CN111001003B - Application of POU2F1 gene expression inhibitor in the preparation of medicaments for the treatment of tissue fibrosis diseases - Google Patents

Abstract

The invention provides a new application of POU2F1 as a new important target point for treating fibrosis. Specifically, the invention provides a treatment method, which is characterized in that the expression of a transcription factor POU2F1 is interfered by small interfering RNA, so that the inhibition effect of the transcription factor POU2F1 on downstream genes is blocked, a plurality of downstream fibrosis inhibition factors are enabled, and the occurrence of cardiac fibrosis is inhibited.

Description

Application of POU2F1 gene expression inhibitor in preparation of medicine for treating tissue fibrosis diseases

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to an application of a POU2F1 gene expression inhibitor in preparation of drugs for treating tissue fibrosis diseases.

Background

Cardiac fibrosis is a common pathological process, and is very common in heart diseases such as myocardial infarction, aortic stenosis, dilated cardiomyopathy and diabetic heart disease. The main pathological changes of cardiac fibrosis are that the extracellular matrix of the intercellular substance of the heart tissue is increased, the hardness of the heart tissue is increased, the parenchymal cells are reduced, and the continuous development can cause the structural damage and the function reduction of the heart, finally cause the heart failure and seriously threaten the health and the life of the human beings. The ever-increasing extracellular matrix and stiffness of cardiac tissue are not only a result of cardiac fibrosis, but can further exacerbate cardiac fibrosis. Myofibroblasts are the main cell type that synthesizes and secretes extracellular matrix. It is mainly differentiated from cardiac fibroblasts abundantly present in the heart. Cardiac fibroblasts are therefore the major members controlling the homeostasis of the extracellular matrix. Differentiation of cardiac fibroblasts into myofibroblasts alpha-smooth actin and fibronectin (fibronectin) were used as markers. The mechanistic study on cardiac fibrosis and the discovery of new important members associated with cardiac fibrosis are the basis and important pathways for preventing and blocking cardiac fibrosis-induced heart failure.

Currently, there have been many advances in research on the promotion of fibrosis in response to changes in the stiffness of cardiac tissue resulting from cardiac fibrosis. From the cellular structure, integrins (integrins), toll-like receptors (toll-like receptors) and syndecans (syndecans) are involved in responses to altered extracellular matrix stiffness at the cardiac fibroblast plasma membrane; in the cytoplasm of cardiac fibroblasts, many members of the TGFB signaling pathway, ROCK signaling pathway, HIPPO signaling pathway, MAPK signaling pathway, and AGE-RAGE signaling pathway have been previously reported to be involved in the response to changes in extracellular matrix stiffness and in the regulation of cardiac fibrosis. In the nucleus of cardiac fibroblasts, LINC complexes (linkers of nucleosokeleton and cytoskeleton) are involved in response to extracellular mechanical signals. The rigidity of the extracellular matrix can also affect the expression of downstream genes by altering the structure of chromatin. For example, Bromodemin-stabilizing protein 4(BRD4) localized to the nuclear membrane interacts with the SUN protein of the LINC complex, and knocking down BRD4 with small interfering RNA lowers the protein levels of alpha-smooth actin, fibronectin, and collagen type 1. In addition, transcription factors in the nucleus can also be influenced by extracellular matrix stiffness to regulate cardiac fibrosis. NESPLIN-2 is a component that can promote the function of the transcription factor Smad3 in response to extracellular forces. There are a number of studies on cardiac fibrosis, but the components found in the past are not ideal for clinical use, and it is therefore an important research direction to find new members capable of modulating numerous cardiac fibrosis signaling pathways and thereby preventing the onset of heart failure.

The transcription factor, POU2F1(POU domain 2transcription factor 1, POU2F1), is a member of the POU factor family, and is the only transcription factor in this family that is widely expressed in various tissues and organs. POU2F1 can be involved with different cofactors in mediating the activation, inhibition and arrest of downstream genes. It has been reported that the mRNA level and protein level of POU2F1 increase in various cancers. The role previously reported by POU2F1 was primarily involved in regulating transcription of genes associated with proliferation and immune regulation. Subsequent POU2F1 related researches found that downstream genes which can be regulated and controlled relate to oxidative and cytotoxic stress resistance, metabolism regulation and stem cell function regulation. In the cardiovascular field, POU2F1 can also bind to a promoter of a heart failure marker beta-myostatin heavy chain and regulate the expression of the heart failure marker beta-myostatin heavy chain, and the POU2F1 is suggested to possibly participate in the occurrence and development of cardiac remodeling.

Disclosure of Invention

In order to provide a new medicine for treating tissue fibrosis diseases, the invention adopts the following technical scheme:

the invention relates to an application of POU2F1 gene expression inhibitor in preparation of drugs for treating tissue fibrosis diseases. Surprisingly, the inventors of the present invention found that intervention of POU2F1 significantly reduces the process of transdifferentiation of fibroblasts including cardiac fibroblasts, thereby inhibiting the occurrence of fibrosis.

In a preferred embodiment of the present invention, the tissue fibrosis disease comprises cardiac fibrosis and pulmonary fibrosis.

The POU2F1 gene expression inhibitor of the present invention is not particularly limited, but a preferred POU2F1 gene expression inhibitor is selected from the group consisting of a small interfering RNA and a POU2F1 gene knock-out reagent.

In another aspect, the invention relates to the use of an inhibitor of POU2F1 gene expression for inhibiting cell fibrosis or promoting fibroblast transdifferentiation in vitro.

In another aspect, the invention relates to the use of the POU2F1 gene expression inhibitor in promoting transcription of downstream genes IL1R2, CD69 and/or TGIF2 in vitro fibrotic cells.

In a preferred embodiment of the invention, the POU2F1 gene expression inhibitor is selected from the group consisting of a small interfering RNA, a POU2F1 gene knock-out agent, for inhibiting cell fibrosis in vitro or promoting transdifferentiation of fibroblasts or promoting transcription of downstream genes IL1R2, CD69 and/or TGIF2 in fibrotic cells in vitro.

In a preferred embodiment of the invention, the cell is a cardiac or pulmonary cell for inhibiting fibrosis of the cell in vitro or promoting transdifferentiation of fibroblasts or promoting transcription of the downstream genes IL1R2, CD69 and/or TGIF2 in a fibrotic cell in vitro.

Drawings

FIG. 1A-FIG. 1C: immunofluorescence staining was used to analyze the effect of pathological extracellular matrix stiffness on the promotion of POU2F1 protein expression levels in cardiac fibroblasts. FIG. 1A: fluorescently labeled POU2F1 protein levels. FIG. 1B: quantification of the fluorescence level of POU2F1 and statistical analysis results. FIG. 1C: simulating the corresponding relation between the hardness of the pathological extracellular matrix and the pathological process of the heart tissue in vitro.

Fig. 2A-2B: the western blot method verifies the promotion effect of the pathological extracellular matrix hardness on the POU2F1 protein expression level in the cardiac fibroblasts. FIG. 2A: western blotting the protein level of POU2F1 was measured in cardiac fibroblasts cultured at different extracellular matrix hardnesses using POU2F1 antibody, GAPDH as an internal control. FIG. 2B: and (3) quantitative and statistical analysis results of the protein content detected by the POU2F1 western blotting method.

FIG. 3: the real-time fluorescent quantitative PCR detects the promotion effect of the pathological extracellular matrix hardness on the POU2F1mRNA level in the cardiac fibroblasts.

FIGS. 4A-C: protein imprinting measures protein levels of POU2F1 in heart tissue at different time periods in the infarct and distant regions of the mouse myocardial infarct model. FIG. 4A: mouse myocardial infarction model diagram. FIG. 4B: protein imprinting was performed using POU2F1 antibody to detect POU2F1 protein levels at 1 day, 4 days, and 7 days post-operatively in the infarct zone, distant zone. FIG. 4C: and (3) quantitative and statistical analysis results of the protein content detected by the POU2F1 western blotting method.

Fig. 5A-5C: immunofluorescence staining detects the influence of pathological extracellular matrix hardness on fibroblast transdifferentiation. FIG. 5A fluorescence shows that myofibroblast markers fibronectin and alpha SMA protein levels, respectively, are modulated by extracellular matrix stiffness. FIG. 5B: quantitative and statistical analysis of the fluorescence level of fibrinectin. FIG. 5C: quantitative and statistical analysis of alpha SMA fluorescence levels.

Fig. 6A to 6C: protein imprinting detection verifies the promoting effect of pathological extracellular matrix hardness on the expression level of fibronectin and alpha SMA protein in cardiac fibroblasts. FIG. 6A: western blotting was performed using antibodies to fibronectin and α SMA as internal control for detecting fibronectin and α SMA protein levels in cardiac fibroblasts cultured at different extracellular matrix hardnesses. FIG. 6B: and (3) the quantitative and statistical analysis results of the protein content detected by the fibrinectin western blotting method. FIG. 6C: and (3) quantifying the protein content detected by the alpha SMA western blotting method and carrying out statistical analysis on the result.

Fig. 7A to 7F: chromatin co-immunoprecipitation experiments tested the ability of the transcription factor POU2F1 to bind to the promoter of its potential downstream genes. Fig. 7A-7C: schematic representation of the transcription factor POU2F1 binding to the promoters of IL1R2, CD69, TGIF 2. Fig. 7D-7F: chromatin co-immunoprecipitation experiments were performed using control IgG and POU2G1 antibodies to examine the binding ability of POU2F1 protein to the promoters of IL1R2, CD69, TGIF 2.

Fig. 8A to 8C: the real-time fluorescent quantitative PCR detects the inhibition effect of pathological extracellular matrix hardness on the mRNA levels of fibrosis inhibition factors IL1R2, CD69 and TGIF2 in the cardiac fibroblasts.

Fig. 9A to 9F: real-time fluorescent quantitative PCR measures the effect on downstream gene mRNA levels when POU2F1 is reduced in the cell. FIG. 9A: under pathological extracellular matrix hardness conditions, cardiac fibroblast POU2F1mRNA levels were knocked down using antisense oligonucleotides, and the efficiency of knocking down POU2F1mRNA levels was examined using real-time fluorescent quantitative PCR. FIG. 9B: protein level knockdown efficiency for POU2F1 was determined using western blotting. FIG. 9C: and (4) quantifying and statistically analyzing the protein content detected by POU2F1 western blotting after the POU2F1 is knocked down. Fig. 9D-F: after the real-time fluorescent quantitative PCR is used for detecting the regulation effect on the mRNA level of downstream genes IL1R2, CD69 and TGIF2 after the POU2F1 is knocked down.

Fig. 10A to 10F: the western blot method examined the effect on the protein level of downstream genes when POU2F1 decreased in the cells. Fig. 10A to 10C: the protein levels of the downstream genes IL1R2, CD69 and TGIF2 after the POU2F1 was knocked down were detected by western blotting using IL1R2, CD69 and TGIF2 antibodies, respectively. Fig. 10D-fig. 10F: and (3) quantitative and statistical analysis results of protein content detected by IL1R2, CD69 and TGIF2 protein chromatography after POU2F1 is knocked down.

Fig. 11A to 11D: immunofluorescent staining examined the effect on fibrosis promoted by transdifferentiation of cardiac fibroblasts when the growth of cardiac fibroblasts was reduced by endogenous POU2F1 under pathological extracellular matrix stiffness conditions. Fig. 11A and 11C: after POU2F1 was knocked down, myofibroblast markers fibrinectin and alpha SMA protein levels were represented by fluorescence, respectively. Fig. 11B and 11C: quantification of fibrinectin and alpha SMA fluorescence levels and statistical analysis results.

Fig. 12A to 12C: protein imprinting measures myofibroblast markers fibronectin and alpha SMA protein levels in pathological extracellular matrix stiffness conditions when intracellular POU2F1 is reduced. FIG. 12A: after POU2F1 was knocked down in the presence of pathological extracellular matrix stiffness, fibronectin and α SMA antibodies were immunoblotted using fibronectin and α SMA antibodies, respectively, and protein levels were detected. Fig. 12B and 12C: quantification and statistical analysis of fibrinectin and α SMA protein levels.

Detailed Description

The invention will be further elucidated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not specified, in the following examples were carried out according to the routine procedures in the art or according to the conditions suggested by the manufacturers.

Example 1, in vitro experiments, using different matrix stiffness to simulate the effect of extracellular matrix stiffness changes in different pathological states on the expression levels of transcription factor POU2F1, and myofibroblast markers fibronectin and α SMA in cardiac fibroblasts.

Isolation and culture of cardiac fibroblasts: cardiac fibroblasts were obtained from 8 to 10 week male C57BL/6 mice. The heart tissue from which the atrial appendage was removed was minced and digested at 37 deg.C with 0.1% collagenase type II (300U, 17101-. After centrifugation at 1000 rpm for 5 minutes at room temperature, the supernatant was discarded, the cell pellet was resuspended in DMEM (12800-058, Gibco, Carlsbad, Calif., USA) containing 10% fetal bovine serum (15140-122, Gibco, Carlsbad, Calif., USA), and then transferred to a 10cm dish and cultured at 37 ℃ in 5% carbon dioxide. This is primary cardiac fibroblast P0. Subsequent experiments were generally performed using P2 fibroblasts.

Preparation of polyacrylamide gels of different hardness: a glass cover glass with the thickness of 25mm multiplied by 25mm is firstly treated by sodium hydroxide, aminopropyl trimethoxy silane and glutaraldehyde respectively, then four hardness degrees of 7.5KPa (10% acrylamide, 0.03% methylene acrylamide), 13KPa (10% acrylamide, 0.07% methylene acrylamide), 19.5KPa (10% acrylamide, 0.13% methylene acrylamide), 35KPa (10% acrylamide, 0.26% methylene acrylamide) are prepared according to different proportions of acrylamide and methylene acrylamide, and a crosslinking agent Sulfo-SANPAH (Thermo Fisher Scientific, Rockford, IL, USA) is added on the surface of the glue after the glue is formed, and the crosslinking is promoted by ultraviolet irradiation. Different hardness of the substrate gels were incubated overnight at 4 degrees celsius with 100 μ g/ml rat tail collagen (Sigma, st. Before use, the substrate glue is put into a 6-well plate, soaked in sterile PBS and sterilized by ultraviolet. Thereafter, P2 generation cardiac fibroblasts were spread on the base gel, and one day later, the cells were fixed or harvested for subsequent experiments.

Immunofluorescence staining experiment: cells were fixed with 37 ℃ warm 4% paraformaldehyde for 15 minutes at 37 ℃, washed 3 times with warm PBS, and then disrupted with 0.2% Triton X-100 for 20-30 minutes. After 3 washes with warm PBS, blocking was added (5% BSA) for 30 min. Primary anti- α SMA (ab32575, abcam, Cambridge, MA, USA), fibrinectin (ab2413, abcam, Cambridge, MA, USA), POU2F1(ab178869, abcam, Cambridge, MA, USA) was then used for overnight incubation at 4 degrees celsius. After recovery of the primary antibody, PBS washes were 3 times followed by incubation of the secondary antibody Alexa Fluor4881 hours at room temperature. Nuclei were stained with Hoechst (Invitrogen, Carlsbad, Calif., USA) for 8 minutes at room temperature. Fluorescence intensity was counted and analyzed using the morpholinology Explorer BioApplication module of the high content screening imaging system Cellomics array Scan VTI HCS Reader (Thermo Fisher Scientific, Rockford, IL, USA). Images collected using a confocal microscope were collected with a 20X objective excitation light wavelength 488. Images were captured and processed using ZEN 2012 software.

Western blot experiments: cells were first digested with pancreatin from the background gel, centrifuged and the supernatant was washed three times with cold PBS, followed by lysis with cell lysate (20mmol/L Tris-HCl PH7.4,150mmol/L NaCl,2.5mmol/L EDTA,50mmol/L NaF,0.1mmol/L Na4P2O7,1mmol/L Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 1% deoxyglycolic acid,1 mmol/LPMSF, and 1mg/ml aprotinin), sonicated and centrifuged at 12000g for 15 min at 4 ℃. And (6) collecting the supernatant. After taking 5 microliters for protein quantification, the remaining supernatant was added to 5 Xgel loading buffer at 100 ℃ for 5 minutes to ensure protein denaturation. Using 10% SDS-PAGE gel to perform electrophoresis, then transferring the nitrocellulose membrane, sealing 5% skim milk at room temperature for 1 hour, and performing cold room overnight incubation at 4 ℃ for the first time, wherein the first product number is respectively as follows: fibrinectin (ab2413, abcam, Cambridge, MA, USA), α SMA (ab32575, abcam, Cambridge, MA, USA), POU2F1(ab178869, abcam, Cambridge, MA, USA), IL1R2(sc-376247, Santa Cruz Biotech, CA, USA), CD69(ab202909, abcam, Cambridge, MA, USA), TGIF2(ab190152, abcam, Cambridge, MA, USA), GAPDH (2118S, CST), TBST washing three times followed by exchanging the corresponding species secondary antibody, Corporation for 1 hour at room temperature, TBST washing the membrane, developing the membrane into a developer (Millipore), and then controlled drying and placing into a luminescence detection machine for exposure. The intensity of the bands was quantified using NIH ImageJ software.

Real-time fluorescent quantitative PCR: experiments total RNA from cardiac fibroblasts was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). cDNA was then synthesized by Reverse Transcription using a 20. mu.l Reverse Transcription System (M-MLV Reverse Transcription System, Promega Corporation, Fitchburg, Wis., USA) and fluorescent quantitative PCR was performed using SYBR Green Mix (TransGen Biotech, Beijing, China) with the instrument model Mastercycler ep realplex2 Real-Time PCR System (Eppendorf). The relative expression levels of POU2F1, IL1R2, CD69, and TGIF2 genes were calculated from the ratio of the CT values of the genes and the housekeeping gene GAPDH.

We explore the influence of transcription factor POU2F1 under different pathological environments by using an in vitro method for simulating the hardness of extracellular matrix of heart tissues under different pathological states. Firstly, directly fixing cardiac fibroblasts cultured on a continuous substrate hardness, and detecting the protein level of endogenous POU2F1 by using immunofluorescence. The fluorescence in fig. 1A indicates the location and content of POU2F1 and the location of the nucleus, and thus can suggest that the location of the transcription factor POU2F1 is mainly within the nucleus, and is consistent with its cellular location that performs a biological function of transcriptional activity. The green fluorescence of POU2F1 was the weakest at 13KPa, with an undesirable increase at 7.5KPa and 19.5KPa, 35KPa and directly on the glass. The quantitative results of the green fluorescence intensity of POU2F1 in FIG. 1B show that the intracellular POU2F1 content of cardiac fibroblasts significantly increases when cultured under the condition of hard pathological extracellular matrix. Fig. 1C shows the corresponding pathological state of the heart as simulated by the hardness of the basal glue in vitro, 13KPa for the hardness of the extracellular matrix of healthy heart tissue, 7.5KPa for the hardness of the extracellular matrix of softer infarct zone in the early onset of myocardial infarction, 19.5KPa for the hardness of the extracellular matrix of heart zone where fibrotic events occurred, and 35KPa for the hardness of the extracellular matrix of late infarct scar zone of the myocardial infarction.

Thereafter, protein levels of POU2F1 were measured by Western blot precipitation experiments using total cardiac fibroblast proteins cultured at different substrate hardnesses. The experimental results are shown in fig. 2, and the results are consistent with those of the immunofluorescence experiment, wherein the protein level of the transcription factor POU2F1 is lowest under the physiological extracellular matrix stiffness and gradually increases under the pathological extracellular matrix stiffness. Quantitative results statistical analysis showed that POU2F1 was significantly elevated in cardiac fibroblasts cultured on pathological extracellular matrix compared to POU2F1 in cardiac fibroblasts cultured on physiological extracellular matrix.

The cardiac fibroblasts cultured under different substrate hardnesses are collected by directly using TRIZOL treatment, RNA is extracted for reverse transcription, and the influence of the different substrate hardnesses on the POU2F1mRNA level is detected by using real-time fluorescent quantitative PCR. Fig. 3 shows that the mRNA level of POU2F1 was also affected by the basal stiffness, and that the promotion of mRNA of POU2F1 for pathological basal stiffness was significant.

The above suggests that pathological extracellular matrix stiffness has a promoting effect on the expression of POU2F1, and may influence the protein level by promoting the mRNA level of POU2F 1.

Meanwhile, the expression levels of the myofibroblast markers, namely fibronectin and alpha SMA, in the cardiac fibroblasts cultured under different basal stiffness are detected. The experimental results suggest that similar to the trend of substrate stiffness regulation for POU2F1, immunofluorescent staining labeled with antibodies for fibrinectin and α SMA, respectively, the results of fig. 5A show that the green fluorescence of fibrinectin and α SMA is also the darkest under physiological conditions, followed by an increase in green fluorescence at pathological substrate stiffness, and that the change in green fluorescence is statistically significant and significantly increased in subsequent quantitative and statistical analyses. We then verified the results using western immunoblotting, the results in fig. 6A confirm the results observed with immunofluorescence staining, and the quantitative and statistical results in fig. 6B and 6C suggest that the protein levels of myofibroblast markers fibronectin and α SMA are significantly elevated at the pathological basal stiffness. The results indicate that the pathological extracellular matrix can promote the transdifferentiation of fibroblasts, so that myofibroblasts are increased. However, the above results do not allow to determine whether there is a direct causal link between the increase of the transcription factor POU2F1 promoted by the pathological extracellular matrix and the transdifferentiation of the fibroblasts promoted. Still further exploration.

Example 2 POU2F1 protein levels were measured in infarct and distant regions at different time points of heart tissue surgery in a mouse myocardial infarction model.

10 week male C57BL/6 mice were used to perform the myocardial infarction model building surgery. Mice were randomly divided into myocardial infarction groups and random control groups. The myocardial infarction group induced the development of myocardial infarction using left coronary stenosis. Sham surgery was performed on the control group. The procedure was performed during aeroanesthesia, and mice were subjected to gas anesthesia by inhalation of 2% isoflurane. Experiment cardiac tissue was collected from the infarcted and distant areas of mouse cardiac tissue at 1, 4, and 7 days post-surgery, respectively.

We detected the protein levels of the transcription factor POU2F1 endogenous to cardiac tissues in infarct and distant regions 1 day, 4 days, 7 days after the mouse myocardial infarction model surgery by western blotting. Figure 4A shows specific locations of the infarcted and distant areas of a cardiac infarct. Fig. 4B shows that POU2F1 levels were elevated in the infarcted area of the heart one day after coronary stenosis surgery and continued to be elevated for 4 days, 7 days after surgery, compared to the sham group. However, no increase in POU2F1 was observed in the distant regions of the heart after 1, 4, and 7 days of surgery. Quantitative and statistical analysis of the immunoblot results, which gave fig. 4C, suggested that the protein level of the transcription factor POU2F1 was not significantly changed in the distant region of the myocardial infarction, but was significantly increased 1 day after the infarct area, and fell back to some extent at 4 and 7 days, but the increased levels were still significantly different from those in the control sham group. Previous studies reported that the extracellular matrix stiffness of the early-onset myocardial infarct region of the myocardial infarct exhibited a softer stiffness as compared to the physiological state. Fibrosis occurs in the late stage of the myocardial infarction, and the hardness of extracellular matrix is harder than that in a physiological state. In the results, the hardness of the heart tissue in the infarct area after one day of the myocardial infarction model operation, and the hardness of the heart tissue after 4 days and 7 days of the myocardial infarction model represent the hardness of the softer extracellular matrix and the harder extracellular matrix respectively under the physiological condition, and the POU2F1 protein level in the heart tissue is increased in the environment. This result, in concert with the results of example 1, suggests that pathological extracellular matrix stiffness promotes elevated levels of the transcription factor POU2F1 protein, whether in vivo or in vitro experiments mimic different cardiac extracellular matrix stiffness.

Example 3 determination of the ability of the transcription factor POU2F1 to bind to the promoter of its potential downstream gene using chromatin co-immunoprecipitation.

Chromatin co-immunoprecipitation experiments: 1% formaldehyde was used to crosslink proteins and DNA associated with them in living cardiac fibroblasts. After cell fixation, cell lysate was disrupted by ultrasonication, and the size of the DNA fragment was determined to be 500-600 bases by means of agarose gel electrophoresis. Antibodies to POU2F1(ab178869, abcam, Cambridge, MA, USA) and control rabbit monoclonal IgG (ab172730, abcam, Cambridge, MA, USA) were used for immunobinding, followed by immunoprecipitation with the addition of A/G beads. After the sediment is centrifuged and washed by rinsing liquid with different salt concentrations, elution and crosslinking are carried out, and then the levels of the IL1R2, CD69 and TGIF2 promoters are detected by using real-time quantitative PCR.

The results in vitro and in vivo suggest that the rigidity of the pathological extracellular matrix promotes the expression of the transcription factor POU2F1 in cardiac fibroblasts. Whether the POU2F1 is involved in the process of changing the rigidity of the extracellular matrix to transdifferentiate the cardiac fibroblasts is an important question to answer. Our bioinformatics analysis results have previously predicted that POU2F1 may play an important role in this process. And predicting that the transcription factor POU2F1 can regulate three important fibrosis inhibiting factors IL1R2, CD69 and TGIF 2. The binding sites of POU2F1 predicted using TRANSFAC are shown in FIGS. 7A-C. Therefore, we designed and carried out the chromatin co-immunoprecipitation experiment, and the results of the experiment are shown in fig. 7D-F, using the antibody of POU2F1 to immunoprecipitate POU2F1 in the total protein, and using real-time fluorescent quantitative PCR to detect the promoter content of IL1R2, CD69, TGIF2 bound to POU2F 1. The protein precipitated with the POU2F1 antibody did bind more of the promoters of IL1R2, CD69, TGIF2 than the control IgG group. We also compared IL1R2, CD69, TGIF2 levels in cardiac fibroblasts grown under pathological and physiological basal stiffness using real-time fluorescent quantitative PCR. The results of the experiment in fig. 8 found that pathological basal stiffness was able to significantly inhibit the mRNA levels of these three fibrotic suppressors. The above results suggest that the transcription factor POU2F1 can be bound to the promoters of the three fibrosis suppressor genes IL1R2, CD69 and TGIF2, but the effect of POU2F1 on promoting or inhibiting the binding of the promoters of the three genes and the effect of cardiac fibrosis transdifferentiation still need to be further investigated.

Example 4 knock down of POU2F1 in cardiac fibroblasts with small interfering RNA, in pathological extracellular matrix stiffness, thereby reducing the mRNA, protein levels of POU2F1 in fibroblasts. Detecting the expression level of the downstream regulatory gene and the expression level of the myofibroblast marker.

Cardiac fibroblasts transfected with small interfering RNA: the heart fibroblast cells P2 were passaged from P1 to 6-well plates overnight before the night of experimental transfection of small interfering RNA, and cultured overnight at 37 ℃ in a 5% carbon dioxide environment to ensure that the fibroblast cells were morphologically spread and that no extracellular matrix was produced that would have affected the transfection efficiency. Transfection experiments the day early fibroblast cells in 6-well plates were gently washed in warm PBS at 37 degrees Celsius and washed three times repeatedly to ensure thorough washing of the culture Medium, after which 500. mu.l OPTI-MEM (Opti-MEM I Reduced Serum Medium,31985070, Life) was added per well, followed by 80nmol/L POU2F1 siRNA (sc-36120, Santa Cruz Biotech, Calif., USA)) or control siRNA (sc-37007, Santa Cruz Biotech, Calif., USA) and 3. mu.l HiPerFect transfection reagent (301705, QIAGEN, Beijing, China), respectively. After 6 hours of transfection, DMEM medium containing 10% fetal bovine serum was added for culture. The protein detection experiment uses the transfected small interfering RNA three days of cell samples, and the mRNA detection experiment uses the transfected small interfering RNA one day of samples.

In order to clarify the effects of the transcription factor POU2F1 on potential downstream genes IL1R2, CD69 and TGIF2 and the functions of the transcription factor POU2F1 in the cardiac fibroblast transdifferentiation process, the POU2F1 expression level in the cardiac fibroblast is reduced by using a technology of silencing gene expression by small interfering RNA, firstly, the POU2F1 knockdown efficiency is determined by using a western blot experiment and real-time fluorescence quantitative PCR, and then, the effects of POU2F1 on the downstream genes and the cardiac fibroblast transdifferentiation process are clarified by using an immunofluorescence staining and a western blot experiment.

The efficiency of knock-down of mRNA levels one day after POU2F1 knockdown by cardiac fibroblasts transfected with small interfering RNA is shown by fig. 9A, with the mRNA levels of POU2F1 being around 30% of the basal levels one day after transfection. Thereafter, FIG. 9B shows the protein level of POU2F1 three days after transfection of small interfering RNA detected by Western blotting, and it can be seen that the protein level of POU2F1 is also significantly decreased. The results of quantification clearly demonstrated that the protein level of POU2F1 was about 30% of basal level three days after transfection.

After the transfection and knockdown efficiency of the small interfering RNA is clarified, the expression levels of potential downstream genes IL1R2, CD69 and TGIF2 of the transfected small interfering RNA are detected by using real-time fluorescent quantitative PCR (polymerase chain reaction) one day. From the results of FIGS. 9D-9F, it can be seen that the mRNA levels of the downstream genes IL1R2, CD69 and TGIF2 were significantly increased after the knock-down of POU2F 1. This result suggests that the binding effect of POU2F1 to the promoters of the downstream genes IL1R2, CD69 and TGIF2 functions by inhibiting their transcription. At the same time we also examined changes in protein levels of these three downstream genes using western blot experiments. Results of the experiments FIGS. 10A-10C show that the protein levels of the downstream regulatory genes IL1R2, CD69, TGIF2 were increased after the knock-down of POU2F 1. Fig. 10D-10F show the quantitative results, confirming that there is significance in the degree of protein elevation of the downstream regulatory gene after knockdown of POU2F 1. These results suggest that POU2F1 may inhibit the transcription of a downstream gene by binding to a promoter of the downstream regulatory gene, thereby inhibiting the downstream gene from performing its function.

To confirm the effect of POU2F1 on cardiac fibroblast transdifferentiation, we examined the protein levels of the myofibroblast markers fibronectin and α SMA in cardiac fibroblasts grown in pathologic basal stiffness after knockdown of POU2F1 using immunofluorescence staining experiments as well as western blot experiments. The results of the immunofluorescent staining experiments are shown in fig. 11. Three days after POU2F1 knockdown, representing a decrease in green fluorescence of fibrinectin and α SMA, the results of fluorescence quantification showed that the fluorescence level of fibrinectin decreased to around 20% of the level before knockdown and the fluorescence level of α SMA decreased to around 60% before knockdown. Western blot experiments figure 12 also shows that the protein levels of fibrinectin and alpha SMA are significantly reduced. The above experiments suggest that the transcription factor POU2F1 promotes the transdifferentiation process of cardiac fibroblasts by inhibiting the transcription of the downstream genes IL1R2, CD69 and TGIF2, and further inhibiting the three fibrosis-inhibiting factors from playing their functions. The intervention of POU2F1 can obviously reduce the transdifferentiation process of cardiac fibroblasts, thereby inhibiting the occurrence of cardiac fibrosis.

The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims (5)

1. The application of POU2F1 gene expression inhibitor in preparing medicine for treating tissue fibrosis diseases, wherein the tissue fibrosis diseases refer to cardiac fibrosis.
2. 2. The use according to claim 1, wherein the POU2F1 gene expression inhibitor is selected from the group consisting of a small interfering RNA, a POU2F1 gene knock-out agent.
3. Use of an inhibitor of POU2F1 gene expression for inhibiting cell fibrosis or promoting transdifferentiation of fibroblasts in vitro, said cells being cardiac cells.
4. Use of an inhibitor of POU2F1 gene expression for promoting transcription of downstream genes IL1R2, CD69 and/or TGIF2 in vitro fibrotic cells, said cells being cardiac cells.
5. 5. The use according to claim 3 or4, wherein the POU2F1 gene expression inhibitor is selected from the group consisting of a small interfering RNA, a POU2F1 knock-out agent.

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