbit IgG Antibody and 10 mu L of Anti-ESR1 Antibody into each tube in sequence, and incubating the mixture in a shaking table at 4 ℃ for overnight; thirdly, adding 20 mu L of ChIP agarose magnetic beads, incubating for 1h at 4 ℃ in a shaking table, sleeving a column in a 2mL collecting pipe, and centrifuging for 30s at 3000 rpm; fourthly, discarding the liquid, adding 0.5mL of IP Wash Buffer 1, incubating for 5min at a shaking table at 4 ℃, and centrifuging for 30s at 3000 rpm; fifthly, discarding the liquid, adding 0.5mL of IP washing liquid 2 and 3 in sequence, repeating the washing steps for 2 times respectively, and leaving at 3000rpm for 1 min;

(4) IP elution: adding 150 mu L of 1 × IP Elution Buffer into the column, and incubating at 65 ℃ for 40 min; while blowing and beating the heavy suspension beads once every 10 min; ② after incubation at 65 ℃, placing the column in a new 1.5mL sterilized centrifuge tube (containing 6 μ L of 5M NaCl and 2 μ L of 20mg/mL protease K), and centrifuging at 6000rpm for 1 min; thirdly, discarding the column, mixing evenly by vortex, and incubating for 1.5h at 65 ℃; unfreezing 5 mu L of input control group on ice, adding 150 mu L of 1 × IP precipitation Buffer, 6 mu L of 5M NaCl and 2 mu L of 20mg/mL protease K, uniformly mixing by vortex, and incubating for 1.5h at 65 ℃;

(5) and (3) DNA purification and recovery: adding 750 mu L of DNA Binding Buffer into each eluted sample tube, and uniformly mixing; secondly, sleeving the column in a 2mL collecting pipe, transferring the liquid to the column (less than or equal to 500 mu L each time), and centrifuging for 1min at 10000 rpm; thirdly, abandoning the filtrate, adding 750 mu L of DNACELUM Wash Buffer, centrifuging, abandoning the filtrate, and performing air separation for 2 min; fourthly, the Column is placed in a 1.5mL centrifuge tube, 50 mu L of DNA Column Solution is added, the centrifugation is carried out for 1min, the filter Column is discarded, and the DNA is stored in a refrigerator at the temperature of 20 ℃ below zero.

(6) And (3) PCR amplification: according to the binding site of ESR1 in the promoter region of the MMP2 gene, ChIP primers are designed by using an NCBI website, and whether ESR1 is bound to the promoter region of the MMP2 gene is analyzed through PCR.

The ChIP primers used in the invention are as follows:

MMP2(-1317～-1274bp)F：5′-CATCGCCCTGACTTCCAAGG-3′(SEQ ID NO.17)；

MMP2(-1317～-1274bp)R：5′-GAGGAGATGGGACTGGGAGT-3′(SEQ ID NO.18)。

and (4) analyzing results:

1. the potential binding site of the transcription factor ESR1 in the promoter region (-2142/+1, +1 is the transcription initiation site) of MMP2 gene is predicted by bioinformatics website. As shown in fig. 1, the MMP2 gene promoter region has 6 binding sites for ESR1, which are located: -2078 to-2071 bp, -2060 to-2053 bp, -1317 to-1310 bp, -1274 to-1267 bp, -406 to-399 bp, -392 to-385 bp.

2. According to the binding site of a transcription factor ESR1 in a promoter region of an MMP2 gene, recombinant plasmids (P1-P4) of an MMP2 gene promoter deletion fragment are constructed, after the recombinant plasmids are transfected to granular cells, a multifunctional enzyme-labeling instrument is used for detecting the relative activity of the MMP2 gene promoter deletion fragment, and the activity of an MMP2 gene promoter deletion fragment carrier P3 is found to be remarkably higher than that of P2(P <0.001), while the activity of a recombinant carrier P4 is remarkably lower than that of P3(P < 0.001). Based on the potential binding site of the transcription factor ESR1 in the promoter region of MMP2 gene, it was preliminarily speculated that ESR1 might bind to the promoter P2-P3 region (-1594 to-718) sequence of MMP2 gene to inhibit the transcription of MMP2, and might bind to the promoter P3-P4 region (-718 to-323) sequence to promote the transcription of MMP2 gene (FIG. 1).

3. Primers were designed to amplify the CDS region of the transcription factor ESR 1: searching the sequence of a transcription factor ESR1(NCBI Gene ID:397435) from NCBI, determining enzyme cutting sites (the enzyme cutting site of ESR1 is EcoRI and XbaI), and designing a specific primer by using an NCBI website; the target fragment is specifically amplified, purified and enzyme-cut by PCR, then is connected to a pcDNA3.1 expression vector, and finally the overexpression vector pcDNA3.1-ESR1 of ESR1 is successfully constructed. The subsequent expression level of ESR1 mRNA was detected by transfecting overexpression vectors with different concentrations (100, 400 and 800ng) in granular cells through qRT-PCR, and it was found that the higher the concentration of the overexpression vector transfected with pcDNA3.1-ESR1, the higher the expression level of ESR1 mRNA, and the difference in the expression levels was significant. When 400ng of ESR1 overexpression vector is transfected in granular cells and the expression level of ESR1 protein is detected by using Western Blot, the expression level of ESR1 protein is remarkably increased after the ESR1 overexpression vector is transfected compared with a control group (figure 2). The constructed ESR1 overexpression vector is proved to be normally expressed in granular cells, and the transfection concentration of 400ng pcDNA3.1-ESR1 is selected in subsequent research.

siRNA-ESR1-1：5′-GCCTTCTTCAAGAGAAGTA-3′(SEQ ID NO.19)；

siRNA-ESR1-2：5′-GGATTTAAGCCTCCATGAT-3′(SEQ ID NO.20)；

siRNA-ESR1-3：5′-GCATTCCTTGCAAATGTAT-3′(SEQ ID NO.21)。

5. pcDNA3.1-ESR1 or ESR1-siRNA-2 is transfected into granular cells respectively, expression levels of mRNA and protein of MMP2 genes are detected by qRT-PCR and Western Blot respectively, overexpression of ESR1 remarkably reduces the expression level of mRNA and protein of MMP2 genes (P <0.01), and interference of ESR1 remarkably increases the expression level of mRNA and protein of MMP2 genes (P <0.01) (figure 4). The detection result of luciferase activity combined with the MMP2 gene promoter deletion fragment shows that the transcription factor ESR1 is mainly combined with the P2-P3(-1594 to-718) region sequence of the MMP2 gene promoter to inhibit the transcription of the MMP2 gene and the synthesis of protein, and other transcription factors or regulatory elements possibly combined with the P3-P4 region (-718 to-323) and in positive regulation can be other transcription factors or regulatory elements.

6. To further demonstrate that ESR1 can inhibit MMP2 gene expression, we treated granulosa cells with estrogen (E2) and an E2 antagonist and examined the effect of E2 and E2 antagonists on MMP2 gene mRNA expression levels by qRT-PCR. The results showed that E2 significantly reduced MMP2 gene mRNA expression levels (P <0.05), while E2 antagonists significantly increased MMP2 gene mRNA expression levels (P <0.05) (fig. 5). The results show that E2 can inhibit the transcription of MMP2 gene, and E2 antagonist can promote the transcription of MMP2 gene, and the results can laterally verify that ESR1 plays a role in inhibiting the expression of MMP2 gene.

7. In order to verify whether the transcription factor ESR1 is combined with the promoter region P2-P3(-1594 to-718 bp) of the MMP2 gene, whether the transcription factor ESR1 is combined with the promoter region sequence of the MMP2 gene is detected by a ChIP experiment. The results showed that the experimental group (Anti-ESR1) and the positive control (Anti-PolyII) lanes of the MMP2 gene promoter (-1317 to-1274 bp) had distinct single bands, while the negative control (Anti-IgG) lane had no distinct band (FIG. 6). Indicating that the transcription factor ESR1 can be combined with the promoter (-1317 to-1274 bp) region of MMP2 gene.

As described above, the transcription factor ESR1 can bind to the promoter (-1317 to-1274 bp) region of MMP2 gene and inhibit the expression of MMP2 gene.

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.

Sequence listing

<110> southern China university of agriculture

<120> method for regulating and controlling pig ovarian granulosa cell MMP2 gene expression

<160> 21

<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<223> P1 R

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gaagatctta ttcgctcggt cgcctc 26

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<213> Artificial Sequence (Artificial Sequence)

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Claims

1. A method for regulating and controlling the expression of pig ovarian granulosa cell MMP2 gene is characterized in that: the transcription factor ESR1 is used as the transcription factor of the promoter region of the MMP2 gene, and the regulation and control of the transcription activity of the MMP2 gene in the porcine ovarian granulosa cells are realized by regulating the expression level of the transcription factor ESR 1.

2. The method of claim 1, wherein:

the expression of the transcription factor ESR1 is inhibited by combining with a promoter-1317-1274 bp region of an MMP2 gene; increasing an exogenous transcription factor ESR1, and reducing the transcriptional activity of MMP2 gene; the expression of a transcription factor ESR1 is inhibited, and the transcriptional activity of an MMP2 gene is increased.

3. The method of claim 2, wherein:

the increase of the exogenous transcription factor ESR1 is realized by a gene overexpression technology, and the adopted gene overexpression vector is prepared by the following method: (1) extracting RNA of the pig ovarian granulosa cells, carrying out reverse transcription on the RNA to form cDNA, and carrying out PCR amplification by taking the cDNA as a template to obtain a target fragment; (2) the objective fragment was ligated to pcDNA3.1 vector digested with restriction enzymes EcoRI and XbaI to obtain a recombinant vector.

4. The method of claim 3, wherein:

the primers used for PCR amplification in step (1) are as follows:

ESR1 F：

ESR1 R：

5. The method of claim 2, wherein:

the expression of the transcription factor ESR1 is inhibited by an RNA interference technology, and the adopted siRNA is as follows:

siRNA-ESR1-2：5′-GGATTTAAGCCTCCATGAT-3′。

6. a core promoter functional regulatory region of the MMP2 gene of ovarian granulosa cells, characterized in that: is a promoter-1317 to-1274 bp region of the MMP2 gene.

7. A nucleic acid fragment for regulating and controlling the expression of an ovarian granulosa cell MMP2 gene, which is characterized in that: an siRNA for inhibiting expression of transcription factor ESR1, the siRNA is as follows:

siRNA-ESR1-2：5′-GGATTTAAGCCTCCATGAT-3′。