CN114874993B

CN114874993B - Method for regulating and controlling MMP2 gene expression of porcine ovarian granulosa cells

Info

Publication number: CN114874993B
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: 2023-08-15
Anticipated expiration: 2042-05-30
Also published as: CN114874993A

Abstract

The invention discloses a method for regulating and controlling MMP2 gene expression of porcine ovarian granulosa cells, belonging to the technical fields of cell engineering and genetic engineering. The invention takes MMP2 gene and transcription factor ESR1 as research objects, and utilizes the technologies of molecular biology, cell biology and the like to construct an overexpression vector of the transcription factor ESR1 and design an interference fragment thereof, so as to research the influence of the transcription factor ESR1 on the expression of the MMP2 gene. The technical scheme of the invention has detailed design and reliable result. The transcription factor ESR1 can be combined with a (-1317 to-1274 bp) region of an MMP2 gene promoter, and inhibit the expression of the MMP2 gene. By exploring the application of the transcription factor ESR1 to the transcription expression mechanism of MMP2 genes, the method has good application value for researching the molecular mechanism of the MMP2 genes affecting ovarian follicle locking, sow primordial initiation and the like.

Description

Method for regulating and controlling MMP2 gene expression of porcine ovarian granulosa cells

Technical Field

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

Background

Gene expression refers to the process by which a gene converts genetic information contained in the gene into RNA, polypeptides, and proteins by transcription and translation. A plurality of regulatory elements exist in the 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 role in maintaining the stability of mRNA, nuclear transport, RNA splicing, cell proliferation and the like in the regulation of gene expression.

Matrix metalloproteinase 2 (Matrix metalloproteinases, MMP 2) is one of the important members of the MMPs family, and can decompose gelatin protein, fibrin and IV type collagen in follicular fluid, and influence proliferation and apoptosis of granulosa cells. In the closed 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 primary aim of the invention is to provide a method for regulating and controlling the MMP2 gene expression of porcine ovarian granulosa cells.

In order to achieve the above object, the present invention adopts the following technical scheme:

a method for regulating and controlling the expression of MMP2 genes in porcine ovarian granulosa cells adopts a transcription factor ESR1 as a transcription factor of a promoter region of the MMP2 genes, and realizes the regulation and control of the transcriptional activity of the MMP2 genes in the porcine ovarian granulosa cells by regulating the expression level of the transcription factor ESR1.

Further, the transcription factor ESR1 inhibits the expression thereof by binding to the region from-1317 to-1274 bp of the MMP2 gene promoter; increasing exogenous transcription factor ESR1, MMP2 gene transcription activity is reduced; inhibiting the expression of transcription factor ESR1 and increasing the transcriptional activity of MMP2 gene.

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 steps: (1) Extracting RNA of porcine ovary granulosa cells, reversely transcribing the RNA into cDNA, and carrying out PCR amplification by taking the cDNA as a template to obtain a target fragment; (2) The desired 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:

ESR1 F：5′-CG GAATTCATCGGTGTCAAACGAAGGTGG-3′；

ESR1 R：5′-GC TCTAGATGAAGTGCGACAGGGATTCT-3′；

note that: the underlined part is the protecting base, and the italic 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 functional regulation region of the core promoter of the ovarian granulosa cell MMP2 gene is the region of-1317 to-1274 bp of the MMP2 gene promoter.

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

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

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

The present invention predicts the existence of estrogen receptor 1 (Estrogen receptor, ESR 1) binding site in MMP2 promoter region through bioinformatics website. ESR1 is one of the important members of the nuclear receptor family of transcription factors, and ESR1 is in an inactive state in the absence of hormone, and after being combined with estrogen, ESR1 becomes homodimer, and then through interaction with a regulatory element combined with a gene promoter, the gene transcription level is regulated, and the developmental maturation of the mammalian reproductive system can also be regulated by receiving signals transmitted by E2 and regulating the expression of key genes of the E2 signal pathway. In summary, the transcription factor ESR1 may bind to the MMP2 promoter region and regulate the expression of MMP2, thereby affecting the function of granulosa cells.

The verification result of the invention is as follows:

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

2. According to the combination site of transcription factor ESR1 in MMP2 gene promoter region, recombinant plasmids (P1-P4) of MMP2 gene promoter deletion fragments are constructed, after transfection into granulosa cells, the relative activity of the MMP2 gene promoter deletion fragments is detected by using a multifunctional enzyme-labeled instrument, the activity of the carrier P3 of the MMP2 gene promoter deletion fragments is found to be extremely higher than that of P2 (P < 0.001), and the activity of the recombinant carrier P4 is extremely lower than that of P3 (P < 0.001) (figure 1).

3. Designing a primer for amplifying a transcription factor ESR1 CDS region: the sequence of transcription factor ESR1 (NCBI Gene ID: 397435) was searched from NCBI, the cleavage site (EcoRI and XbaI for ESR 1) was determined, and the NCBI website was used to design specific primers; specifically amplifying, purifying and enzyme cutting target fragments by PCR, then connecting to a pcDNA3.1 expression vector, and finally successfully constructing an ESR1 super-expression vector pcDNA3.1-ESR1. Subsequently, expression levels of ESR1mRNA were detected by qRT-PCR by transfecting the overexpressing vectors at different concentrations (100, 400 and 800 and ng) in the granulosa cells, and it was found that the higher the concentration of the transfected pcDNA3.1-ESR1 overexpressing vector, the higher the expression level of ESR1mRNA and the difference was significant. 400 ng of ESR1 overexpression vector was transfected into the granulosa cells, and the expression level of ESR1 protein was detected by Western Blot, and it was found that the expression level of ESR1 protein was significantly increased after transfection of the ESR1 overexpression vector compared with the control group (FIG. 2). Proved that the constructed ESR1 overexpression vector can be normally expressed in granulosa cells, and the subsequent research selects 400 ng as the transfection concentration of pcDNA3.1-ESR1.

4. 3 pairs of small interfering RNAs/controls (ESR 1-siRNA/siRNA-NC) interfering with ESR1 were synthesized, followed by transfection of different concentrations (30 nmol, 50nmol, and 100 nmol) of small interfering RNAs in the granulosa cells, whose interfering efficiency was detected by qRT-PCR. As can be seen from the results of the figures, the effect of the ESR1-siRNA-2 small interfering RNA was best, 50nmol of ESR1-siRNA-2 was then transfected into the granulosa cells, and the expression level of ESR1 protein was detected by Western Blot, and it was found that the expression level of ESR1 protein was significantly reduced after transfection of ESR1-siRNA-2 small interfering RNA compared with the control group (FIG. 3). Proved by the experiment, the synthesized small interfering RNA can interfere the expression of ESR1 in granulosa cells, has high efficiency, and finally selects 50nmol ESR1-siRNA-2 small interfering RNA for subsequent experiments.

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

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

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

5. The expression levels of MMP2 gene mRNA and protein were detected by respectively transfecting pcDNA3.1-ESR1 or ESR1-siRNA-2 into granulosa cells, respectively using qRT-PCR and Western Blot, and overexpressing ESR1 significantly reduced the expression levels of MMP2 gene mRNA and protein (P < 0.01), while interfering ESR1 significantly increased the expression levels of MMP2 gene mRNA and protein (P < 0.01) (FIG. 4).

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

7. To verify whether transcription factor ESR1 binds to the P2-P3 (-1594/-718 bp) region of the MMP2 gene promoter region, this study examined whether transcription factor ESR1 binds to the MMP2 gene promoter region sequence by the ChIP assay. The results showed that the experimental group (Anti-ESR 1) and the positive control (Anti-PolyII) lanes of the MMP2 gene promoter (-1317-1274 bp) region both had a distinct single band, while the negative control (Anti-IgG) lanes had no distinct band, indicating that the transcription factor ESR1 could bind to the MMP2 gene promoter (-1317-1274 bp) region (FIG. 6).

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

the technical scheme of the invention has detailed design and reliable result. MMP2 gene and transcription factor ESR1 are directly or indirectly involved in growth and differentiation of granulosa cells, follicular development and primordial state initiation, and the research takes the MMP2 gene and the transcription factor ESR1 as research objects, and adopts molecular and cell biology technology to research the influence of the transcription factor ESR1 on the expression of the MMP2 gene. To confirm that transcription factor ESR1 inhibits the expression of MMP2 genes, the present invention validated at multiple levels, multiple angles, first at mRNA level and protein level, then further through drug treatment, and finally specific binding sites through ChIP. The method has good application value for researching the expression regulation mechanism of MMP2 genes in ovarian granulosa cells.

Drawings

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

FIG. 2 is a graph showing the detection of the efficiency 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 a is the efficiency detection of ESR1 small interfering RNA at mRNA level, and b is the efficiency detection of ESR1 small interfering RNA at protein level;

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

FIG. 5 is a graph of the effect 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 antagonists on MMP2 gene expression;

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

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. The experimental procedure, in which specific conditions are not noted in the examples below, is generally followed by conventional conditions.

The results of 3 independent experiments in each example were analyzed using a statistical method in the present invention, and "mean ± standard deviation" was calculated, respectively, and a single factor analysis of variance was used for the analysis of significance of the differences (in the figure, "x" represents P <0.05, "x" represents P < 0.01).

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

(1) Designing primers by using NCBI website, and amplifying promoter regions P1 (-2142- +191), P2 (-1594- +191), P3 (-718- +191) and P4 (-323- +191) of MMP2 gene by using extracted DNA of pig granulosa cells as a template; the amplified fragment was purified, recovered, ligated to pMD18T vector (purchased from Takara Co.), transformed, screened, and sequenced to identify the correct plasmid. The BioEdit software analyzes the distribution of the restriction sites of the MMP2 gene promoter region, and finally selects two restriction enzymes KpnI and BglII as the restriction sites. The upstream and downstream primers are added with the enzyme cleavage site sequences of KpnI and BglII enzymes respectively. Then, PCR amplification is carried out by using each recombinant pMD18T common plasmid as a template and each enzyme digestion primer; the fragment was purified, recovered, digested with two enzymes, ligated to pGL3-basic vector, transformed, screened, sequenced and identified correctly, and then the endotoxin-free plasmid was extracted (endotoxin-free plasmid miniprep kit was purchased from Magen, USA), with pGL3-basic as negative control and pGL3-control as positive control.

MMP2 gene promoter deletion fragment primer used in the invention:

P1 F：5′-GG GGTACCTACAGAGGGACGGGTTGCT-3′（SEQ ID NO.1）；

P1 R：5′-GA AGATCTTATTCGCTCGGTCGCCTC-3′（SEQ ID NO.2）；

P2 F：5′-GG GGTACCTGCTGCCATGTGGAAATT-3′（SEQ ID NO.3）；

P2 R：5′-GA AGATCTTATTCGCTCGGTCGCCTC-3′（SEQ ID NO.4）；

P3 F：5′-GG GGTACCTGCGTGCCAGGCAAGCC-3′（SEQ ID NO.5）；

P3 R：5′-GA AGATCTTATTCGCTCGGTCGCCTC-3′（SEQ ID NO.6）；

P4 F：5′-GG GGTACCTGCTGGGGTTCCCTTCT-3′（SEQ ID NO.7）；

P4 R：5′-GA AGATCTTATTCGCTCGGTCGCCTC-3′（SEQ ID NO.8）；

(2) The detection of luciferase reporter gene activity is carried out by referring to the description of a double luciferase detection kit of Shanghai, assist in san Biotech Co., ltd, and comprises the following steps: after 24h transfection, the medium was removed, washed 2 times with PBS, 80. Mu.L of cell lysate was added, the plates were gently rotated to allow complete coverage of cells, and incubated on ice for 5 min; centrifuging at 13000 rpm for 1 min at 4deg.C, collecting supernatant, and placing in a new 1.5. 1.5 mL sterilized centrifuge tube; 25. Mu.L of the above cell lysate was added to a 96-well plate and 6 wells were repeated for each group; adding 100 mu L of firefly luciferase reaction solution, standing for 5 min, and detecting the luminescence value of firefly luciferase; then adding 100 mu L of Renilla luciferase reaction solution, standing for 5 min, and detecting the luminescence value of Renilla luciferase; and (3) calculating: relative activity of firefly luciferase = luminescence value of firefly luciferase/luminescence value of renilla luciferase.

Example 2: construction of ESR1 overexpression vector

(1) Designing a specific primer for amplifying an ESR1 (Gene ID: 397435) CDS region by using NCBI, and performing amplification by using extracted cDNA of the porcine ovary granular cells as a template; the amplified fragment was purified, recovered, ligated to pMD18T vector (purchased from Takara Co.), transformed, screened, and sequenced to identify the correct plasmid.

(2) Analysis by using BioEdit software found that the CDS region sequence of ESR1 gene had no EcoRI and XbaI restriction sites, whereas pcDNA3.1 vector had EcoRI and XbaI restriction sites. The EcoRI and XbaI restriction site sequences were added to the primers upstream and downstream, respectively. Performing PCR amplification by taking CDS region recombinant pMD18T common plasmid of ESR1 as a template; the fragment was purified, recovered, double digested, ligated to pcDNA3.1 vector, transformed, screened, sequenced and identified correctly and then the endotoxin-free plasmid was extracted (endotoxin-free plasmid miniprep kit was purchased from Magen, USA) and designated pcDNA3.1-ESR1.

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

ESR1 F：5′-CG GAATTCATCGGTGTCAAACGAAGGTGG-3′（SEQ ID NO.9）；

ESR1 R：5′-GC TCTAGATGAAGTGCGACAGGGATTCT-3′（SEQ ID NO.10）；

Example 3 qRT-PCR

The qRT-PCR detection of the gene of the present invention was performed using a Maxima SYBR Green qPCR Master Mix (2X) kit (Shanghai assist Saint Biotech Co., ltd.). The experiment adopts a Ct value comparison method to detect the content of the sample genes, and the specific calculation formula is as follows:

gene expression level=2- { < the experiment set target gene Ct value- > (the experiment set reference gene Ct value- > (the control set target gene Ct value- > (the control set reference gene Ct value)

GAPDH is used as an internal reference for detecting genes, and qRT-PCR primers used in 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 Magen corporation) as follows: about 1×107 cell samples were collected in a 1.5 mL enzyme-removing centrifuge tube, and 100 μl of PBS buffer was added to blow the resuspended cells; add 500. Mu.L of cell lysate (CRL), vortex 13 s, and rest for 1 min; transferring the liquid to a column, centrifuging, removing the liquid, adding 600 mu L Buffer CW, centrifuging for 1 min, removing the liquid, and then air-separating for 2 min; the column was placed in a 1.5 mL enzyme-removing centrifuge tube, 60. Mu. L RNase Free Water was added, left to stand for 2 min, and RNA was eluted by centrifugation and stored at minus 80 ℃.

PrimeScript using TaKaRa Corp ^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 commercial sow healthy ovaries at a Guangzhou city Kong Wang slaughterhouse, cleaning for 2 times, placing the ovaries in PBS containing 1% double-antibody autoclaving, and rapidly taking the ovaries back to a laboratory in an ice box;

(2) Washing ovary 3 times with PBS containing 1% double-antibody autoclaving, transferring ovary into cell room super clean bench (wiping with alcohol cotton in advance and irradiating with ultraviolet for 30 min);

(3) Preparing a complete culture medium: 89% of DMEM, 10% of serum and 1% of diabody, and mixing the components upside down;

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

(5) Pouring out the supernatant, and adding 5mL of PBS buffer solution for washing twice; centrifuging at 1000 rpm for 3min each time, and finally re-suspending the cell pellet with 3 mL complete medium;

(6) Adding 12 mL complete culture medium into 75 mL culture bottle, and adding the heavy suspension;

(7) Microscopic observation is carried out, and then the mixture is placed at 37 ℃ and 5 percent CO ₂ Culturing in incubator, observing growth condition of granulosa cells after 24 and h, and keeping granulosa cells to 90%About, the medium was decanted and washed 2 times with pre-warmed PBS containing 1% diabody;

(8) Adding 5mL trypsin, placing in an incubator for digestion for about 5 min, observing that most cells float under a microscope, immediately adding an equal amount of complete culture medium, and stopping digestion;

(9) Transferring to a centrifuge tube of 15 mL, and centrifuging at 1000 rpm for 5 min; the supernatant was discarded, washed 2 times with PBS containing 1% of diabody, and centrifuged at 1000 rpm for 5 min;

(10) Gently resuspending the cell pellet with complete medium, uniformly dividing into each well, supplementing volume with complete medium, gently shaking, and culturing in incubator;

(11) About 24 to h, observing the cell state, and carrying out transfection when the cell confluence reaches about 80%;

(12) The transfection method was performed according to the Lipofectamine 3000 kit instructions of Invitrogen company, 3 replicates were set per group;

(13) The transfected well plate was placed at 37℃with 5% CO ₂ Culturing in an incubator, carrying out transfection for 24-72 h, observing the cell state, and collecting the cells after good growth.

The double antibodies are penicillin and streptomycin.

Example 5: western Blot

(1) Extraction and quantification of total protein of ovarian granulosa cells: the culture medium is discarded, the cells are washed for 2 times by precooled PBS, and a proper amount of lysate (5-10 multiplied by 10) containing 0.2 percent protease inhibitor is added ⁶ Adding 500 μL of individual cells), mixing, oscillating at 4deg.C for 20 min, centrifuging at 13000 rpm for 10 min at 4deg.C, and collecting supernatant in a precooled 1.5. 1.5 mL sterilizing centrifuge tube;

(2) Protein sample concentration was determined using BCA method: (1) firstly, preparing BCA working solution (reagent A: reagent B=50:1) according to the number of samples; (2) taking 10 mu L of protein standard substance, putting the protein standard substance into a 1.5 mL sterilization centrifuge tube, and adding PBS to dilute the protein standard substance to a final concentration of 0.5 mg/mL; (3) adding diluted protein standard substances into 96-well plates according to 0, 1, 2, 4, 8, 12, 16 and 20 mu L, and sequentially adding 20, 19, 18, 16, 12, 8, 4 and 0 mu LPBS into corresponding wells; (4) adding 2 μl of protein sample and 18 μl LPBS to the sample well; 0.2 mL BCA working solution is added to each well, and incubated for 30 min at 37 ℃; (5) the absorbance was measured by an enzyme-labeled instrument (wavelength: 570 nm), and the protein concentration was calculated.

(3) SDS-PAGE electrophoresis: 20 mug total protein and 5 Xloading buffer were mixed in 5:1 and boiled for 5 min. SDS-PAGE electrophoresis is carried out until bromophenol blue just goes out of the bottom of the gel;

(4) Transferring: activating a PVDF film with methanol for 3min, balancing with PVDF film balancing solution for 2 min, sequentially stacking a sponge, the PVDF film, gel and the sponge on the positive electrode of a film transfer clamp, covering the negative electrode of the film transfer clamp, and inserting the film transfer clamp into a channel of a rapid film transfer instrument for film transfer (using default parameters of the instrument);

(5) Immunoblotting: taking off PVDF membrane, cleaning with TBST for 3 times, 10 min each time, and sealing with 6% skimmed milk powder at room temperature for 2.5 h; excess skim milk powder was washed off with TBST, and antibody diluted with TBST (MMP 2:10373-2-AP, proteontech, 1:1000; ESR1:04-820-25UL, merck, 1:2000; GAPDH:10494-1-AP, proteontech, 1:30000) was incubated overnight at 4 ℃; secondary antibody incubation: taking out the membrane, washing the membrane 3 times with TBST for 10 min/time, diluting the secondary antibody with TBST in proportion (murine source is 1:3000, rabbit source is 1:5000), and incubating at room temperature for about 2 h; after incubation of the secondary antibodies, the membranes were removed, washed 3 times with TBST for 10 min/time, then the luminescent solutions A and B were mixed at 1:1, the front and back sides of the membranes were soaked in the mixed solution 30: 30 s, developed with a chemiluminescent apparatus, photographed and stored, and finally the protein bands were analyzed using Image Plus software.

Example 6: chromatin co-immunoprecipitation (ChIP)

The chromatin co-immunoprecipitation protocol was performed with reference to Pierce Agarose ChIP Kit instructions, and the specific steps are as follows:

(1) Crosslinking and GCs separation: (1) adding a proper amount of 37% formaldehyde into each culture dish containing 8 mL cell culture medium to make the final concentration of formaldehyde be 1%, uniformly mixing, and incubating for 10 min; (2) adding a proper amount of 10 Xglycine to the final concentration of 1X, uniformly mixing, incubating for 5 min at room temperature, thoroughly sucking off the culture medium, and washing the cells for 2 times by using a precooled PBS buffer solution; (3) 10. Mu.L of Hall Cocktail was added to 1 mL precooled PBS, the mixture was added to a petri dish, the cells were scraped with a cell scraper, and the cells were transferred to a 1.5 mL centrifuge tube; 3000 Centrifuging at rpm for 5 min, removing liquid, and storing cell sediment at-80deg.C or directly proceeding to the next step;

(2) Cleavage and digestion: (1) thawing frozen cells on ice, adding 100 μl of Lysis Buffer 1 (prepared in advance on ice), mixing well by vortex 15 s, incubating on ice for 10 min, and centrifuging at 9000 rpm for 3 min; (2) removing supernatant, adding 100 μ L MNase Digestion Buffer Working Solution, blowing to resuspend cell pellet, adding 0.25 μ L Micrococcal Nuclease (ChIP Grade) (10U/. Mu.L), vortex mixing, water bath at 37deg.C for 15 min, and mixing once every 5 min; (3) adding 10 mu l MNase Stop Solution vortex to terminate the reaction, incubating for 5 min on ice, centrifuging for 5 min at 9000 rpm, and discarding the supernatant to leave a precipitate; (4) adding 50 μl of Lysis Buffer 2 to resuspend cell pellet, incubating on ice for 15 min, vortex mixing for 15 s every 5 min, centrifuging at 9000 rpm for 5 min, transferring supernatant to a new 1.5 mL sterilizing centrifuge tube, and immunoprecipitation, or storing the sample in a-80deg.C refrigerator;

(3) Immunoprecipitation reaction: (1) transferring 5 μl of the supernatant to a 1.5 mL centrifuge tube, storing at-20deg.C as Input control group, adding 450 μl of 1× IP Dilution Buffer to the rest supernatant, mixing, and transferring to 3 columns on average; (2) sequentially 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, and incubating the mixture to be overnight at 4 ℃; (3) adding 20 μl of ChIP agarose magnetic beads, incubating in a shaker at 4deg.C for 1 h, sleeving the column in a 2 mL collection tube, and centrifuging at 3000 rpm for 30 s; (4) discarding the liquid, adding a shaking table at 0.5mL IP Wash Buffer 1,4 ℃ for incubation for 5 min, and centrifuging at 3000 rpm for 30 s; (5) discarding the liquid, sequentially adding 0.5mL of IP washing liquid 2 and 3, repeating the step (4) for 2 times, and carrying out air separation at 3000 rpm for 1 min;

(4) IP elution: (1) 150. Mu.L of 1X IP Elution Buffer was added to the column and incubated at 65℃for 40min; the resuspension beads were blown once every 10 min during the period; (2) after incubation at 65℃the column was placed in a fresh 1.5. 1.5 mL sterile centrifuge tube (containing 6. Mu.L 5M NaCl and 2. Mu.L 20mg/mL protease K) and centrifuged at 6000 rpm for 1 min; (3) discarding the column, mixing by vortex, and incubating at 65 ℃ for 1.5 h; (4) thawing 5 μL of input control group on ice, adding 150 μL of 1×IP ElutionBuffer, 6 μL of 5M NaCl and 2 μL of 20mg/mL protease K, mixing by vortex, and incubating at 65deg.C for 1.5 h;

(5) And (3) DNA purification and recovery: (1) adding 750 mu L DNA Binding Buffer into each eluted sample tube, and uniformly mixing; (2) the column was sleeved in a 2 mL collection tube, the liquid was transferred to the column (500. Mu.L each time), and centrifuged at 10000 rpm for 1 min; (3) discarding the filtrate, adding 750 mu L DNA ColumnWash Buffer, centrifuging, discarding the filtrate, and carrying out air separation for 2 min; (4) the column was placed in a 1.5 mL centrifuge tube, 50. Mu. L DNAColumn Elution Solution was added, centrifuged for 1 min, the filter column was discarded, and the DNA was stored in a-20℃refrigerator.

(6) And (3) PCR amplification: the ChIP primer was designed based on the binding site of ESR1 in the MMP2 gene promoter region using NCBI website, and PCR was used to analyze whether ESR1 binds to the MMP2 gene promoter region.

The ChIP primers used in the invention are as follows:

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

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

analysis of results:

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

2. According to the combination site of transcription factor ESR1 in MMP2 gene promoter region, constructing recombinant plasmids (P1-P4) of MMP2 gene promoter deletion fragments, and after transfection into granulosa cells, detecting the relative activity of the MMP2 gene promoter deletion fragments by using a multifunctional enzyme-labeling instrument, the activity of the carrier P3 of the MMP2 gene promoter deletion fragments is found to be extremely higher than that of P2 (P < 0.001), and the activity of the recombinant carrier P4 is extremely lower than that of P3 (P < 0.001). Based on the potential binding site of transcription factor ESR1 in the promoter region of MMP2 gene, it can be speculated that ESR1 may bind to the P2-P3 region (-1594 to-718) sequence of the promoter of MMP2 gene, inhibit the transcription of MMP2, and may bind to the P3-P4 region (-718 to-323) sequence, and promote the transcription of MMP2 gene (FIG. 1).

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. The expression levels of MMP2 gene mRNA and protein were detected by respectively transfecting pcDNA3.1-ESR1 or ESR1-siRNA-2 into granulosa cells, respectively using qRT-PCR and Western Blot, and overexpressing ESR1 significantly reduced the expression levels of MMP2 gene mRNA and protein (P < 0.01), while interfering ESR1 significantly increased the expression levels of MMP2 gene mRNA and protein (P < 0.01) (FIG. 4). The luciferase activity detection result of the deletion fragment of the promoter combined with the MMP2 gene shows that the transcription factor ESR1 is mainly combined with the P2-P3 (-1594 to-718) region sequence of the promoter of the MMP2 gene, inhibits the transcription of the MMP2 gene and the synthesis of protein, and is possibly other transcription factors or regulatory elements combined with the P3-P4 region (-718 to-323) and positively regulated.

6. To further demonstrate that ESR1 can inhibit the expression of MMP2 genes, we treated granulosa cells with estrogen (E2) and E2 antagonists and examined the effect of E2 and E2 antagonists on the mRNA expression level of MMP2 genes by qRT-PCR. The results indicate 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). It is shown that E2 can inhibit the transcription of MMP2 genes, while E2 antagonists can promote the transcription of MMP2 genes, and these results can verify from the side that the transcription factor ESR1 has an inhibitory effect on the expression of MMP2 genes.

7. To verify whether transcription factor ESR1 binds to the P2-P3 (-1594 to-718 bp) region of the MMP2 gene promoter region, this study examined whether transcription factor ESR1 binds to the MMP2 gene promoter region sequence by ChIP experiments. The results show that both the experimental group (Anti-ESR 1) and the positive control (Anti-PolyII) lanes of the MMP2 gene promoter (-1317-1274 bp) region have a distinct single band, while the negative control (Anti-IgG) lanes have no distinct band (FIG. 6). It was shown that the transcription factor ESR1 can bind to the MMP2 gene promoter (-1317-1274 bp) region.

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

The embodiments described above are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be made in the equivalent manner, and are included in the scope of the present invention.

Sequence listing

<110> agricultural university of south China

<120> method for regulating and controlling MMP2 gene expression in porcine ovarian granulosa cells

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<213> Artificial sequence (Artificial Sequence)

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<213> Artificial sequence (Artificial Sequence)

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Claims

1. A method for in vitro regulation of gene expression of porcine ovarian granulosa cell MMP2 is characterized in that: the transcription factor ESR1 is adopted as a transcription factor of an MMP2 gene promoter region, and the regulation of the transcriptional activity of the MMP2 gene in the pig ovarian granulosa cells is realized by regulating the expression level of the transcription factor ESR 1; wherein, the transcription factor ESR1 inhibits the expression thereof by binding to the region of MMP2 gene promoter-1317-1274 bp; increasing exogenous transcription factor ESR1, MMP2 gene transcription activity is reduced; inhibiting the expression of transcription factor ESR1 and increasing the transcriptional activity of MMP2 genes; and the exogenous transcription factor ESR1 is increased by a gene over-expression technology, and the inhibition of the expression of the transcription factor ESR1 is realized by an RNA interference technology.

2. The method according to claim 1, characterized in that:

the adopted gene overexpression vector is prepared by the following steps: (1) Extracting RNA of porcine ovary granulosa cells, reversely transcribing the RNA into cDNA, and carrying out PCR amplification by taking the cDNA as a template to obtain a target fragment; (2) The desired fragment was ligated to pcDNA3.1 vector digested with restriction enzymes EcoRI and XbaI to obtain a recombinant vector.

3. The method according to claim 2, characterized in that:

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

ESR1 F：5′- CGGAATTCATCGGTGTCAAACGAAGGTGG-3′；

ESR1 R：5′- GCTCTAGATGAAGTGCGACAGGGATTCT-3′。

4. the method according to claim 1, characterized in that:

the sirnas employed were as follows:

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