CN108559750B - Application of STAT3 in porcine ovarian granulosa cells - Google Patents
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Abstract
The invention discloses an application of STAT3 in a porcine ovary granular cell, which takes STAT3 as a research object, finds a core promoter region of STAT3 by constructing a porcine STAT3 gene promoter dual-luciferase reporter gene recombinant plasmid and through the expression activity of the porcine STAT3 gene promoter dual-luciferase reporter gene recombinant plasmid in the porcine ovary granular cell, verifies the interaction between a transcription factor C/EBP β and a STAT3 core promoter region, then constructs a C/EBP β overexpression vector and synthesizes small interfering RNA (C/EBP β -siRNA), detects the influence of the C/EBP β on STAT3, and finally respectively transfects the C/EBP β overexpression vector and the C/EBP β -siRNA to the granular cell to detect the apoptosis and proliferation of the cell.
Description
Technical Field
The invention belongs to the technical field of cell engineering and genetic engineering, and particularly relates to application of STAT3 in porcine ovarian granulosa cells.
Background
The ovary is an important organ determining the fertility of female animals and one of its main functions is follicular development and ovulation. Ovarian dysfunction, which may lead to decreased reproduction, includes Premature Ovarian Failure (POF), polycystic ovary syndrome (PCOS), and ovarian cancer. Among them, the main causes of premature ovarian failure: one is that enough primordial follicles are not formed during the embryonic period (congenital deficiency); secondly, the activation and inhibition of primordial follicles are influenced, and researches show that the acceleration of follicular atresia can also cause premature ovarian failure.
During the development of the follicle, the granulosa cells change from flat to cubic and then differentiate, and a series of changes play important roles in the growth and development of the oocyte, the initiation of primordial follicle growth, the regulation and control of follicle development in the growth period and follicle atresia. Furthermore, granulosa apoptosis is also an important mechanism for initiating follicular atresia.
The STAT3(The signal transducer and activator of transcription 3) protein, a member of The STAT family, has various biological functions including cell proliferation, differentiation, and apoptosis in various cell types. Studies have shown that the STAT3 gene is expressed in heart, lung, kidney, ovary, fallopian tube, and uterus tissues, particularly in reproductive tissues, at relatively high levels. In addition, high levels of STAT3 and proteins were detected in oocytes, granulosa cells, membrane cells, and stromal cells. The STAT3 pathway is activated by several ligands such as leptin and interleukin 6, and in mice, activated STAT3 (phosphorylated STAT3, pSTAT3) is involved in the physiological process of mammary gland degeneration caused by apoptosis. Another study showed that STAT3 is activated during atresia of bovine follicles. Leptin modulates steroid production by porcine ovarian granulosa cells, while addition of leptin to the cell culture medium increases the level of phosphorylated STAT 3. Epidermal growth factor is a regulator of granulosa cell function, and STAT3 is activated by EGF and then transferred from the cytoplasm to the nucleus of granulosa cells.
The current research only proves on the level of cells and tissues, but does not prove on live pigs, the main reason is that the change of certain phenotype of animals is caused by various factors, and in addition, the cost of live pig experiments is high under the condition of no high-confidence successful experiments.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides the application of STAT3 in porcine ovarian granulosa cells.
Another objective of the invention is to provide application of C/EBP β in regulation and control of STAT3 gene transcription.
It is still another object of the present invention to provide RNA small interfering fragments (siRNA) that inhibit C/EBP β.
The purpose of the invention is realized by the following technical scheme: application of STAT3 in porcine ovarian granulosa cells.
The application of C/EBP β in regulating STAT3 gene transcription, wherein transcription factor C/EBP β promotes the transcription activity of STAT3 core promoter.
The invention provides siRNA for inhibiting C/EBP β, which has the following sequence:
siRNA-C/EBPβ-1：5′-CCATGGAAGTGGCCAACTT-3′；
siRNA-C/EBPβ-2：5′-CCTCGCAGGTCAAGAGTAA-3′；
siRNA-C/EBPβ-3：5′-GGAACTTGTTCAAGCAGCT-3′；
the transcription factor C/EBP β promotes the transcription and protein synthesis of STAT3 gene, and the siRNA-C/EBP β inhibits the transcription and protein synthesis of STAT3 gene.
The application of C/EBP β in inhibiting the apoptosis of the porcine ovarian granulosa cells and/or promoting the proliferation of the porcine ovarian granulosa cells.
The verification results of the invention are as follows:
1. the specific values of firefly luciferase and renilla luciferase are measured by a multifunctional microplate reader, and the corresponding ratio is calculated to be the activity of each recombinant plasmid, the activity values of each deletion fragment in the 5' regulatory region of the Stat3 gene are shown in the figure, the results of the figure show that the activity of P3(-1034/+375) is remarkably lower than that of P2(-1532/+375) and P4(-587/+375), and the difference is very remarkable (P0.000178 and P0.000964), when the fragment-1532/-is deleted, the activity is remarkably reduced 1034, so that the fragment-1532/-1034 can be known to have a binding site of a forward regulatory element, when the fragment-1034/-587 is deleted, the activity is remarkably increased, so that the fragment-1537/-587 has a binding site of a reverse regulatory element, and the fragment-1532/-3 is a core gene region.
2. Firstly, the transcription factors of a-1532/-1034 region are predicted by using some bioinformatics websites such as TFBIND, JASPAR, Bimas and the like, and then verified by ChIP, the transcription factors of C/EBP β are mainly concerned, ChIP results show that an experimental group Ip and an Input group have strips and basically consistent brightness, a positive control group H3 has strips but a negative control group IgG has no strips, and in conclusion, C/EBP β identifies a-1397/-1387 region bound in a STAT3 promoter region.
3. The results of the figure show that the small interfering gene fragment is transfected into the particle cells, and finally C/EBP β -siRNA-2 small fragments with better interference effect are screened by qRT-PCR means for subsequent experiments.
siRNA-C/EBPβ-1：5′-CCATGGAAGTGGCCAACTT-3′；
siRNA-C/EBPβ-2：5′-CCTCGCAGGTCAAGAGTAA-3′；
siRNA-C/EBPβ-3：5′-GGAACTTGTTCAAGCAGCT-3′。
4. To study the effect of C/EBP β on STAT3 gene expression, we first co-transfected recombinant plasmid P2(-1532/+375) of the STAT3 forward regulatory region with C/EBP β overexpression vector (pcDNA3.1-C/EBP β) or small interfering RNA (C/EBP β 1-siRNA) to the granulosa cells to explore the effect of C/EBP β on STAT3 core promoter region transcription, then we transfected pcDNA3.1-C/EBP β or C/EBP β -siRNA to the granulosa cells, respectively, and analyzed the effect of C/EBP β siRNA on STAT3 gene expression at transcription and translation levels using Q-PCR and WB to analyze the effect of C/EBP β on STAT3 gene expression at the Q-PCR and WB NC. siRNA levels. β group fluorescence activity was significantly higher than that of the control group DNA3.1, C/EBP 2-siRNA group fluorescence activity was significantly lower than that of the control group siRNA, and the inhibition of the expression of PCDNAcDNAcDNA3/EBP 8672 gene transcription of STAT NC. was significantly lower than that of the PCDNA3-EBP 8672-siRNA, and the synthesis of STAT gene synthesis of PCDNAprotein.
5. We transfect pcDNA3.1-C/EBP β or C/EBP β -siRNA to particle cells respectively, and detect the influence of C/EBP β on particle cell apoptosis and proliferation by Annexin V-FITC method and Edu method respectively, the results show that the apoptosis rate (early apoptosis + late apoptosis) of pcDNA3.1-C/EBP β group is obviously lower than that of control group pcDNA3.1, the apoptosis rate of C/EBP β -siRNA group is obviously higher than that of control group siRNA-NC., the results of the other part show that the proliferation rate of pcDNA3.1-C/EBP β group is obviously higher than that of control group pcDNA3.1, the proliferation rate of C/EBP β -siRNA group is obviously lower than that of control group siRNA-NC., and C/EBP β can inhibit the apoptosis and promote proliferation of ovary particle cells.
Compared with the prior art, the invention has the following advantages and effects:
1. the invention firstly proves the influence of the transcription factor C/EBP β on the transcriptional control of the STAT3 gene, finds out the core promoter region of the STAT3 gene through a dual-luciferase report system, further finds out and verifies the influence of the transcription factor C/EBP β on the transcription of the STAT3 by using a chromatin immunoprecipitation technology (ChIP), a real-time quantitative PCR (Q-PCR) and a Western Blot (WB), and finally researches the influence of the C/EBP β on the apoptosis and proliferation of ovarian granulosa cells.
2. STAT3 may directly or indirectly participate in gonad development, oocyte maturation, ovum formation and follicle atresia, the invention takes STAT3 as a research object, and adopts molecular and cell biological methods to research the application of the STAT3 in porcine ovarian granulosa cells, C/EBP β can inhibit apoptosis of the ovarian granulosa cells and promote proliferation, and the STAT3 has good application value in the research of ovarian follicle atresia mechanism and the like.
3. In order to verify the transcriptional regulation and control effect of the transcription factor C/EBP β on STAT3 gene and the influence of the transcription factor C/EBP β on cell functions, the invention verifies from multiple levels and multiple angles, verifies the transcriptional activity, the mRNA level and the protein level, and finally verifies the functions in granular cells.
Drawings
FIG. 1 is a graph showing the dual fluorescence activity analysis of the recombinant vector of STAT3 promoter deletion fragment.
FIG. 2 is a graph showing the binding of ChIP-verified transcription factor C/EBP β in the STAT3 core promoter region, where M is 1500bpmarker, lane 1 is Input group, lane 2 is experimental group Ip, lane 3 is H3, and lane 4 is IgG.
FIG. 3 is a graph of small fragment interference efficiency of qRT-PCR detection 3 on C/EBP β interference.
FIG. 4 is a diagram for verifying transcriptional control of STAT3 by C/EBP β from three aspects of transcriptional activity, mRNA and protein level, wherein a is a diagram for promoting transcriptional activity of STAT3 core regulatory region by over-expressing C/EBP β, a diagram B is a diagram for promoting generation of mRNA of STAT3 by over-expressing C/EBP β, a diagram C is a diagram for inhibiting transcriptional activity of STAT3 core regulatory region by interfering C/EBP β, a diagram D is a diagram for inhibiting generation of mRNA of STAT3 by interfering C/EBP β, a diagram E is a diagram for promoting protein synthesis of STAT3 gene by over-expressing C/EBP β, and a diagram F is a diagram for inhibiting protein synthesis of STAT3 gene by interfering C/EBP β.
FIG. 5 is a graph showing the result of detecting apoptosis of granulosa cells by transcription factor C/EBP β by Annexin V-FITC method, wherein panels A-C show that overexpression C/EBP β inhibits granulosa cell apoptosis, and panels D-F show that C/EBP β is interfered to promote granulosa cell apoptosis.
FIG. 6 is a graph of Edu staining method for detecting the effect of transcription factor C/EBP β on the proliferation of granulosa cells, wherein, graphs A and B show that overexpression of C/EBP β promotes the proliferation of granulosa cells, and graphs C and D show that interference of C/EBP β inhibits the proliferation of granulosa cells.
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.
EXAMPLE 1 construction of STAT3 promoter reporter recombinant vector
(1) Designing a Primer by using Primer5, and amplifying a 5' regulatory region of STAT3 by using extracted pig ear DNA as a template; the amplified fragment was purified, recovered, ligated to pMD18T vector (purchased from Takara), transformed, screened, sequenced and identified as correct, and then a common plasmid was extracted and named T-STAT 3.
(2) The BioEdit software analysis finds that the STAT3 gene 5 'regulatory region sequence has no restriction enzyme cutting sites of MIu I and Xho I, and pG L-basic Vector has MIu I and Xho I cutting sites, Primer5 designs 5 primers for deleting fragments of the STAT 567 gene 5' regulatory region, the MIu I and Xho I cutting sites are respectively added on the upstream and downstream primers, T-STAT3 common plasmid is taken as a template, PCR amplification is carried out, and after the deletion fragments are purified, recovered, double-cut, connected with pG L3 Vector, transformed, screened and sequenced to identify the right, an endotoxin-free plasmid is extracted (the endotoxin-free plasmid miniprep kit is purchased from American Magen company and is respectively named as P1(-2199/+375), P2(-1532/+375), P3(-1034/+375), P4(-587/+375) and P5(-167 + 375).
The STAT3 gene 5' regulatory region primer used in the invention is as follows:
STAT3(-2199/+375)Forward：5′-TCCTCAACCCACCAAGAAAG-3′；
Reverse：5′-CTCCCGGTCTCTTCGTATCC-3′。
the deletion fragment primers used in the invention are as follows:
reverse: the same as above;
reverse: the same as above;
reverse: the same as above;
reverse: as above.
Note: the black bold font is the protecting base, the underlined is the restriction site.
Example 2 culture and transfection of ovarian granulosa cells
(1) Collecting ovaries of healthy sows in a Jiang He slaughterhouse of Guangzhou, placing the ovaries in a PBS (on ice) buffer solution containing 1% (v/v) double antibody, and quickly transporting the ovaries back to a laboratory for treatment;
(2) firstly, washing the ovary outside a cell chamber by PBS (phosphate buffer solution) containing double antibodies, placing the ovary in a beaker, and placing the beaker into a transfer window;
(3) wiping a cell room superclean bench with alcohol, clamping an ovary with forceps, sucking follicular fluid with an injector, and pumping into a centrifuge tube containing 5m L DMEM culture fluid, wherein each tube extracts follicular fluid to 9m L;
(4) centrifuging at 1000rpm for 5min, discarding the supernatant, adding 5m L PBS, blowing, beating, mixing, and washing twice;
(5) preparing a complete culture medium: 89% DMEM, 10% serum and 1% double antibody are mixed by turning upside down;
(6) add 5m L complete medium to resuspend the cell pellet;
(7) adding 10m L of complete culture medium into a 75m L culture flask, and then adding the resuspension;
(8) observing with microscope, and standing at 37 deg.C and 5% CO2Culturing in an incubator, observing the growth condition of granular cells after 24h, pouring out the culture medium when the granular cells grow to about 90%, and washing for 3 times by using preheated PBS (phosphate buffer solution) containing 1% (v/v) of double antibody;
(9) 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;
(10) washing with DMEM for 2 times, and centrifuging at 1000rpm/min for 5 min;
(11) 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;
(12) observing the cell state after about 24 hours, and preparing transfection when the confluence degree of the cells reaches about 80%;
(13) transfection method Invitrogen
3000 kit instructions, each set 3 replicates;
(14) the transfected well plates were placed at 37 ℃ in 5% CO2Culturing 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 3qRT-PCR
In the present invention, the qRT-PCR detection of the gene was performed using Maxima SYBR Green qPCR Master Mix (2X) kit (Thermo Scientific Co.). 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-STAT3Forward：5′-GGGCTTTATCAGTAAGGAGA-3′；
Reverse：5′-GGAATGTCAGGGTAGAGGTA-3′；
qRT-PCR-C/EBPβForward：5′-CGGACTGCAAGCGGAAGGAGGA-3′；
Reverse：5′-GGCTGGACGACGAGGATGTGGA-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 following steps were performed:
(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 0.2m L chloroform (every 1m L TRIzol) and violently shaking for 15-30 s, standing at room temperature for 5min, and centrifuging at 12000g at 4 ℃ for 15 min;
(4) absorbing the upper aqueous phase and placing the upper aqueous phase in a new RNase-free EP tube;
(5) adding 0.5m L isopropanol (per 1m L TRIzol), gently mixing by turning upside down, standing at room temperature for 10min, and centrifuging at 4 deg.C 12000g for 10 min;
(6) discarding the supernatant, placing at room temperature, adding 1m L75% ethanol-DEPC (L TRIzol per 1 m) 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.
PrimeScript from TaKaRa was usedTMThe RT Master Mix (Perfect Real Time) cDNA reverse transcription kit reverse transcribes total RNA.
Example 4 chromatin Co-immunoprecipitation (ChIP)
(1) Collecting ovaries of healthy sows in a slaughterhouse, extracting follicular fluid for culturing, and performing cell crosslinking treatment when the cell growth density reaches 70%;
(2) and (3) crosslinking: removing the original culture medium in the culture bottle, treating the cells with a mixed solution of formaldehyde, glycine and PBS-HaltCocktail (PBS buffer solution containing protease inhibitor Halt Cocktail), and centrifuging to collect cell precipitates;
(3) lysis Digestion, adding L sys Buffer 1(ABClonal-ChIP Assay Kit purchased from ABClonal) for heavy suspension precipitation, centrifuging at 10000g for 10min, collecting the precipitate, adding MNase digest Buffer working Solution, Micrococcus nucleic acid, MNase Stop Solution, L sys Buffer 2, centrifuging at 10000g for 10min, and collecting the supernatant;
(4) performing immunoprecipitation, namely taking 45 mu l of supernatant, diluting with 450 mu l of 1 × IP Dilution Buffer, transferring to a binding column, adding an antibody, and correspondingly incubating, wherein ChIP Grade Protein A/G resin, IP Wash Buffer 1, IPWash Buffer 2 and IP Wash Buffer 3 are respectively added into the binding column, and residual liquid is removed by emptying;
(5) IP and DNA recovery: adding IP Elution Buffer, NaCl and proteinase K into the combined column respectively for heat shock treatment; after DNA Binding Buffer, Column Wash Buffer, ddH2O, recovering DNA;
(6) and (3) qualitative PCR: a PCR primer is designed by taking an area within 1.5kb before a transcription initiation site of STAT3 gene as a template sequence, and the product range is 100-160 bp. The primer sequence and the PCR reaction system are as follows;
the qualitative PCR primers used in the invention are as follows:
STAT3Forward：5′-ATAGCTATCCTTGGGGAGG-3′；
Reverse：5′-AAGGGCCTGTTATCTCAC-3′；
GAPDH Forward：5′-GATGTCCTGAGCCCCTACAG-3′；
Reverse：5′-GGTAGGTGATGGGGACTGAG-3′；
the reaction system is as follows: ddH2O7. mu.l, upstream and downstream primers 0.5. mu.l, DNA 2. mu.l, 2xPCR Premix 10. mu.l.
Example 5Western Blot
(1) Extracting total protein of monolayer adherent cells (ovary granular cells in example 2), namely pouring out cell culture solution, adding a proper amount of precooled PBS (phosphate buffer solution) to wash the cells for three times to wash out the culture solution, abandoning the PBS and placing a culture bottle on ice, adding 100-fold protein lysate of 200 mu L and PMSF of 10 mu L100 mM into each hole of 6-hole plate cells, cracking the cells for 30min, collecting the cell lysate, transferring the cell lysate into a 1.5m L centrifuge tube, centrifuging at 14000rpm at 4 ℃ for 5min, adding part of supernatant into a sample buffer, boiling for 10min, slowly recovering the room temperature, slightly centrifuging, and storing at-20 ℃;
(2) SDS-PAGE electrophoresis, wherein after the protein sample is preliminarily quantified by the BCA method, 20 mu g of total protein and 5 × sample buffer solution are mixed according to the ratio of 5:1 in each group, and the mixture is boiled for 5 min;
(3) protein transfer: and (3) pretreating the PVDF membrane for 3-5 s by using methanol, and soaking the PVDF membrane in a transfer printing liquid for 30 min. Taking out the gel, and placing the gel on filter paper to form a sandwich structure of a gel transfer printing accumulation layer. This operation must remove the bubbles completely. Constant pressure of 100V for 60-120 min;
(4) immunoblotting: the hybridization membrane was removed, rinsed for 5min in TBST and repeated three times. 5% (w/w) skimmed milk powder solution was blocked at room temperature for 90min, and TBST rinsed for 5min, repeated three times. STAT3 primary antibody (STAT3 primary antibody, purchased from Aibisin) (1:500) was added and incubated overnight at 4 ℃ and the membranes were washed for 5min in TBST three times. Add secondary antibody dilution (secondary antibody: Goat anti-rabbitIgG-HRP), buy from SANTA CRUZ) (1:5000) incubate for 1h at room temperature, TBST membrane washing 5min, three times. The membrane was rinsed with distilled water for 2min, three times. Wiping off the liquid on the PVDF film, adding a chemiluminescent agent, placing on a film, placing in an X-ray cassette, and placing in a dark room. Opening the film box in the darkroom, putting in the film, opening the film box after 5min, and taking out the film. Protein bands in the films were analyzed using Image Plus software.
Example 6 granular cell apoptosis assay
The Annexin V-FITC technology for detecting the Apoptosis refers to the Annexin V-FITC Apoptosis Detection Kit operating instruction of BioVision, and comprises the following specific operating steps:
(1) placing the cell culture plate at room temperature, gently rinsing the cells in the culture plate with 2m L PBS solution, and removing the PBS solution;
(2) adding pancreatin-digested cells without EDTA, and gently resuspending the cells in the medium of step (1) to a density of about 1 × 106Cells/m L;
(3) 0.5m L cell suspension was removed from the cell culture plate (about 5 × 10)5Individual cells) were transferred into a clean centrifuge tube and 500 μ L1 × Binding Buffer was added;
(4) adding 5 mu L Annexin V-FITC and 5 mu L propidium iodide at room temperature;
(5) reacting at room temperature in dark for 5 min;
(6) analysis was immediately performed using a FACSCalibur flow cytometer (triplicates per group).
Example 7 granulosa cell proliferation assay
The invention uses EdU method to detect Cell proliferation, refer to Cell-L light of Ruibo corporationTMThe EdU Apollo 567Invitro Kit detection Kit comprises the following specific operation steps:
(1) preparation of 50 μ M EdU medium: culturing the cells in a cell culture medium at a temperature of 1: diluting the EdU solution at a ratio of 1000;
(2) discarding the culture solution when the cell fusion degree is 50-80%, and adding 100 mu L50 mu M EdU culture medium to incubate for 2 h;
(3) cell fixing, namely removing the culture solution, adding 100 mu L of cell fixing solution (4% paraformaldehyde PBS) into each hole, and incubating for 15-30 min at room temperature;
(4)2mg/m L glycine incubation for 10min, PBS washing 2 times;
(5) discarding the supernatant, adding 100 μ L penetrating agent (0.5% (v/v) TritonX-100 PBS) to permeabilize the cells, and washing with PBS for 1 time;
(6) EdU test with addition of 100. mu. L
Dyeing reaction liquid, incubating for 30min at room temperature in a dark place, washing for 1 time by PBS, precipitating cells, and removing supernatant;
(7) DNA staining, namely adding 100 mu L DAPI reaction solution into each hole, and incubating for 30min at room temperature in a dark place;
(8) adding 100 mu L penetrating agent (0.5% (v/v) TritonX-100 PBS) to wash for 3 times, and eluting DAPI reaction solution;
(9) fluorescence microscopy (triplicates per group).
Example 8 luciferase reporter Activity assays
The luciferase reporter gene activity detection refers to a Dual-L luciferase reporter assay System kit of Promega corporation, 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 Glo L ysis Buffer was added to each well of cells, shaken slightly for 5min at room temperature, and cell lysates were collected;
(2) after 30. mu. L cell lysate was added to a 96-well plate, 75. mu. L was added thereto
L mutual Assay Reagent, standing for 15-30 min at 20-25 ℃ after mixing, detecting a luminescence value on a Synergy 2 multifunctional microplate reader of BioTek company, wherein the luminescence value corresponds to the expression level of firefly luciferase;
(3) then 75 mu L Stop was added&
And uniformly mixing the reagents, 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. the specific values of firefly luciferase and renilla luciferase are measured by a multifunctional microplate reader, and the corresponding ratio is calculated, namely the activity of each recombinant plasmid, the activity values of each deletion fragment in the 5' regulatory region of the Stat3 gene are shown in fig. 1, the results of the figure show that the activity of P3(-1034/+375) is remarkably lower than that of P2(-1532/+375) and P4(-587/+375), the difference is very remarkable (P ═ 0.000178 and P ═ 0.000964), when the fragment-1532/-1034 is deleted, the activity is remarkably reduced, so that the fragment-1532/-1034 has a binding site of a forward regulatory element, when the fragment-1034/-587 is deleted, the activity is remarkably increased, so that the fragment-587/-587 has a binding site of a reverse regulatory element, the fragment-1532/-1537 is a core region of the gene/-15378, and the further research is carried out on the activity of the fragment-1532/-1034.
2. We first used TFBIND, JASPAR, Bimas, etc. bioinformatics websites to predict the transcription factor of the-1532/-1034 region, and then verified by ChIP, we mainly focused on the transcription factor C/EBP β, ChIP results show (FIG. 2) that the experimental group Ip and Input groups both have bands and have basically consistent brightness, the positive control group H3 has bands but the negative control group IgG has no bands, and together, C/EBP β identifies the-1397/-1387 region bound to the STAT3 promoter region.
3. Synthesizing 3 pairs of small interfering C/EBP β fragments/controls (C/EBP β -siRNA/siRNA-NC), screening and detecting the interference efficiency, and finally screening C/EBP β -siRNA-2 fragments with better interference effect for subsequent experiments by transfecting gene interfering fragments into particle cells and using a qRT-PCR method as shown in the result of figure 3.
siRNA-C/EBPβ-1：5′-CCATGGAAGTGGCCAACTT-3′；
siRNA-C/EBPβ-2：5′-CCTCGCAGGTCAAGAGTAA-3′；
siRNA-C/EBPβ-3：5′-GGAACTTGTTCAAGCAGCT-3′。
The small interference fragments are synthesized by Ribo Biotech, Inc., Guangzhou; control siRNA-NC was from Ribo Biotech, Inc., Guangzhou.
4. To study the effect of C/EBP β on STAT3 gene expression, we first co-transfected the recombinant plasmid P2(-1532/+375) of the forward regulatory region of STAT3 with the C/EBP β overexpression vector (pcDNA3.1-C/EBP β) or small interfering RNA (C/EBP β -siRNA) to the particle cells to search the effect of C/EBP β on STAT3 core promoter region transcriptional activity, wherein the construction method of the C/EBP β overexpression vector is to amplify CDS region (protein coding region) of C/EBP β by PCR, extract the normal plasmid after verifying the correctness of the sequencing, extract the endotoxin-free plasmid after verifying the correctness of the sequencing by double digestion, ligation, transformation, and single clone sequencing of the normal plasmid and pcDNA3.1 vector, finally perform double digestion on the endotoxin-free plasmid extracted, verify whether the construction of the C/EBP 2 overexpression vector succeeds, and the expression of the transfection of the gene expression vector, and the expression of the mRNA of pcDNA3.7-EBP 8672-cDNA group mRNA showed that the expression of the PCR-EBP promoter protein is significantly lower than that the PCR-HBP- β gene synthesis of the PCR-HBCP-HBP promoter region and/siRNA promote the expression of the PCR-EBP β gene synthesis of the PCR-HBCP-HBP gene synthesis of the PCR-HBP- β group mRNA showed significant inhibition group mRNA-7-HBeB-7-HBeB-7 gene expression.
5. We transfected pcDNA3.1-C/EBP β or C/EBP β -siRNA to the granular cells respectively, and detected the influence of C/EBP β on granular cell apoptosis and proliferation by Annexin V-FITC method and Edu method respectively, the results showed that the apoptosis rate (early apoptosis + late apoptosis) of pcDNA3.1-C/EBP β group was significantly lower than that of the control group pcDNA3.1 (FIG. 5), the apoptosis rate of C/EBP β -siRNA group was significantly higher than that of the control group siRNA-NC (FIG. 5), and the other results showed that the proliferation rate of pcDNA3.1-C/EBP β group was significantly higher than that of the control group pcDNA3.1 (FIG. 6), and the proliferation rate of C/EBP β -siRNA group was significantly lower than that of the control group siRNA-NC (FIG. 6).
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 <120> STAT3 in porcine ovarian granulosa cells
<160>25
<170>SIPOSequenceListing 1.0
<210>1
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>siRNA- C/EBPβ-1
<400>1
ccatggaagt ggccaactt 19
<210>2
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>siRNA- C/EBPβ-2
<400>2
cctcgcaggt caagagtaa 19
<210>3
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>siRNA- C/EBPβ-3
<400>3
ggaacttgtt caagcagct 19
<210>4
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>STAT3（-2199/+375）Forward
<400>4
<210>5
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>STAT3（-2199/+375）Reverse
<400>5
<210>6
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>P1（-2199/+375）Forward
<400>6
cgacgcgttc ctcaacccac caagaaag 28
<210>7
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>P1（-2199/+375）Reverse
<400>7
ccctcgagct cccggtctct tcgtatcc 28
<210>8
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>P2（-1532/+375）Forward
<400>8
cgacgcgtct ccaagtcatt gattttct 28
<210>9
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>P2（-1532/+375）Reverse
<400>9
ccctcgagct cccggtctct tcgtatcc 28
<210>10
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>P3（-1034/+375）Forward
<400>10
cgacgcgtta ctaaacaaac acaataaa 28
<210>11
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>P3（-1034/+375）Reverse
<400>11
ccctcgagct cccggtctct tcgtatcc 28
<210>12
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>P4（-587/+375）Forward
<400>12
cgacgcgtct gaggttcaaa gcaggcgg 28
<210>13
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>P4（-587/+375）Reverse
<400>13
ccctcgagct cccggtctct tcgtatcc 28
<210>14
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>P5（-167/+375）Forward
<400>14
cgacgcgtct ctcctcattg gcaagtgg 28
<210>15
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>P5（-167/+375）Reverse
<400>15
ccctcgagct cccggtctct tcgtatcc 28
<210>16
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>qRT-PCR-STAT3 Forward
<400>16
<210>17
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>qRT-PCR-STAT3 Reverse
<400>17
<210>18
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>qRT-PCR-C/EBPβ Forward
<400>18
cggactgcaa gcggaaggag ga 22
<210>19
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>qRT-PCR-C/EBPβ Reverse
<400>19
ggctggacga cgaggatgtg ga 22
<210>20
<211>17
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>qRT-PCR-GAPDH Forward
<400>20
tcccgccaac atcaaat 17
<210>21
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>qRT-PCR-GAPDH Reverse
<400>21
cacgcccatc acaaacat 18
<210>22
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>STAT3 Forward
<400>22
atagctatcc ttggggagg 19
<210>23
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>STAT3 Reverse
<400>23
aagggcctgt tatctcac 18
<210>24
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>GAPDH Forward
<400>24
<210>25
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>GAPDH Reverse
<400>25
Claims (4)
- Use of C/EBP β for inhibiting apoptosis and/or promoting proliferation of porcine ovarian granulosa cells in vitro.
- 2. The use according to claim 1, wherein C/EBP β is inhibited by siRNA, wherein the sequence of the siRNA inhibiting C/EBP β is as follows:siRNA- C/EBPβ-1：5′- CCATGGAAGTGGCCAACTT -3′。
- 3. the use according to claim 1, wherein C/EBP β is inhibited by siRNA, wherein the sequence of the siRNA inhibiting C/EBP β is as follows:siRNA- C/EBPβ-2：5′- CCTCGCAGGTCAAGAGTAA -3′。
- 4. the use according to claim 1, wherein C/EBP β is inhibited by siRNA, wherein the sequence of the siRNA inhibiting C/EBP β is as follows:siRNA- C/EBPβ-3：5′- GGAACTTGTTCAAGCAGCT -3′。
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