CN108441494B - Application of sow MMP2 gene in promotion of E2 generation in ovarian granulosa cells - Google Patents
Application of sow MMP2 gene in promotion of E2 generation in ovarian granulosa cells Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6489—Metalloendopeptidases (3.4.24)
- C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/24—Metalloendopeptidases (3.4.24)
- C12Y304/24007—Interstitial collagenase (3.4.24.7), i.e. matrix metalloprotease 1 or MMP1
Abstract
The invention discloses an application of sow MMP2 gene in promotion of generation of E2 in ovarian granulosa cells, and belongs to the technical field of genetic engineering and cell engineering. The invention takes MMP2 gene as a research object, and adopts a cell biology method to research the expression application of the MMP2 gene in the porcine ovarian granulosa cells. The invention proves the expression condition of the pig MMP2 gene in the pig ovarian granulosa cells for the first time; and effects on sow ovarian granulosa cells after overexpression or interference of MMP2 in pig ovarian granulosa cells; the activity of the promoter of MMP2 gene was deleted in granulosa cells.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and cell engineering, and particularly relates to application of a sow MMP2 gene in promotion of generation of E2 (estradiol) in ovarian granulosa cells.
Background
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. The development process of the follicle is accompanied by the growth, development and differentiation of the granulosa cell, and the proliferation and apoptosis of the granulosa cell are closely related to the growth and development of the follicle-developing oocyte, the initiation of primordial follicle growth, the regulation of follicle development in the growth phase and follicle atresia.
Matrix Metalloproteinases (MMPs) are important zinc ion hydrolases, mainly function in degrading extracellular matrix (ECM), regulating cell adhesion, acting on extracellular components or other protein components to initiate potential biological functions, and directly or indirectly participate in normal physiological processes such as embryonic development, corneal repair, bone growth, tissue remodeling, angiogenesis and wound healing, and pathological processes such as inflammation and tumor invasion and metastasis. At least a large number of these have been found in humans to date, constituting the superfamily of MMPs. MMPs are classified into five classes based on their specific substrates: (1) collagenase, the substrate of which is mainly interstitial collagen; (2) gelatins, whose action substrates are mainly type IV gelatin and gelatin; (3) the matrix sol can degrade proteoglycan and glycoprotein in III, IV, V, collagen and matrix and can activate certain MMP; (4) model gums, which degrade several ECM components, some which activate other MMPs; (5) others, more complex and some substrates are not detailed. Tissue metalloproteinase inhibitors (TIMPs) are both natural inhibitors of metalloproteinases and play an important role in the process of tumorigenesis.
MMP2 is an important member of the MMPs family, is located in human chromosome 16q21, consists of 13 exons and 12 introns, and can hydrolyze not only denatured collagen and the major component type iv collagen in the extracellular basement membrane, and the regulation of MMP2 expression and function occurs at various levels of transcription, secretion, activation of preproenzymes, cell surface binding, and interaction with inhibitors derived from tumor or host cells. MMP2 plays an important role in proliferation, angiogenesis, infiltration and metastasis of tumor cells, and in addition to degrading basement membrane collagen, it can also decompose various important bioactive molecule binding proteins, and free bioactive molecules to play a role.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide the application of the sow MMP2 gene in promoting the generation of E2 in ovarian granulosa cells.
It is another object of the present invention to provide a small interfering RNA fragment (siRNA) that inhibits the MMP2 gene.
Another object of the present invention is to provide the core promoter region of the MMP2 gene.
Still another object of the present invention is to provide the use of the core promoter region of the MMP2 gene.
Constructing deletion fragments of the promoter region of the gene by a gene engineering technology, analyzing the activity of the deletion fragments of each promoter region, and finding out the core promoter region of the gene.
The purpose of the invention is realized by the following technical scheme:
the invention provides application of a sow MMP2 gene in promotion of generation of E2 in ovarian granulosa cells. The over-expression of MMP2 gene can promote the generation of E2, and the interference of MMP2 gene can inhibit the generation of E2.
The invention provides siRNA for inhibiting MMP2 gene, which has the following sequence:
MMP2-siRNA-2：5′-GCGACAAGAAGUACGGCUU-3′；
MMP2-siRNA-3：5′-GCAAACAGGACAUCGUCUU-3′；
the invention provides a core promoter region of MMP2 gene, the position of which is located in-1145 to-686 bp region.
The verification results of the invention are as follows:
1. constructing a eukaryotic expression vector pcDNA3.1-MMP2 containing an MMP2 gene CDS region, and detecting the expression condition of an MMP2 gene after overexpression of an MMP2 gene by qRT-PCR.
2. 3 interfering small fragment/control (MMP 2-siRNA/Scambred-siRNA) of MMP2 gene were synthesized, screened and tested for their interference efficiency.
3. MMP2 gene was overexpressed or disrupted in porcine ovarian granulosa cells, and apoptosis was detected using the FITC Annexin VApoptosis Detection Kit with PI.
4. The MMP2 gene is over-expressed or interfered in the ovarian granulosa cells of the pigs, and the content of E2 in the supernatant of the ovarian granulosa cells is detected by ELISA.
5. Constructing a dual-luciferase reporter gene recombinant containing a deletion fragment of the MMP2 gene promoter region, transiently transfecting into ovarian granulosa cells of a pig, and analyzing the activity of the deletion fragment of the MMP2 gene promoter region by using a dual-luciferase reporter system and determining a core promoter region.
The invention takes MMP2 gene as a research object, and researches the expression regulation and control of the MMP2 gene in the porcine ovarian granulosa cells by adopting a molecular and cell biological method. The key points are as follows:
(1) detecting the expression of MMP2 gene in each tissue;
(2) the influence of overexpression or interference of sow MMP2 gene on apoptosis of sow ovarian granule cells;
(3) the influence of overexpression or interference of sow MMP2 gene on E2 level in the supernatant of sow ovarian granulosa cells;
(4) constructing a dual-luciferase reporter gene recombinant containing a deletion fragment of the promoter region of the MMP2 gene, and carrying out promoter activity analysis to find out a core promoter region.
The mechanism of the invention is as follows:
the invention mainly detects the expression condition of the MMP2 gene of the growing up binary hetero sow on each tissue of the sow through qRT-PCR, constructing a eukaryotic expression vector containing a CDS region sequence of the MMP2 gene, synthesizing three pairs of siRNA of MMP2 genes, the overexpression and interference condition of MMP2 gene are detected by qRT-PCR, the influence of the overexpression or interference MMP2 gene on the ovarian granulosa cells of the sow is detected by a flow cytometer, and an ELISA kit is used for detecting the concentration change of E2 in the supernatant of the sow ovarian granule cells after overexpression or interference of MMP2 gene, then a PCR technology is used for obtaining the sequence of the promoter region of the sow MMP2 gene, a dual-luciferase reporter gene recombinant containing the deletion fragment of the promoter region of the MMP2 gene is constructed and transiently transfected into the ovary granule cells of the pig, and a dual-luciferase reporter system is used for analyzing the activity of the deletion fragment of the promoter region of the MMP2 gene and determining the core promoter region of the dual-luciferase reporter gene.
Compared with the prior art, the invention has the following advantages and effects:
the invention takes MMP2 gene as a research object, and adopts a cell biology method to research the expression application of the MMP2 gene in the porcine ovarian granulosa cells. The invention proves the expression condition of the pig MMP2 gene in the pig ovarian granulosa cells for the first time; and effects on sow ovarian granulosa cells after overexpression or interference of MMP2 in pig ovarian granulosa cells; the activity of the promoter of MMP2 gene was deleted in granulosa cells.
Drawings
FIG. 1 is a diagram showing the expression of porcine MMP2 gene in each tissue.
FIG. 2 is a graph of optimal transfection concentration of MMP2 gene in ovarian granulosa cells measured by qRT-PCR.
FIG. 3 is a graph of interference efficiency of qRT-PCR detection of MMP2 gene 3 on interfering small fragments.
FIG. 4 is a graph of the change in E2 levels following overexpression or interference with the MMP2 gene.
FIG. 5 is a diagram showing the analysis of the activity of the promoter deletion fragment of MMP2 gene.
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 state of the granular cells after about 24 hours, and preparing transfection when the confluence degree of the cells reaches about 80%;
(6) the transfection method is as followsInvitrogen corporation
3000 kit instructions; 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 3 qRT-PCR
The qRT-PCR detection of the gene in the invention adopts SYBR SYBR Green qPCRMix reagent kit of Thermo company in USA. 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 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- < - >
Wherein GAPDH is used as an internal reference for gene detection, and qRT-PCR primers used by the invention are as follows:
qRT-PCR-MMP2 Forward：5′-GCGAACTTGACCAGAGCACC-3′；
Reverse：5′-GAGCGAAGGCATCATCCACC-3′；
qRT-PCR-GAPDH Forward：5′-TCCCGCCAACATCAAAT-3′；
Reverse：5′-CACGCCCATCACAAACAT-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.
Reverse transcription PCR of mRNA was performed using the Thermo Scientific RevertAID first Strand cDNA Synthesis Kit from Thermo Co.
Example 4 FITC Annexin V Apoptosis Detection Kit with PI Detection
(1) The cells were collected by centrifugation at 1000rpm for 5min, the supernatant was discarded, and the cells were washed twice with pre-cooled PBS.
(2) 1 × Binding Buffer was added to resuspend the cells to a cell concentration of 1.0 × 106one/mL.
(3) And (3) sucking 100 mu L of the cell suspension into another centrifuge tube, sequentially adding 5 mu L of FITC-annexin V and 5 mu L of PI, lightly mixing uniformly, and reacting for 15min at room temperature (25 ℃) in a dark place.
(4) 400 μ L of 1 × Binding Buffer was added to each tube and the machine was used for flow analysis. The appropriate channel was selected (FL1 channel for FITC, FL3 or FL2 channel for PI).
Example 5 determination of E2 content in porcine ovarian granulosa cell supernatant samples by ELISA
The ELISA method for detecting the content of E2 in the porcine ovarian granulosa cell supernatant sample refers to a PigE2ELISA kit of CUSABIO company, and comprises the following specific operation steps:
(1) after 48h of transfection in 24-well plates, the cell culture fluid was aspirated into sterile 1.5mL centrifuge tubes and stored at-20 ℃ until use.
(2) Preparing a sample and a reagent;
(3) setting a blank contrast;
(4) add 50. mu.L of sample per well;
(5) adding 50 μ L of HRP-conjugate to each well of the sample group, adding no HRP-conjugate to the control group, adding 50 μ L of antibody to each well, and mixing well;
(6) incubating at 37 ℃ for 1 h;
(7) washed three times with 200. mu.L of Wash Buffer;
(8) add 50. mu.L of Substrate A and 50. mu.L of Substrate B to each well, mix well, incubate for 15min at 37 ℃;
(9) add 50 μ L of Stop Solution into each well, blow gently, mix well, measure the OD value of the complete sample within 10 min.
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 the lysate was added thereto
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. qRT-PCR detects the expression of MMP2 gene in each tissue of the large and large binary hetero sow, and the result is shown in figure 1. The expression of the MMP2 gene in the ovary is obviously higher than that of other tissues, which indicates that the MMP2 gene possibly plays an important role in the development process of ovarian follicles.
2. qRT-PCR detects the overexpression and interference effect of MMP2 gene in pig ovarian granulosa cells. The results are shown in FIGS. 2 and 3. The overexpression concentration selected in the research is 200ng, and the interference small fragment with the best interference effect is MMP 2-siRNA-2.
MMP2-siRNA-1：5′-GGUGUUCCACCACCUACAA-3′；
MMP2-siRNA-2：5′-GCGACAAGAAGUACGGCUU-3′；
MMP2-siRNA-3：5′-GCAAACAGGACAUCGUCUU-3′；
2. After the over-expression and interference of MMP2 gene, the apoptosis of ovary granular cells is detected by a flow cytometer, and after the over-expression and interference of MMP2 gene, the apoptosis of the ovary granular cells of the sow is not influenced.
3. The results of the change of the concentration of E2 in the culture solution of ovarian granulosa cells after the overexpression and the interference of MMP2 gene are detected by using an ELISA kit are shown in FIG. 4, and the result is that the production of E2 is promoted after the overexpression of MMP2 gene, and the production of E2 is inhibited after the interference of MMP2 gene.
4. Culturing the porcine ovarian granular cells, transiently transfecting porcine MMP2 gene promoter deletion fragment reporter gene recombinant plasmids into the porcine ovarian granular cells, taking pRL-TK as an internal reference, wherein the pRL-TK can provide constitutively expressed renilla luciferase, an internal reference value is provided for normalization of experimental firefly luciferase reporter genes, and dual-luciferase activity detection is carried out, wherein the result is shown in figure 5, and the detection result is obtained by analyzing the fluorescence activity of the porcine MMP2 gene promoter deletion fragment: the activity of the recombinant plasmids P0 and P1 is not obviously different, the difference between P1, P2 and P3 is obvious, and the activity of the promoter region of the porcine MMP2 gene is obtained by combining the situation of the CPG island of the porcine MMP2 gene promoter.
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
Application of MMP2 gene of <120> sow in promotion of generation of E2 in ovarian granulosa cells
<160>7
<170>SIPOSequenceListing 1.0
<210>1
<211>19
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>MMP2-siRNA-1
<400>1
gguguuccac caccuacaa 19
<210>2
<211>19
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>MMP2-siRNA-2
<400>2
gcgacaagaa guacggcuu 19
<210>3
<211>19
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>MMP2-siRNA-3
<400>3
gcaaacagga caucgucuu 19
<210>4
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>qRT-PCR- MMP2 Forward
<400>4
<210>5
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>qRT-PCR- MMP2 Reverse
<400>5
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<211>17
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>qRT-PCR- GAPDH Forward
<400>6
tcccgccaac atcaaat 17
<210>7
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>qRT-PCR- GAPDH Reverse
<400>7
cacgcccatc acaaacat 18
Claims (1)
1. SowMMP2A core promoter functional regulatory region of a gene characterized by:
the sowMMP2The core promoter function regulating region of the gene is located in the region of-1145 to-686 bp.
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