Application of transcription factor YY1 in endothelial cell injury repair

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of a transcription factor YY1 in endothelial cell injury repair. The research shows that YY1 can regulate the expression of KDR and promote the repair process after endothelial cell injury by regulating an HIF-1alpha / VGEF / KDR signal. The invention provides a new therapeutic target for repairing endothelial cell injury, and further explores the relationship and potential action mechanism between YY1 and cardiovascular diseases, thereby providing a new direction and potential therapeutic target for prevention and treatment of cardiovascular diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of transcription factor YY1 in repairing endothelial cell damage. Background Art

[0002] Endothelial cells in the heart undergo apoptosis due to hypoxia, which may lead to myocardial dysfunction. Endothelial cell damage caused by hypoxia is one of the common pathogenesis of cardiovascular disease (CVD). Coronary heart disease, heart failure, and hypertension are all common types of cardiovascular diseases. CVD is one of the diseases with the highest morbidity and mortality rates in the world, ranking at the forefront of all diseases. According to statistics, a large number of people die from cardiovascular diseases every year. Although the diagnosis and treatment technologies of cardiovascular diseases have been continuously improved in recent years, the treatment effect is still not ideal, the recurrence rate and complication rate of cardiovascular diseases are still high, and the quality of life and life expectancy of patients are significantly affected.

[0003] Endothelial cells are an important part of the cardiovascular system, and their integrity plays a key role in maintaining normal vascular function. Endothelial cells will produce oxidative stress response, inflammatory response and other damage under hypoxic conditions, which will affect the normal function of cells, such as limiting cell proliferation and changing migration ability. The imbalance of vascular structure and the destruction of functional homeostasis caused by hypoxia are one of the key pathophysiological mechanisms that cause various functional disorders in organs and systems. The repair process after endothelial cell damage plays an important role in angiogenesis, regulating inflammatory response, and maintaining its normal physiological function. When the oxygen content is reduced, the oxygen level in the cell decreases, causing endothelial cells to activate a series of survival pathways to adapt to hypoxia. These include increased expression of hypoxia-inducible factor-1A (HIF-1A) and vascular endothelial growth factor (VEGF), thereby activating downstream pathways and promoting the proliferation and migration of endothelial cells. Therefore, studying the molecular mechanism of repair of vascular endothelial cells (VECs) after hypoxia-induced injury is of great significance for understanding the pathogenesis of cardiovascular diseases and discovering new treatments.

[0004] Vascular endothelial growth factor type II receptor (KDR), also known as VEGFR-2 (Vascular Endothelial Growth Factor Receptor-2), plays a key role in the physiological functions and pathological reactions of endothelial cells. It is mainly expressed on the surface of endothelial cells and is an important target for VEGF to exert its biological effects. When endothelial cells are damaged and stimulated, the level of VEGF in the local microenvironment increases, and VEGF binds to KDR on endothelial cells to activate a series of complex signaling pathways. This not only promotes the proliferation of endothelial cells and helps repair damaged endothelium, but also enhances the migration ability of endothelial cells, prompting them to move to the site of injury, thereby restoring the integrity of the endothelium to a certain extent. At the same time, the activation of the KDR signaling pathway is also involved in the process of new blood vessel formation, which is of positive significance for establishing collateral circulation and improving myocardial perfusion in cases such as ischemic cardiovascular disease.

[0005] Transcription factor YY1 (Yin Yang 1) is a multifunctional zinc finger protein, a member of the GLI-Kruppel family, and part of the polycomb complex. YY1 has a dual transcriptional effect, promoting or inhibiting gene expression depending on the target gene and other auxiliary factors it binds to. YY1 has been shown to be associated with angiogenesis, but its mechanism of action in the repair of endothelial cell damage is not clear. Summary of the invention

[0006] The purpose of the present invention is to provide an application of transcription factor YY1 in repairing endothelial cell damage in view of the above problems.

[0007] In order to achieve its purpose, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides the use of transcription factor YY1 as a drug target in screening drugs for repairing endothelial cell damage.

[0009] The second aspect of the present invention provides the use of transcription factor YY1 as a drug target in screening drugs for treating cardiovascular diseases induced by endothelial cell injury.

[0010] The endothelial cell injury is hypoxia-induced endothelial cell injury.

[0011] The drug promotes the expression of transcription factor YY1.

[0012] YY1 promotes the repair process of endothelial cells after injury by regulating the HIF-1α / VEGF / KDR signaling pathway.

[0013] YY1 positively regulates VEGF and KDR.

[0014] The third aspect of the present invention provides the use of an agent for promoting the expression of transcription factor YY1 in the preparation of a drug for repairing endothelial cell damage.

[0015] A fourth aspect of the present invention provides use of an agent for promoting the expression of transcription factor YY1 in the preparation of a drug for treating cardiovascular diseases induced by endothelial cell injury.

[0016] The reagent for promoting the expression of transcription factor YY1 is an overexpression vector of YY1.

[0017] Preferably, the overexpression vector is a lentivirus pLV3-CMV-YY1(human)-3×FLAG-Puro recombinant vector.

[0018] The beneficial effects of the present invention are:

[0019] In the present invention, bioinformatics methods are used to predict the binding sites of YY1 and KDR promoter regions, and molecular biology experiments are used to verify that the two are in a positive regulatory relationship. By constructing a hypoxia-induced endothelial cell injury model, the important role of YY1 in the repair process of endothelial cell injury is further verified. Studies have found that YY1 is upregulated in hypoxia-induced human umbilical vein endothelial cells (HUVECs). Under hypoxic conditions, downregulation of YY1 can inhibit the cell migration of HUVECs, while overexpression of YY1 has no significant effect on the cell migration of HUVECs. Mechanistically, we found that YY1 induced increased expression of VEGFA and KDR. Therefore, our data show that YY1 can regulate the expression of KDR and promote the repair process of endothelial cells after injury by regulating HIF-1α / VGEF / KDR signals. The present invention provides a new therapeutic target for repairing endothelial cell damage, and further explores the relationship and potential mechanism of action between YY1 and cardiovascular disease, thereby providing new directions and potential therapeutic targets for the prevention and treatment of cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The results showed that KDR was directly regulated by transcription factor YY1: (A) Through bioinformatics analysis, YY1 was determined from four transcription factor databases to be one of the prospective transcription factors that promote KDR expression; (B) The Jaspar database was used to predict the YY1 binding site in the KDR promoter; (C) qRT-PCR technology was used to analyze the situation after YY1 knockdown in HUVECs, and the mRNA expression levels of YY1 and KDR were measured. Statistical analysis showed that the two showed a linear positive correlation; (D) WB data showed that the protein expression level of KDR in HUVECs was also significantly reduced after YY1 knockdown; the data of three independent experiments are expressed as the mean ± SD in (CD), *P < 0.05, **P < 0.01, ***P < 0.001.

[0021] Figure 2 The construction of the endothelial cell hypoxic injury and repair model is shown: (A) The expression of the hypoxia marker HIF-1α gradually increased within 12 hours of hypoxia stimulation (n=3); (B) The proliferation ability of cells was detected by CCK-8, and the proliferation ability of cells under hypoxia stimulation tended to decrease compared with that under normoxic treatment conditions (n=3); (C) After 12 hours of hypoxia treatment, the cells were reoxygenated, and it was observed that the proliferation ability of cells gradually recovered within 36 hours of reoxygenation (n=3); (D) Flow cytometry data showed that hypoxia stimulation increased the number of early and late apoptotic cells in HUVECs compared with normoxic conditions (n=3); (E) In the wound healing assay, HUVECs were treated under normoxic or hypoxic conditions for 24 hours, and photographed at 0, 12 and 24 hours (n=5); The data of three independent experiments are expressed as the mean ± SD in (AE), *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 3 The expression levels of YY1 and KDR in HUVECs under hypoxic conditions are shown: (AC) Expression of VEGFA, YY1 and KDR mRNA after hypoxic stimulation (n=3); (D) Western blot analysis of YY1 and KDR protein expression within 12 hours of hypoxia treatment (n=3); The data of three independent experiments are expressed as the mean ± SD in (AD), *P<0.05, **P<0.01, ***P<0.001.

[0023] Figure 4The role of YY1 in the repair of hypoxia-induced endothelial cell injury is shown: (AB) qPCR detection of YY1 and KDR mRNA expression after YY1 gene downregulation and overexpression (n=3); (CD) CCK-8 detection of cell proliferation after YY1 gene knockout and overexpression in HUVECs treated with hypoxia-reoxygenation or normoxia (n=6); (E) In the wound healing assay, HUVECs treated with si-NC / siYY1 or CMV / OE-YY1 were Cs for 24 hours, and photographed at 0, 12 and 24 hours (n=6), respectively. It was found that after knocking down YY1, the migration ability of cells was significantly reduced, while overexpression of YY1 had no significant effect on it; (F) Transwell experiment showed that after knocking down YY1, the cell migration phenomenon was significantly weakened compared with the normoxic group and hypoxic group (n=3); The data of three independent experiments are expressed as the mean ± SD in (AF), *P<0.05, **P<0.01, ***P<0.001.

[0024] Figure 5 The results show that YY1 promotes the repair process after endothelial cell injury by regulating the VEGFA / KDR signaling pathway: (A and C) qRT-PCR was used to detect the changes in VEGFA\YY1\KDR mRNA expression in HUVECs after hypoxia / reoxygenation with si-NC / si-YY1 and CMV / OE-YY1 (n=3); (B and D) qRT-PCR was used to detect the changes in VEGFA\YY1\KDR mRNA expression in normal HUVECs in the si-NC / si-YY1 and CMV / OE-YY1 groups (n=3); the data of three independent experiments are expressed as the mean ± SD in (AD), *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0027] The research route of the present invention is:

[0028] (1) Transcriptional regulation of YY1 on KDR in human umbilical vein endothelial cells

[0029] In order to explore the transcriptional regulatory mechanism of KDR, the transcription factors in the promoter region of KDR were predicted and identified by bioinformatics analysis combined with experimental verification.

[0030] (2) Establishment of a model of endothelial cell repair after hypoxic injury

[0031] To construct an endothelial cell hypoxia injury and repair model to simulate the effects of hypoxia on endothelial cells and the subsequent repair process in vivo, and to provide a suitable cell model for studying related molecular mechanisms. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia, and then the cells were transferred to normal culture conditions for reoxygenation culture, and a corresponding control group was set up. In terms of detection indicators, Western blotting technology was used to detect the protein expression of the hypoxia marker HIF-1α to determine whether hypoxia effectively activated the relevant signaling pathways; flow cytometry combined with Annexin V-FITC and PI Apoptosis Detection Kit was used according to standard procedures to detect early and late cell apoptosis data and evaluate the effects of hypoxia on cell survival status; CCK-8 detection kit was used to observe the changes in cell proliferation ability during hypoxia treatment and reoxygenation; wound healing experiments were used to evaluate the effects of hypoxia on cell migration ability and the degree of recovery of cell migration ability after reoxygenation.

[0032] (3) Detection of the expression changes of YY1 and KDR in HUVEC under hypoxic conditions

[0033] In order to further explore the expression changes of YY1, VEGFA and KDR in HUVECs under hypoxia and the regulatory effect of YY1 on KDR expression, we first set up normoxic culture group and hypoxic culture group, and used qRT-PCR to detect the changes in mRNA levels of molecules. At the same time, Western blotting technology was used to verify the expression change pattern of YY1 in hypoxia stress at the protein level, and the dynamic changes of related molecules in the hypoxia process were comprehensively analyzed by combining the detection results of mRNA and protein levels, thereby confirming the regulatory effect of YY1 on KDR expression under hypoxia.

[0034] HUVECs were exposed to hypoxia and normoxic environments for 3h, 6h, 9h and 12h, respectively. The detection found that during the 12-hour hypoxia, the mRNA level of VEGFA gradually increased, and the protein and mRNA levels of YY1 were upregulated under hypoxia. At the same time, the mRNA expression levels of KDR and YY1 were dynamically upregulated during the hypoxia process, further confirming the regulatory role of YY1 on KDR expression under hypoxic conditions.

[0035] (4) To investigate the role of YY1 in the repair of hypoxia-induced endothelial cell damage

[0036] First, we constructed YY1 knockdown (siYY1) and overexpression (oeYY1) models. Subsequently, we detected the expression levels of related molecules in cells under different treatment conditions. In HUVECs with YY1 knockdown, KDR expression was found to be significantly reduced; while in cells overexpressing YY1, KDR expression increased, which is consistent with the regulatory effect of YY1 on KDR. Next, we used CCK8, scratch and Transwell experimental methods to detect cell proliferation and migration ability.

[0037] (5) YY1 promotes endothelial cell injury and repair by regulating VEGF / KDR signaling

[0038] To elucidate the potential mechanism of YY1 in the repair of hypoxia-induced endothelial cell injury, the mRNA expressions of YY1, VEGFA and KDR in the hypoxia / reoxygenation model and normoxic conditions were detected by knocking down and overexpressing YY1, respectively.

[0039] Through the above research, the following results were obtained:

[0040] (1) YY1 binds to the KDR promoter and regulates its expression.

[0041] (2) The endothelial cell hypoxia injury and repair model was successfully constructed.

[0042] (3) Expression levels of YY1 and KDR in HUVECs under hypoxic conditions.

[0043] (4) The role of YY1 in the repair of hypoxia-induced endothelial cell damage.

[0044] (5) YY1 promotes the repair process of endothelial cells after injury by regulating the VGEF / KDR signaling pathway.

[0045] The specific research process is as follows:

[0046] Example 1

[0047] 1 Materials and methods

[0048] 1.1 Cells

[0049] Human umbilical vein endothelial cell line (HUVEC-C, CVCL_2959) was used for the experiment.

[0050] 1.2 Methods

[0051] 1.2.1 Bioinformatics prediction analysis

[0052] The sequence of -200-2000bp of KDR gene was downloaded from NCBI as the promoter region. Bioinformatics databases such as PROMO and hTFtarget were used to predict the transcription factors that regulate the transcription of KDR gene. The transcription factor YY1 was identified through subsequent screening and analysis. The binding site of YY1 in the promoter region of KDR gene was predicted using Jaspar database, and it was found that YY1 was most likely to bind to the 1612bp-1617bp region in KDR promoter. Gene ID of YY1 in NCBI: 7528.

[0053] 1.2.2 RNA extraction and reverse transcription

[0054] Conventional method.

[0055] 1.2.3 qPCR reaction

[0056] 1.2.3.1 qPCR reaction system:

[0057]

[0058] 1.2.3.2 qPCR reaction conditions:

[0059]

[0060] The 2-ΔΔct method was used for quantitative analysis. Three replicate wells were set for each sample, and the experiment was repeated three times. GADPH was used as an internal reference. The primer sequences are as follows:

[0061]

[0062]

[0063] 1.2.4 Model establishment

[0064] 1.2.4.1 Establishment of endothelial cell hypoxia injury model

[0065] In order to establish a model of hypoxia-induced cell injury, the cells were replaced with sugar-free serum-free DMEM medium and exposed to hypoxia containing 1% O2, 5% CO2 and 94% N2 for 12 hours. The cells in the control normoxic group were also cultured in sugar-free serum-free DMEM medium at 37°C, 5% CO2 and 95% air. Under an optical microscope, compared with the normoxic environment, the number of cells attached to the culture plate and the number of suspended dead cells increased significantly under hypoxia.

[0066] 1.2.4.2 Establishment of the repair model of endothelial cells after hypoxic injury

[0067] After the cells were subjected to hypoxia injury for 12 h, the cells were replaced with DMEM medium containing 10% FBS and cultured in an incubator at 37° C. and 5% CO 2 .

[0068] 1.2.5 Western Blotting Experiment

[0069] Each group of cells was lysed in RIPA lysis buffer containing 1% PMSF, and then the cells were broken by ultrasound, and the supernatant was collected by high-speed centrifugation. After the protein concentration was determined by BCA protein detection kit, 20μg of protein was separated by 10% SDS-PAGE and then transferred to PVDF membrane. Blocked with 5% skim milk at room temperature for 3 hours, the strips were washed three times with TBST for 10 minutes each time, placed in primary antibody dilution, and incubated overnight on a shaker at 4℃. After primary antibody incubation, the strips were washed three times with TBST for 10 minutes each time. The strips were placed in HRP-coupled secondary antibody dilution and incubated at room temperature for 1 hour. The strips were removed and washed three times with TBST for 10 minutes each time. The protein expression on the cell membrane was detected by ECL chemiluminescence system. The gray value of the strips was analyzed using Image J software analysis system, and the relative expression of the target protein was normalized to the relative expression of β-actin.

[0070] 1.2.6 CCK-8 assay

[0071] The CCK-8 method was used to detect the in vitro proliferation of cells. Trypsin-digested HUVECs were seeded into 96-well plates at a density of 5000 cells per well. After the cells were treated differently, the spent culture medium was removed. Subsequently, 200 μL of fresh culture medium was added to each well, followed by 10 μL of CCK-8 solution. After incubation at 37°C for 30 min, the absorbance at 450 nm was measured using a microplate reader.

[0072] 1.2.7 Scratch test

[0073] Trypsinized cells were seeded into six-well plates and treated with different treatments according to the experimental design. Subsequently, a 200 μl sterile micropipette tip was used to make a scratch on the cell surface in the culture dish. The wound healing was monitored at different time points using an inverted microscope and photographed.

[0074] 1.2.8 Transwell experiment

[0075] First, the chamber was placed in a 24-well plate, and each group of cells was trypsinized and then 5The cells were resuspended in serum-free medium at a density of 100 cells / mL. Subsequently, 150 μL of the cell suspension was carefully added to the upper chamber, and 600 μL of complete growth medium supplemented with 10% fetal bovine serum was added to the lower chamber. The culture dish was then placed in a tri-gas incubator and cultured under hypoxic or normoxic conditions at 37°C and 5% CO2 atmosphere for 8 hours to promote the migration of cells through the holes. At the end, the non-migrated cells in the upper chamber were removed with a cotton swab. The migrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Finally, the chamber was inverted to air dry overnight and photographed under an inverted microscope.

[0076] 1.2.9 Cell apoptosis experiment

[0077] When using the Annexin-V FITC / PI cell apoptosis detection kit, first take an appropriate amount of phosphate buffer to gently and thoroughly wash each group of cells twice, then digest with trypsin, discard the trypsin, add 1 mL PBS to resuspend and centrifuge at 1000 rpm for 5 minutes, discard the supernatant to collect the cells; then resuspend the cells with 100 μl 1xBinding Buffer, add 10 μl PI and 5 μl Annexin-V FITC to mix, incubate on ice away from light for 15 minutes, and finally add 400 μl 1xBinding Buffer to mix, and use flow cytometry to detect the apoptosis rate of each group of cells.

[0078] 1.2.10 Cell transfection

[0079] 1.2.11 Plasmid extraction

[0080] 1.2.11.1 Construction of vector

[0081] (1) YY1 overexpression was carried out using the lentivirus pLV3-CMV-YY1 (human)-3×FLAG-Puro. The backbone plasmid was provided by Wuhan Miaoling Biotechnology Co., Ltd.

[0082] (2) siRNA for knockdown of YY1 was synthesized by Shanghai Bioengineering Co., Ltd. The sequence is shown in the table below.

[0083] name sequence SEQ ID NO si-YY1#1 5'-UUGUUUCAAUGUAGUCGUCGUC-3' SEQ ID NO.11 si-YY1#2 5'-GACGACGACUACAUUGAACAA-3' SEQ ID NO.12 si-NC#1 5'-UUCUCCGAACGUGUCACGUTT-3' SEQ ID NO.13 si-NC#2 5'-ACGUGACACGUUCGGAGAATT-3' SEQ ID NO.14

[0084] 1.2.11.2 Cultivation and screening of monoclonal colonies

[0085] After the dry powder plasmid is centrifuged at 5000rpm for 1min, then add 20μl sterile water to dissolve the plasmid; thaw the corresponding competent medium on an ice box, take 2μl of plasmid and add it to 100μl competent medium, and place it on ice for 30min; heat shock at 42 degrees for 60s, place it on ice for 2min, add 900μl of LB medium without resistance, and culture it with shaking at 180rpm for 45min; centrifuge at 6000rpm for 3min, keep 100μl of supernatant and resuspend it, and then apply it to the LB plate with corresponding resistance; after inverting and culturing overnight, if there are too many colonies, dilute the plasmid for transformation, if there are no colonies, take 10ul of plasmid for transformation; pick a single colony and place it in the LB liquid medium with corresponding resistance for shaking culture overnight, and extract the plasmid as needed.

[0086] 1.2.11.3 Plasmid extraction

[0087] (1) Pick a transformed monoclonal colony and inoculate it into 6 mL of LB liquid culture medium containing ampicillin. Set the temperature in a 37°C constant temperature shaker at 200 rpm and culture for 12-16 h until the liquid culture medium becomes turbid visibly.

[0088] (2) Transfer 1.5 mL of the culture to a 2 mL centrifuge tube and centrifuge at 12,000 rpm for 30 seconds. Discard the culture medium and aspirate as much residual liquid as possible.

[0089] (3) Resuspend the bacterial pellet in 100 μL of pre-cooled alkaline lysis solution I and shake vigorously.

[0090] (4) Add 200 μL of freshly prepared alkaline lysis solution II to each tube of bacterial suspension, cap the tube tightly, invert rapidly several times to mix thoroughly, and place the centrifuge tube on ice for 3 min.

[0091] (5) Add 150 μL of pre-cooled alkaline lysis solution III and invert the tube several times to allow the alkaline lysis solution III to decompose evenly in the viscous bacterial lysate. Place the tube on ice for 3 min.

[0092] (6) Centrifuge at 12,000 rpm at 4°C for 5 min and transfer the supernatant to a new centrifuge tube.

[0093] (7) Add an equal volume of phenol chloroform, shake vigorously to mix the organic phase and aqueous phase, let stand for 3 min, centrifuge at 12,000 rpm at 4°C for 2 min, and transfer the supernatant to a new centrifuge tube.

[0094] (8) Add 2 volumes of anhydrous ethanol, pipette and mix well, let stand at room temperature for 2 min, centrifuge at 12,000 rpm at 4°C for 5 min, discard the supernatant, and collect the precipitate.

[0095] (9) Add 1 mL of 75% ethanol, invert several times, let stand for 2 min, centrifuge at 12,000 rpm at 4°C for 2 min, carefully discard the supernatant, and leave the tube with the lid open for 7 min to dry any remaining liquid.

[0096] (10) Redissolve the precipitate with 100 μL of ddH2O and take 20 μL to send to Sangon Biotech for sequencing. 1.2.12 Lentivirus packaging and stable transfection screening

[0097] (1) One day in advance, plate HEK 293T cells in four wells of a six-well plate. When the growth rate reaches 70-80% on the next day,

[0098] (2) Using a 3-plasmid system, 3. Target gene plasmid (pLV3-CMV-YY1): PxpAx2: PMD2g = 4:3:1. The total mass of plasmid required for one well of a six-well plate is 3ug, so the masses of plasmids required are 1.5ug: 1.13ug: 0.375ug respectively;

[0099] (3) Before the experiment, the culture medium in the six-well plate was replaced with 2 ml of fresh complete culture medium. The total mass of 12 μg of plasmid (4 wells) was added to a total volume of 250 μL of DMEM serum-free and double-antibody-free culture medium, recorded as liquid A, and mixed 20 times with a pipette;

[0100] (4) Take 24ul of LIPO8000 (6ul per well) and add it to a total volume of 250μL of DMEM serum-free and double antibody-free medium, recorded as solution B, and mix with a pipette 20 times;

[0101] (5) Add solution B drop by drop into solution A, mix gently with a pipette, and let stand at room temperature for 20 min;

[0102] (6) Slowly add the rested transfection reagent (A+B) to the HEK 293T cells, 125ul per well.

[0103] (7) Record the time. After 24 hours, replace the culture medium in the six-well plate with 2 ml of fresh complete culture medium. Collect the supernatant of each well into a 15 ml centrifuge tube between 12 and 24 hours. Centrifuge at 800 g for 3 min. Filter the supernatant with a filter and divide it into 2 ml centrifuge tubes. This is the virus. Handle it with care. It can be stored at -80°C when not in use. Alternatively, add it directly to the target cells to be transfected.

[0104] (8) Inoculate HUVEC into T25 cells. The density should be 50%.

[0105] (9) Wash twice with PBS, add virus: 5ug / ml polybrene complete medium at a ratio of 3:1 (about 2 ml in total) to T25, and add 2 ml of complete medium after 6 hours.

[0106] (10) After 24 hours, replace the culture medium with fresh complete medium. After one day of culture, add the medium containing the drug to which the plasmid is resistant (puromycin, G418) for drug screening. The concentration should be explored in advance.

[0107] (11) Change the medium every 2 days. When the cells no longer float, they are stable (5-7 days). Then extract the protein for identification and statistical analysis.

[0108] All quantitative data were expressed as mean ± standard deviation and analyzed using GraphPad Prism 9.0 software. For comparisons between two groups, T test was used. In the case of comparisons involving three or more groups, one-way analysis of variance (ANOVA) and multiple tests were used for significant analysis of differences between groups. p-value < 0.05 indicated a statistically significant difference.

[0109] 2 Results

[0110] 2.1 YY1 binds to the KDR promoter and enhances KDR expression in HUVECs

[0111] KDR, as a receptor for vascular endothelial growth factor, plays a crucial role in mediating endothelial cell-dependent angiogenesis. To elucidate the potential mechanism of YY1-induced angiogenesis, we examined the effect of YY1 on KDR expression. To explore the transcriptional regulatory mechanism of KDR, we conducted an in-depth analysis of the data from the hTFtarget, GTRD, HumanTFDB, and PROMO databases, predicted transcription factors in the KDR promoter region, and integrated the prediction data from the four databases. We predicted that YY1 is a potential transcription factor that regulates KDR ( Figure 1 A). Subsequently, we used the Jaspar database to predict the potential YY1 binding sites in the KDR promoter ( Figure 1 B). After knocking down YY1 in HUVECs, WB and qRT-PCR results showed that knocking down YY1 significantly reduced the expression of KDR in endothelial cells, and the mRNA and protein expressions of KDR and YY1 were linearly positively correlated. In addition, there was a significant linear correlation between the expression of these two genes ( Figure 1 C and D). Analysis of the direct positive regulatory effect of YY1 on KDR.

[0112] 2.2 Construction of endothelial cell hypoxia injury and repair model

[0113] The expression of HIF-1α protein, a marker of hypoxia, and the data of early and late apoptosis of cells were detected to verify whether the endothelial cell injury and repair model caused by hypoxia was successfully constructed. The expression level of HIF-1α protein was significantly increased after 3h, 6h, 9h, and 12h of hypoxia induction in HUVECs ( Figure 2 A). At the same time, previous studies have shown that hypoxia affects the proliferation of endothelial cells. In our experiment, the cells were treated with the CCK-8 method for 12 hours. It was observed that the cell proliferation ability was significantly weakened in the short-term hypoxia interval. After 36 hours of reoxygenation, the cell proliferation ability was tested every 12 hours to determine the optimal reoxygenation time. We found that within 36 hours of reoxygenation, the proliferation ability of endothelial cells rebounded and gradually entered a recovery state ( Figure 2 B and C). In addition, we subjected the cells to normoxia for 1 hour and hypoxia for 3 hours, respectively, and confirmed that hypoxia treatment led to a significant increase in the rate of cell apoptosis ( Figure 2 D). The scratch healing data showed that after 12h and 24h of hypoxia stimulation, the migration ability of cells was significantly weakened compared with the normoxic treatment group ( Figure 2 E). The above experimental results show that the endothelial cell hypoxia injury repair model was successfully established.

[0114] 2.3 Expression levels of YY1 and KDR in HUVECs under hypoxia

[0115] To investigate the changes in the expression levels of YY1 and KDR in HUVECs under hypoxic conditions, the cells were exposed to hypoxia for 3 h, 6 h, 9 h, 12 h and normoxia for the same period of time. During the 12-hour hypoxia period, the mRNA level of VEGFA increased, indicating that the cells activated the hypoxic response mechanism to adapt to the hypoxic environment ( Figure 3 A). WB and qPCR experiments showed that the protein and mRNA levels of YY1 were upregulated under hypoxia ( Figure 3 B and D). This finding indicates that the expression level of YY1 in HUVECs is significantly altered under hypoxia. Based on the previous experimental results, we demonstrated that YY1 can bind to the promoter region of KDR and regulate its expression. qPCR and WB data analysis showed that under hypoxia, the mRNA and protein expression levels of KDR and YY1 are dynamically upregulated ( Figure 3 C and D). This result confirms our previous validation and further demonstrates the regulatory role of YY1 in regulating KDR expression under hypoxic conditions.

[0116] 2.4 Role of YY1 in the repair of hypoxia-induced endothelial cell damage

[0117] YY1 knockdown (siYY1) and overexpression (oeYY1) models were constructed. After transfection with siYY1, the mRNA expression of YY1 was reduced, while after transfection with the YY1 overexpression vector, the mRNA expression of YY1 was upregulated ( Figure 4 A and B). In our study, we found that the expression of KDR was significantly reduced after knockdown of YY1. Similarly, we found that the expression of KDR increased with the increase of YY1 level ( Figure 4 A and B). This result is consistent with the findings of previous experiments. Through these models, we studied the specific role of YY1 in the reoxygenation phase after 12 hours of hypoxia. CCK8 experiments showed that knocking down YY1 reduced the proliferation capacity of endothelial cells during the repair process after hypoxic injury, while overexpression of YY1 had no significant effect on it ( Figure 4 C). At the same time, the same phenomenon was observed in HUVECs under normal culture conditions ( Figure 4 D). To further verify the role of YY1 in endothelial cell migration, we performed a scratch healing assay. The results showed that knockdown of YY1 significantly impeded the migration ability of HUVECs, while overexpression of YY1 had no significant effect ( Figure 4 E). In addition, the results of the Transwell experiment showed that the cell migration ability was significantly reduced after knocking down YY1, regardless of whether it was under normoxic or hypoxic conditions ( Figure 4 F).

[0118] In summary, the current findings provide strong evidence that YY1 plays a key role in processes related to cell migration. Under normal physiological conditions, it actively participates in the dynamic coordination of cell distribution during tissue development and homeostasis maintenance. Notably, under pathological conditions such as hypoxia, YY1 also becomes an important contributor to cell migration. Therefore, YY1 and KDR play an important role in protecting the normal physiological function of endothelial cells and promoting the repair mechanism after injury. These research results provide valuable clues for in-depth exploration of the role of YY1 and KDR in relevant pathophysiological backgrounds, thus laying a solid theoretical foundation for the identification of potential therapeutic targets.

[0119] 2.5 YY1 promotes the repair process of endothelial cells after injury by regulating the VGEF / KDR signaling pathway

[0120] To elucidate the potential mechanism of YY1 in the repair of hypoxia-induced endothelial cell injury, we studied the changes in the mRNA expression of YY1, VEGFA, and KDR in the hypoxia / reoxygenation model and normoxic conditions. Our study found that during the repair of endothelial cell injury, the mRNA expression level of VEGFA showed a trend of first increasing, then decreasing, and then increasing again ( Figure 5A and C). In the hypoxia and reoxygenation model, knockdown of YY1 resulted in a significant decrease in the mRNA expression of KDR, but had no significant inhibitory effect on the mRNA expression of VEGFA ( Figure 5 A). In contrast, under normoxic conditions, knockdown of YY1 resulted in decreased mRNA expression levels ( Figure 5 In addition, after overexpression of YY1, the expression of KDR was downregulated during hypoxia and returned to normal levels during reoxygenation ( Figure 5 C). It is worth noting that in normal culture, overexpression of YY1 still exerts a positive regulatory effect on KDR ( Figure 5 D).

[0121] 3 Analysis

[0122] Cardiovascular disease is characterized by high morbidity, high disability and high mortality, which seriously endangers human health and is a global health problem that urgently needs attention and solution. In biological systems, hypoxia may induce hypoxic damage to endothelial cells, leading to the deterioration of their normal functions and further aggravating cardiovascular disease. Therefore, hypoxic damage to endothelial cells is often a key factor in the progression of the disease. In-depth research on this is expected to provide new targets for the prevention and treatment of cardiovascular diseases.

[0123] In this study, bioinformatics methods were used to predict the transcription factors of vascular endothelial growth factor receptor type II (KDR) and validated through a series of experiments. This study demonstrated for the first time that YY1 acts as a transcriptional regulator of KDR and positively regulates its expression. Inhibition of YY1 expression directly inhibits the expression of KDR in HUVECs, suggesting that it plays an important role in the repair of endothelial cell hypoxia injury. The results showed that knockdown of YY1 led to decreased cell proliferation and migration ability. This finding demonstrates its important role and provides a basis and perspective for studying cell survival and hypoxia intervention strategies. Based on previous research results and the experimental results of this article, we speculate that the role of YY1 in the repair of endothelial cell hypoxia injury may be mediated through the HIF-1α / VEGF / KDR signaling pathway.

[0124] YY1 is a transcription factor with multiple functions. Previous studies have reported that it is associated with hypoxia and vascular endothelial growth. In the process of angiogenesis, VEGF is a key factor in activating endothelial cell proliferation and migration. The relationship between YY1 and angiogenic factor receptors is still unclear. This study aims to explore how YY1 regulates KDR to promote angiogenesis under hypoxic conditions. This study provides new insights into the molecular mechanism of hypoxia-induced endothelial cell damage and repair, and provides stronger evidence for clarifying the role of YY1. At the same time, in the treatment of cardiovascular diseases and other related diseases, YY1 may be a more targeted and effective potential therapeutic target. By intervening in the direct regulation of KDR by YY1, we can more accurately regulate the damage and repair process of vascular endothelial cells, thereby providing a clearer direction for the development of new therapeutic strategies and drugs.

Claims

1. Application of transcription factor YY1 as a drug target in screening drugs for repairing endothelial cell damage.

2. Application of transcription factor YY1 as a drug target in screening drugs for the treatment of cardiovascular diseases induced by endothelial cell damage.

3. The use according to claim 1 or 2, characterized in that: The endothelial cell injury is hypoxia-induced endothelial cell injury.

4. The use according to claim 1 or 2, characterized in that: The drug promotes the expression of transcription factor YY1.

5. The use according to claim 1 or 2, characterized in that: YY1 promotes the repair process of endothelial cells after injury by regulating the HIF-1α / VEGF / KDR signaling pathway.

6. The use according to claim 5, characterized in that: YY1 positively regulates VEGF and KDR.

7. Use of reagents that promote the expression of transcription factor YY1 in the preparation of drugs for repairing endothelial cell damage.

8. Use of an agent that promotes the expression of transcription factor YY1 in the preparation of a drug for treating cardiovascular diseases induced by endothelial cell damage.

9. The use according to claim 7 or 8, characterized in that: The reagent for promoting the expression of transcription factor YY1 is an overexpression vector of YY1.

10. The use according to claim 9, characterized in that: The overexpression vector is a lentivirus pLV3-CMV-YY1 (human)-3×FLAG-Puro recombinant vector.

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