Application of PXDC1 in prevention and / or treatment of myocardial infarction

Abstract

The invention relates to the technical field of biology, in particular to application of PXDC1 in prevention and / or treatment of myocardial infarction. The invention discloses the core regulation and control effect of PXDC1 in the pathological process of myocardial infarction for the first time, the pathological process of myocardial infarction can be effectively regulated and controlled by specifically inhibiting heart PXDC1, myocardial cell inflammation infiltration is relieved, cardiac hypofunction and myocardial cell fibrosis are inhibited, and ventricular remodeling after myocardial infarction is further improved. The invention not only discloses that PXDC1 is used as a potential therapeutic target of myocardial infarction, but also provides a clear molecular target and an experimental basis for developing gene therapeutic drugs and specific inhibitors for preventing and treating myocardial infarction by intervening multiple pathological links such as inflammatory infiltration, fibrosis and ventricular remodeling; a new research direction and theoretical basis are opened up for a new target spot or a new treatment strategy of cardiovascular diseases, especially prevention or treatment of myocardial infarction.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to use of PXDC1 in preventing and / or treating myocardial infarction. Background Art

[0002] Cardiovascular disease, as a major global cause of death that poses a serious threat to human life and health, has far-reaching and widespread impacts. Among them, myocardial infarction (MI), as a typical and severe type of ischemic heart disease, is not only the core clinical manifestation of acute coronary syndrome (ACS), but also an important pathological form of coronary heart disease (CHD). Although the age-standardized mortality rate has declined in recent years, myocardial infarction is still one of the major and growing causes of death worldwide.

[0003] Myocardial infarction is an irreversible pathological result caused by coronary ischemia, and its fundamental cause lies in coronary atherosclerosis. The formation, erosion and even rupture of atherosclerotic plaques can lead to vascular occlusion, especially after the rupture of vulnerable plaques with thin fibrous caps, the necrotic and pro-inflammatory plaque contents are exposed to the blood circulation, which in turn triggers thrombosis. After myocardial infarction occurs, different types of innate and adaptive immune cell responses are triggered early, triggering a widespread inflammatory response, and the infiltration of monocytes and macrophages reaches a peak on the third day after infarction. Subsequently, it enters the repair stage, and macrophages with anti-inflammatory or repair properties proliferate rapidly. About 10 days after infarction, inflammatory cells in the infarct area gradually dissipate, and the tissue repair process is initiated, and eventually necrotic myocardial cells are replaced by scar tissue. In this process, ischemia-induced energy metabolism disorders, myocardial cell necrosis and programmed death (such as apoptosis and pyroptosis), increased oxidative stress and activated inflammatory responses are also accompanied, which lead to fibrosis and ventricular remodeling, seriously impairing cardiac function and even endangering the patient's life.

[0004] Currently, the treatment options for myocardial infarction are still limited. Although surgical treatment, thrombolytic therapy, and drug therapy can improve ventricular remodeling and protect residual myocardial cells to a certain extent, these methods cannot fundamentally solve key problems such as massive myocardial cell necrosis, excessive inflammatory response, and fibrosis, and are also difficult to effectively restore the proliferation capacity of myocardial cells, so it is difficult to meet the actual clinical needs.

[0005] PX domain-containing protein 1 (PXDC1), as a novel functional protein, is mainly composed of the PX (Phox) functional domain. At present, the research on PXDC1 is still in its infancy. Although some studies have revealed that it is an imprinted gene with a high specificity of paternal allele expression, with paternal expression being more than 8 times higher than maternal expression, and the PX domain is highly conserved in a variety of eukaryotic proteins from yeast to humans. However, in the field of cardiovascular diseases, especially myocardial infarction, there have been no relevant research reports. The present invention discovers the key role of PXDC1 in the occurrence and development of myocardial infarction, providing a new potential target for the treatment of myocardial infarction. Summary of the Invention

[0006] In view of this, based on the core regulatory role of PXDC1 in the pathological process of myocardial infarction, the present invention proposes the use of PXDC1 in the prevention and / or treatment of myocardial infarction.

[0007] The technical solution of the present invention is realized as follows:

[0008] In the first aspect, the present invention provides the use of PXDC1 as a drug target in screening drugs for preventing and / or treating myocardial infarction; specifically, by screening drugs that inhibit the expression of cardiac PXDC1, candidate drugs with the effect of preventing and / or treating myocardial infarction are selected.

[0009] PXDC1, also known as C6orf145. In the human body (Homo sapiens), the Pxdc1 gene is characterized by encoding PX domain-containing protein 1. This gene exhibits genetic diversity, specifically manifested as having 4 transcripts and 214 orthologous genes. In the human gene bank, the full-length cDNA sequence hPxdc1, GenBank reference sequence number NM_183373.4, corresponds to the mRNA transcript of the Pxdc1 gene and is a linear mRNA with a full length of 1878 base pairs (bp).

[0010] In the second aspect, the present invention provides the use of a PXDC1 inhibitor in the preparation of a drug for preventing and / or treating myocardial infarction, wherein the PXDC1 inhibitor specifically inhibits cardiac PXDC1.

[0011] In some specific embodiments, the PXDC1 inhibitor is a reagent for knocking out the cardiac Pxdc1 gene.

[0012] Furthermore, the PXDC1 inhibitor is a CRISPR / Cas9 gene editing system for knocking out the cardiac Pxdc1 gene, which is used to reduce the expression of PXDC1.

[0013] Furthermore, the Cas9 is selected from SpCas9 or SaCas9. The CRISPR / Cas9 gene editing targets the exon2-exon4 region of the Pxdc1-202 transcript (ENSMUST00000053459.15, Ensembl database website: https: / / www.ensembl.org). For the CRISPR / Cas9 gene editing, the CRISPR / Cas9 system and the donor vector are injected into the fertilized eggs of C57BL / 6JGpt mice by microinjection, and then the fertilized eggs are transplanted to obtain positive F0 generation mice, which are verified by PCR and sequencing; further, the positive F0 generation mice are mated with C57BL / 6JGpt mice to finally obtain stable F1 generation Pxdc1 fl / fl mouse model; mating Pxdc1 fl / fl mice with the αMHC-MerCreMer mouse strain carrying CRE enzyme to obtain an inducible heart-specific knockout mouse strain, and after induction with Tamoxifen, Pxdc1 heart-specific knockout mice are obtained.

[0014] In some specific embodiments, the PXDC1 inhibitor is a reagent that inhibits the expression of PXDC1 in the heart, and includes substances that can inhibit the transcription or translation of the PXDC1 gene or block the expression and biological activity of the PXDC1 protein.

[0015] Furthermore, the inhibition can be manifested as partial inhibition (i.e., reducing the expression level and / or functional activity of PXDC1 in the heart), or complete inhibition can also be achieved (i.e., substantially eliminating the expression of PXDC1 in the heart or its biological function).

[0016] Furthermore, the PXDC1 inhibitor includes, but is not limited to, at least one of the following: nucleic acid molecules, small molecule chemical drugs, antibody drugs, polypeptides, proteins, nucleic acid constructs, interfering lentiviruses, and interfering adeno-associated viruses.

[0017] In some specific embodiments, the PXDC1 inhibitor exerts the efficacy of preventing and / or treating cardiovascular diseases through at least one of the following: (1) reducing myocardial cell inflammatory infiltration; (2) inhibiting cardiac function decline; (3) inhibiting myocardial cell fibrosis; (4) improving ventricular remodeling after myocardial infarction.

[0018] The beneficial effects of the present invention at least include the following:

[0019] The present invention first reveals the core regulatory role of PXDC1 in the pathological process of myocardial infarction. Targeted inhibition of cardiac PXDC1 has the following effects: By targeting and inhibiting cardiac PXDC1, cardiac function indicators are significantly improved, including the increase in ejection fraction and fractional shortening, and the decrease in left ventricular end-diastolic volume and internal diameter, indicating enhanced myocardial contractile function, reduced ventricular chamber size, and improved cardiac structure. PXDC1 inhibition leads to a significant reduction in the area of myocardial fibrosis, and at the same time, the expression levels of inflammatory factors and fibrosis markers decrease significantly, effectively regulating the pathological process of myocardial infarction. At the same time, the improvement trend of cardiac structure parameters after PXDC1 inhibition is stable, and the abnormal expression of heart failure-related markers is significantly inhibited, showing a continuous therapeutic effect.

[0020] The present invention not only reveals PXDC1 as a potential therapeutic target for myocardial infarction, but also provides a clear molecular target and experimental basis for the development of gene therapy drugs and specific inhibitors for the prevention and treatment of myocardial infarction by intervening in multiple pathological processes such as inflammatory infiltration, fibrosis, and ventricular remodeling. This discovery opens up new research directions and theoretical bases for new targets or new treatment strategies for cardiovascular diseases, especially for the prevention or treatment of myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 a shows the detection of the expression of Pxdc1 in various tissues and organs by agarose gel electrophoresis in the embodiment of the present invention;

[0023] Figure 1 b shows the schematic diagram of the construction of a Pxdc1 global knockout mouse model by CRISPR-Cas9 technology in the embodiment of the present invention;

[0024] Figure 1 c shows the Mendelian genetic statistical results of the offspring of heterozygous Pxdc1 knockout mice in the embodiment of the present invention;

[0025] Figure 1 d shows the schematic diagram of the construction of a Pxdc1 cardiac-specific knockout mouse model by CRISPR-Cas9 technology in the embodiment of the present invention;

[0026] Figure 1 e shows the detection of cardiac-specific knockout of Pxdc1 by agarose gel electrophoresis in the embodiment of the present invention (experimental group: Pxdc1 fl / fl; cre and the control group Pxdc1 fl / f ) Results;

[0027] Figure 1 f shows the results of the ratio of heart weight to body weight of mice at 9 weeks after MI in the examples of the present invention, n = 7 - 11 (sham: 7, MI: 11); the four groups of mice are Pxdc1 fl / fl -sham group; Pxdc1 fl / fl ; cre-sham group; Pxdc1 fl / fl -MI group; Pxdc1 fl / fl ; cre-MI group (in specific examples, the grouping of each experimental group refers to the description here and the markings in the pictures, and the grouping details will not be repeated in the subsequent content to avoid redundancy);

[0028] Figure 1 g shows the Masson staining pictures of the hearts of Pxdc1 fl / fl and Pxdc1 fl / fl ; cre mice; scale bar 1000μm;

[0029] Figure 2 a shows the echocardiogram of the short axis section of the left ventricle of mice in the examples of the present invention;

[0030] Figure 2 b shows the results of quantitative echocardiogram showing the effects of cardiac-specific deficiency of Pxdc1 on ejection fraction (EF) and fractional shortening (FS), n = 5 - 9 (sham: 5, MI: 9);

[0031] Figure 2 c shows the results of quantitative echocardiogram showing the effects of cardiac-specific deficiency of Pxdc1 on left ventricular end-diastolic volume (LVEDV) and left ventricular internal diameter at end-diastole (LVIDd), n = 5 - 9 (sham: 5, MI: 9);

[0032] Figure 2d shows the effects of cardiac-specific deficiency of Pxdc1 on left ventricular end-systolic volume (LVESE) and left ventricular internal diameter at end-systole (LVIDs) as shown by quantitative echocardiogram results in the embodiments of the present invention, n = 5 - 9 (sham: 5, MI: 9);

[0033] Figure 2 e shows Pxdc1 at 4 weeks, 6 weeks, and 9 weeks after MI in the embodiments of the present invention fl / fl and Pxdc1 fl / fl ; quantitative statistical results of EF and FS in cre mice, n = 5 - 9 (sham: 5, MI: 9);

[0034] Figure 2 f shows Pxdc1 at 4 weeks, 6 weeks, and 9 weeks after MI in the embodiments of the present invention fl / fl and Pxdc1 fl / fl ; quantitative statistical results of LVEDV and LVIDd in cre mice, n = 5 - 9 (sham: 5, MI: 9);

[0035] Figure 2 g shows Pxdc1 at 4 weeks, 6 weeks, and 9 weeks after MI in the embodiments of the present invention fl / fl and Pxdc1 fl / fl ; quantitative statistical results of LVESV and LVIDs in cre mice, n = 5 - 9 (sham: 5, MI: 9);

[0036] Figure 3 a shows the results of qRT-RCR detection of heart failure indicators in the cardiac tissues of Pxdc1 fl / fl and Pxdc1 fl / fl ; cre mice in the sham group and the MI group, n = 5;

[0037] Figure 3 b shows the results of qRT-RCR detection of fibrosis indicators in the cardiac tissues of Pxdc1 fl / fl and Pxdc1 fl / fl ; cre mice in the sham group and the MI group, n = 5;

[0038] Figure 3 c shows the results of qRT-RCR detection of inflammation indicators in the cardiac tissues of Pxdc1 fl / fl and Pxdc1 fl / fl ; cre mice in the sham group and the MI group, n = 5. Detailed implementation manners

[0039] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without any creative efforts belong to the scope of protection of the present invention. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not indicated with the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0040] Unless otherwise specified, the methods used in the following embodiments are all conventional methods. For the specific steps, reference can be made to: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor). The acquisition routes of various biological materials described in the embodiments only provide an experimental acquisition route to achieve the specific disclosure purpose and should not limit the source of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the prompts in the embodiments. The genes, proteins, or their fragments involved in the present invention can be natural purified products, chemically synthesized products, or products produced using recombinant techniques from prokaryotic or eukaryotic hosts (such as bacteria, yeast, plants, etc.).

[0041] Table 1 Sequence Information Table

[0042]

[0043]

[0044] I. Experimental Methods

[0045] All animal experiment procedures in the embodiments of this application have been reviewed and approved by the Animal Ethics Committee of Shenzhen Hospital, Fuwai Hospital, Chinese Academy of Medical Sciences, and are carried out in accordance with the Guide for the Care and Use of Laboratory Animals (8th Edition) published by the National Institutes of Health (USA). All mice are raised in a normal environment at a room temperature of 24 ± 3°C, a humidity of 55 ± 5%, a 12-hour light / 12-hour dark cycle, and fed a normal diet. Male mice of C57BL / 6 are used in this application. The Pxdc1 fl / flMice were bred with αMHC-MercreMer mice to obtain Pxdc1 fl / fl ; male mice with cre were used for subsequent experiments (see Experimental Method 2 for details).

[0046] 1. Construction of a Pxdc1 gene knockout mouse model

[0047] In this application, the CRISPR-Cas9 system was used to construct Pxdc1 knockout mice (see Figure 1 b). The wild-type allele was knocked out by Cas9 cleavage guided by sgRNA1 and sgRNA2 to achieve global knockout. The specific steps are as follows: The exon2-exon5 region of the Pxdc1-202 transcript ENSMUST00000053459.15 was used as the knockout target region. Using CRISPR / Cas9 technology, gRNA was designed and in vitro transcribed, and Cas9 and gRNA were co-injected into mouse fertilized eggs. The Cas9 protein binds to the target site under the guidance of gRNA, causing double-strand breaks in DNA, thereby achieving deletion of the base sequence at the target site and gene knockout. However, during the breeding of Pxdc1-KO mice, we found that homozygous mice could not be obtained. Therefore, we constructed Pxdc1 heart-specific knockout mice.

[0048] 2. Construction of a Pxdc1 gene heart-specific knockout mouse model

[0049] Using the CRISPR-Cas9 system to construct Pxdc1 heart-specific knockout mice (as shown in Figure 1 d), the wild-type allele was introduced with LoxP sites by CRISPR-Cas9 to form Flox alleles; αMHC-MercreMer was activated under Tamoxifen induction to excise the fragment between LoxP sites, resulting in specific knockout of the Pxdc1 gene in the heart. The specific steps are as follows:

[0050] The mouse Pxdc1 gene (NCBI database GENE ID: 66895) has a total of 4 transcripts. According to the structure of the Pxdc1 gene, the region from exon 2 to exon 4 (exon2-exon4) of the Pxdc1-202 transcript (ENSMUST00000053459.15) was selected as the knockout target. This region contains 322bp of coding sequence, which will lead to protein function destruction after knockout. In this project, we used CRISPR / Cas9 technology to edit the Pxdc1 gene. The brief process is as follows: The CRISPR / Cas9 system and the donor vector (Donor) were injected into the fertilized eggs of C57BL / 6JGpt mice by microinjection, and then the fertilized eggs were transplanted to obtain positive F0 mice, and the effectiveness of gene editing was verified by PCR and sequencing; the positive F0 mice were further mated with C57BL / 6JGpt mice, and finally a stable F1 mouse model was obtained (homozygous Pxdc1 was screened by PCR). fl / fl mice). fl / fl The mice were crossed with the αMHC-MerCreMer (MCM) mouse strain carrying the CRE enzyme to obtain an inducible cardiac-specific knockout of Pxdc1 fl / fl ;αMHC-MCM mouse strain, after tamoxifen induction (75 mg / kg), Pxdc1 heart-specific knockout mice were obtained.

[0051] 3. Establishment of mouse myocardial infarction (MI) model

[0052] After skin preparation, the mice were anesthetized, the chest and abdomen of the mice were disinfected, and an oblique skin incision was made along the lower edge of the left pectoralis major muscle of the mice toward the left upper limb of the mice. The pectoralis major and pectoralis minor muscles were separated, and the widest part of the 3rd-4th intercostal space was entered. The intercostal space was opened with hemostatic forceps, and the heart was squeezed out with the thumb and index finger of the left hand. The left anterior descending branch was ligated, and the hemostatic forceps gently clamped the heart and sent it back to the heart cavity, the chest cavity was closed, and the skin was sutured. No ligation was performed in the sham group, and other surgical steps were the same as those in the model group.

[0053] 4. Mouse heart ultrasound detection

[0054] Mice were anesthetized with flurane gas, and the limbs of the mice were fixed to the ultrasound electrode sheet with tape, and coupling agent was applied to the skin of the limbs and heart area to avoid bubbles. The detection was performed by the echocardiography system with a 30-MHz imaging sensor. When detecting the short axis of the left ventricle of the heart, the notch of the ultrasound probe was directed toward the left forelimb of the mouse, with an inclination angle of about 45°, and the probe was adjusted to fit the mouse skin. After turning on the B-mode ultrasound mode, the operation panel was adjusted according to the image until a clear circular cardiogram with two papillary muscles was displayed. Then the M-mode ultrasound mode was turned on, the sampling line was adjusted to the correct position, and the save key was pressed to obtain the M-mode echocardiogram of the short axis of the left ventricle.

[0055] 5. Histopathological staining

[0056] After the mice were sacrificed, the excised heart tissues were rinsed in physiological saline and fixed in 4% paraformaldehyde at room temperature for 24 hours. Subsequently, these heart tissues were embedded in paraffin and cut into 5-μm tissue sections using standard histological methods. Fibrosis was stained by Masson staining, which was completed by Sevier Biotechnology Co., Ltd. Scanning and photographing were performed using a confocal microscope (Olympus FV300).

[0057] 6. Extraction of mouse tissue DNA

[0058] Take 5 mg of mouse heart and liver tissues, soak them in ACL buffer solution containing proteinase K, and the subsequent extraction steps are carried out according to the instructions of the genomic extraction kit (Vazyme, #DC112-02).

[0059] 7. Real-time quantitative PCR

[0060] Total RNA was extracted from heart and liver tissues using RNA extraction solution (Servicebio) according to the manufacturer's instructions.

[0061] RNA was quantified using NanoDrop (Thermo Fisher Scientific). Cells were washed twice with PBS buffer for 3 minutes each time, then Trizol was added. Using a cell scraper, the cells were scraped off the six-well plate; left standing at room temperature for 5 minutes, pre-cooled chloroform at 4°C was added, shaken vigorously for 30 seconds, left standing at room temperature for 3 minutes, centrifuged at 12,000 rpm at 4°C for 15 minutes; the liquid was layered, the upper aqueous layer and the lower organic layer. Carefully aspirate the upper aqueous layer, add an equal volume of isopropanol, mix gently, left standing at room temperature for 10 minutes, centrifuged at 12,000 rpm at 4°C for 60 minutes; RNA formed a precipitate and sank to the bottom of the tube. Add 75% alcohol prepared with DEPC water, wash once, centrifuge at 12,000 rpm at 4°C for 10 minutes; aspirate the 75% alcohol, left standing at room temperature to dry, add an appropriate amount of DEPC water to dissolve the RNA, and measure the concentration with a Nanodrop instrument. RNA was reverse transcribed into cDNA using a reverse transcription kit (RevertAid First Strand cDNA Synthesis kit). The reaction system was as follows: 2 μL of 5×Reaction Buffer, 1 μL of 10 mM dNTP Mix, 1 μL of Random Primer, 0.5 μL of RiboLock RNase Inhibitor, 0.5 μL of RevertAid M-MuLV RT, 1 μg of RNA, and nuclease-free Water was supplemented to 10 μL; PCR reaction: 25°C for 5 minutes; 42°C for 60 minutes; 70°C for 5 minutes. Real-time fluorescence quantitative PCR was performed on a QuantStudio 7 Pro detection instrument (Thermo Fisher Scientific) using specific primers and ChamQ SYBR qPCR Master Mix (Vazyme). The reaction system for qRT-PCR was as follows:

[0062] 2×SYBR Green Master MIX 5 μL Water 3 μL Forward Primer 0.5 μL Reserve Primer 0.5 μL cDNA 1 μL

[0063] First, add the reaction system except cDNA to a 384-well plate, then add 1 μL of cDNA to the corresponding wells, and seal the 384-well plate with a transparent film. After centrifuging the 384-well plate at 1000 rpm at room temperature for 1 min, place it in a real-time quantitative PCR instrument for detection. The reaction program was as follows:

[0064]

[0065] After the reaction, the specificity of the primers was judged by the melting curve, and the content of the target gene was calculated by the Ct value. The PCR primer sequences used in this application are shown in Table 1. Using Gapdh as an endogenous control, the relative expression level of the gene was measured.

[0066] 8. Statistical analysis

[0067] Statistical analysis was performed using GraphPad Prism 8 software. All experimental data were expressed as the mean ± SEM of at least three independent experiments. One-way ANOVA or two-way ANOVA was used for the statistical significance of multiple comparisons, followed by Tukey's test. Bonferroni adjustment was used for post hoc analysis. A t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant.

[0068] II. Experimental results

[0069] 1. Genetic effects of global Pxdc1 knockout and expression of Pxdc1 in different tissues:

[0070] As Figure 1 shown in - / - c, Pxdc1 global knockout mice (Pxdc1 - / - ) failed to produce offspring, suggesting that global Pxdc1 knockout may cause a lethal effect, indicating the importance of the Pxdc1 gene in organism survival. Using qRT-PCR detection with Actb as the internal reference gene, it was found that the Pxdc1 gene was normally expressed in heart and liver tissue samples ( Figure 1 a).

[0071] 2. Verification of cardiac-specific knockout:

[0072] In this example, a mouse model (Pxdc1 fl / fl ; cre mice) carrying loxp sites inserted into two alleles of the Pxdc1 gene and expressing cardiac tissue-specific Cre recombinase was constructed ( Figure 1 d). The Pxdc1 gene in different tissues of mice with different genotypes was detected by agarose gel electrophoresis ( Figure 1 e). The insertion of the two loxp sites spanned exons 2-4. If knockout did not occur, because the two loxp sites were about 10,000 bp apart, the target band could not be obtained by conventional PCR, and the agarose gel electrophoresis showed no band, such as in Pxdc1 fl / fl control and the liver of the Pxdc1 fl / fl ; cre group. Only when Tamoxifen induced the Cre recombinase to recognize the loxp site of αMHC and perform cleavage could the PCR successfully synthesize the target band, and the agarose gel electrophoresis showed a band. Combining these two groups of controls finally demonstrated that Pxdc1 was successfully knocked out specifically in the cardiac tissue.

[0073] 3. Effect of cardiac-specific knockout on myocardial infarction:

[0074] In the mouse model of myocardial infarction, compared with the Pxdc1 cardiac-specific knockout group (Pxdc1fl / fl ; cre) and Pxdc1 fl / fl In a group of mice, it was found that cardiac-specific knockout of Pxdc1 could slow down the increase in the ratio of heart weight to body weight in mice after myocardial infarction ( Figure 1 f), indicating that it has a certain protective effect on the heart. The results of Masson staining showed ( Figure 1 g), Pxdc1 fl / fl ; cre group mice had significantly improved cardiac tissue fibrosis compared to Pxdc1 fl / fl group mice after MI, further confirming the reversal effect of Pxdc1 knockout on myocardial infarction.

[0075] 4. Pxdc1 in myocardial infarction (MI) and its effects on cardiac function and structure

[0076] Pxdc1 fl / fl -sham group, Pxdc1 fl / fl ; cre-sham group, Pxdc1 fl / fl -MI group, and morphological differences in the short-axis cross-sections of the left ventricles of Pxdc1 fl / fl ; cre-MI group mice were shown as Figure 2 shown. Figure 2 b, c, and d Pxdc1 fl / fl ; cre group and Pxdc1 fl / fl group showed that the ejection fraction (EF) and fractional shortening (FS) of Pxdc1 fl / fl ; cre were significantly increased compared to the Pxdc1 fl / fl group, and their values approached the physiological level of the Sham control group, indicating that myocardial contractile function was preserved; the left ventricular end-diastolic volume (LVEDV) and left ventricular end-diastolic diameter (LVIDd) were significantly decreased, indicating that the abnormal dilation of the left ventricular cavity after MI was significantly inhibited; the left ventricular end-systolic volume (LVESV) and left ventricular end-systolic diameter (LVIDs) also showed a decreasing trend, further confirming that Pxdc1 knockout can counteract MI-induced systolic volume accumulation and ventricular structure deterioration. In addition, Figure 2 e, f, and g show the changing trends of the above indexes at three time points of 4 weeks, 6 weeks, and 9 weeks after surgery in the Pxdc1 fl / fl ; cre-MI group. Among them, the EF value of the Pxdc1 fl / fl ; cre-MI group decreased by approximately 15%-20% less than that of the Pxdc1 fl / fl -MI group ( Figure 2 b), and the time-course analysis showed that this difference remained stable at each time point after surgery ( Figure 2e), which confirmed that Pxdc1 knockout could dynamically inhibit the progressive deterioration of systolic function after MI. Pxdc1 fl / fl ; The reduction amplitudes of LVEDV and LVIDd in diastole and LVESV and LVIDs in systole in the cre-MI group were approximately 20%-25% and 18%-22% respectively compared with those in the Pxdc1 fl / fl -MI group ( Figure 2 c-d), and the temporal analysis showed that ventricular dilation continuously improved over time ( Figure 2 f-g), further verifying that Pxdc1 knockout had a cumulative improvement effect on ventricular geometric remodeling after MI.

[0077] The experimental results showed that the improvement trends of various indicators in the experimental group persisted and were statistically significant (P<0.05), indicating that cardiac-specific knockout of the Pxdc1 gene had a long-term effect on the improvement of cardiac function and structure in myocardial infarction mice.

[0078] 5. Effects of Pxdc1 on the gene expression levels of heart failure indicators, fibrosis indicators, and inflammation indicators in mouse heart tissues

[0079] From the expression results of heart failure-related molecular markers such as Nppa, Nppb, Myh6, Myh7, Acta1, etc. ( Figure 3 a), it can be seen that compared with the Pxdc1 fl / fl -sham group, the expression of heart failure-related markers (such as Nppa, Nppb, etc.) in the Pxdc1 fl / fl -MI group was significantly up-regulated, indicating that myocardial infarction led to changes in the expression of heart failure-related genes. Compared with the Pxdc1 fl / fl -MI group, the up-regulation amplitude of heart failure-related genes in the Pxdc1 fl / fl ; cre-MI group was somewhat reduced, suggesting that Pxdc1 gene knockout might have a regulatory effect on the changes in the expression of heart failure-related genes induced by myocardial infarction, which was helpful for alleviating the overexpression of heart failure-related genes. For example, the relative expression value of Nppa in the Pxdc1 fl / fl -MI group was significantly higher than that in the Pxdc1 fl / fl -sham group, while the relative expression value in the Pxdc1 fl / fl ; cre-MI group was lower than that in the Pxdc1 fl / fl -MI group, indicating that Pxdc1 knockout could inhibit its overexpression.

[0080] The results of the detected fibrosis indicators Col1a1, Col3a1, and Postn ( Figure 3 b) showed that compared with the Pxdc1 fl / fl -sham group, the Pxdc1 fl / fl- The expression of fibrosis-related genes in the MI group was significantly increased, indicating that myocardial infarction promoted cardiac tissue fibrosis. Compared with Pxdc1 fl / fl -MI group, Pxdc1 fl / fl ; In the cre-MI group, the increase in the expression of fibrosis-related genes was reduced, indicating that Pxdc1 gene knockout could inhibit the expression of fibrosis-related genes in cardiac tissue induced by myocardial infarction, and thus might reduce the degree of cardiac tissue fibrosis.

[0081] The detection results of the expression levels of inflammatory markers (Il1b, Il6, Tnf, Ccl2, Cxcl1) showed that, compared with Pxdc1 fl / fl -sham group, Pxdc1 fl / fl -MI group, the expression of inflammatory genes was significantly upregulated, indicating that myocardial infarction triggered an inflammatory response in cardiac tissue. Compared with Pxdc1 fl / fl -MI group, Pxdc1 fl / fl ; In the cre-MI group, the upregulation of the expression of some inflammatory genes (such as Il1b, Il6, etc.) was reduced, indicating that Pxdc1 gene knockout could regulate the expression of inflammation-related genes induced by myocardial infarction, and thus might reduce the inflammatory response in cardiac tissue. Taking Il1b as an example, its relative expression value in the Pxdc1 fl / fl -MI group was significantly higher than that in the Pxdc1 fl / fl -sham group, while its relative expression value in the Pxdc1 fl / fl ; cre-MI group was lower than that in the Pxdc1 fl / fl -MI group, indicating that Pxdc1 knockout had a mitigating effect on inflammation.

[0082] The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of PXDC1 as a drug target in screening drugs for preventing and / or treating myocardial infarction.

2. The use according to claim 1, characterized in that, By screening drugs that inhibit the expression of cardiac PXDC1, candidate drugs with the effect of preventing and / or treating myocardial infarction are selected.

3. Use of a PXDC1 inhibitor in the preparation of a drug for preventing and / or treating myocardial infarction, characterized in that, The PXDC1 inhibitor specifically inhibits cardiac PXDC1.

4. The use according to claim 3, wherein The PXDC1 inhibitor is a reagent for knocking out the cardiac Pxdc1 gene.

5. The use according to claim 4, characterized in that, The PXDC1 inhibitor is a CRISPR / Cas9 gene editing system for knocking out the cardiac Pxdc1 gene, which is used to reduce the expression of cardiac PXDC1.

6. The use according to claim 5, characterized in that, The Cas9 selects SpCas9 or SaCas9.

7. The use according to claim 5, characterized in that, The CRISPR / Cas9 gene editing targets the exon2-exon4 region of the Pxdc1-202 transcript.

8. The use according to claim 6, characterized in that, The described CRISPR / Cas9 gene editing involves injecting the CRISPR / Cas9 system and the donor vector into the fertilized eggs of C57BL / 6JGpt mice by microinjection. Subsequently, the fertilized eggs are transplanted to obtain positive F0 generation mice, which are verified by PCR and sequencing. Further, the positive F0 generation mice are mated with C57BL / 6JGpt mice to finally obtain a stable F1 generation of Pxdc1 fl / fl mouse model; crossing the Pxdc1 fl / fl mice with the αMHC-MerCreMer mouse strain carrying the CRE enzyme to obtain a mouse strain with inducible cardiac-specific knockout. After induction with Tamoxifen, Pxdc1 cardiac-specific knockout mice are obtained.

9. The use according to claim 3, characterized in that, The PXDC1 inhibitor is a reagent for inhibiting the expression of cardiac PXDC1.

10. The use according to claim 3, characterized in that, The PXDC1 inhibitor exerts the efficacy of preventing and / or treating cardiovascular diseases through at least one of the following: (1) reducing myocardial cell inflammatory infiltration; (2) inhibiting cardiac function decline; (3) inhibiting myocardial cell fibrosis; (4) improving ventricular remodeling after myocardial infarction.

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