Application of Psmg4 gene in the preparation of drugs for treating heart diseases and related diagnostic products

Abstract

The present invention belongs to the field of gene medicine, and in particular relates to the use of the Psmg4 gene in the preparation of drugs for treating heart diseases or their compositions, as well as kits for detecting heart diseases. The inventors discovered for the first time that the Psmg4 gene is sheared in hypertrophic cardiomyocytes and cardiac tissues, and that overexpression of the Psmg4 gene has an inhibitory effect on pathological hypertrophy of cardiomyocytes. The present invention proposes drugs or their compositions for treating related heart diseases, as well as kits for detecting heart diseases. The drugs or their compositions for preventing and treating heart diseases contain the coding sequence of Psmg4, or its promoter, or a pharmaceutical preparation of a combination thereof, and the drugs can effectively prevent and treat various heart diseases. The kit for detecting heart diseases contains a primer pair for detecting the shearing of the Psmg4 gene.

Description

Technical Field

[0001] The present invention belongs to the field of gene medicine, and specifically relates to the application of Psmg4 gene in the preparation of heart disease therapeutic drugs and related diagnostic products. Background Art

[0002] Cardiovascular disease has become a health problem of widespread concern worldwide. The pathogenesis of cardiovascular disease is extremely complex, affected by genetic and / or environmental factors, and involves various types of cells, such as cardiomyocytes, endothelial cells, fibroblasts, and immune cells. After myocardial damage, cardiomyocytes are widely affected, causing adverse remodeling of the heart. Pathological myocardial hypertrophy is caused by hypertension, myocardial infarction, structural heart disease, etc. The expression levels of hypertrophy-related indicators (such as BNP) in hypertrophic myocardial cells will be significantly increased, and the cell surface area will increase. However, continuous pathological overload will cause maladaptation and cardiac remodeling, leading to heart failure. At present, research on heart diseases related to pathological myocardial hypertrophy is not in-depth enough, and there is still an urgent need to develop new technical methods for the diagnosis and treatment of related heart diseases. In view of the current situation, the present invention is specially proposed. Summary of the Invention

[0003] The inventors have discovered for the first time that the Psmg4 (proteasome assembly chaperone 4) gene is cleaved in hypertrophic cardiomyocytes and cardiac tissue, and that overexpression of the Psmg4 gene has an inhibitory effect on pathological cardiomyocyte hypertrophy. Based on this discovery, the present invention provides the use of the Psmg4 gene in the preparation of drugs for treating heart disease, the use of the Psmg4 gene as a biomarker for diagnosing heart-related diseases, and the preparation of detection kits.

[0004] In a first aspect, the present invention provides the use of the Psmg4 gene for preparing a drug for preventing or treating heart disease, for example, by overexpressing the Psmg4 gene through a vector, gene editing, mRNA translation, etc.

[0005] Based on this application, the second aspect of the present invention provides a drug for preventing or treating heart disease, wherein the drug includes a pharmaceutical preparation that enhances the expression of the Psmg4 gene.

[0006] In some embodiments, the pharmaceutical preparation is a viral vector that overexpresses the Psmg4 gene.

[0007] In some embodiments, the viral vector is selected from one or more of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, or a retroviral vector, preferably an adenoviral vector.

[0008] In some embodiments, the medicament further comprises a drug delivery formulation, and / or excipients.

[0009] In some embodiments, the drug delivery formulation is selected from one or more of liposomes, chitosan, cholesterol, LNPs or nanoparticles, preferably liposomes.

[0010] In some embodiments, the excipient is selected from one or more of phosphate buffer, mannitol, or physiological saline, preferably phosphate buffer; the pH of the phosphate buffer is preferably 6.5 to 8.0.

[0011] The third aspect of the present invention provides the use of the Psmg4 gene as a biomarker in the preparation of a product for diagnosing heart disease. The fourth aspect of the present invention also provides the use of a reagent for detecting a biomarker in the preparation of a product for diagnosing heart disease, wherein the biomarker is the Psmg4 gene.

[0012] The fifth aspect of the present invention also provides a product for diagnosing heart disease, comprising a reagent for detecting a biomarker, wherein the biomarker is the Psmg4 gene.

[0013] In some embodiments, the heart disease is selected from hypertrophic cardiomyopathy, dilated cardiomyopathy, myocardial fibrosis, heart failure, coronary heart disease, myocardial damage caused by ischemia, myocardial infarction caused by ischemia, etc., preferably hypertrophic cardiomyopathy.

[0014] In some embodiments, the product uses cells, tissues or blood as test samples and diagnoses diseases by detecting the splicing status of the Psmg4 gene.

[0015] In some embodiments, the reagent for detecting a biomarker includes a primer pair for amplifying the Psmg4 gene. In some embodiments, the nucleotide sequences of the upstream and downstream primers in the primer pair are shown in SEQ ID NOs: 1-2, respectively.

[0016] In some embodiments, the reagents for detecting biomarkers include reagents for detecting the splicing status of the Psmg4 gene, such as a buffer, a marker for detecting the gene, and the like.

[0017] In some embodiments, the reagent for detecting a biomarker includes a reagent for detecting the ratio of different splicing forms of the Psmg4 gene by RT-qPCR technology.

[0018] In some embodiments, in the present invention, the nucleotide sequence of the Psmg4 gene is shown in SEQ ID NO:7.

[0019] Compared with the prior art, the beneficial effects of the present invention include at least:

[0020] The inventors have discovered for the first time that the Psmg4 gene is cleaved in hypertrophic cardiomyocytes and cardiac tissue, and that overexpression of the Psmg4 gene has an inhibitory effect on pathological cardiomyocyte hypertrophy. The present invention provides the use of the Psmg4 gene in the preparation of a drug for treating heart disease, which effectively prevents and treats a variety of heart diseases, exhibits significant therapeutic effects, and has a wide range of applications. The present invention also provides the use of the Psmg4 gene as a biomarker in the preparation of products for diagnosing heart disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The experimental results of angiotensin II (Ang II)-induced myocardial hypertrophy in mice provided in the embodiments of the present invention;

[0022] Figure 2 The experimental results of cardiomyocytes treated with AngⅡ provided in the embodiments of the present invention;

[0023] Figure 3 Schematic diagram of the original plasmid vector used in Examples 1 and 2 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, intended to be used for explaining the present invention, and do not constitute a limitation of the present invention.

[0025] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0026] The terms involved in the following embodiments and drawings are explained as follows:

[0027] Saline: saline-treated group, intraperitoneal injection of saline group, serving as the control of AngⅡ group;

[0028] AngⅡ (angiotensin II): AngⅡ-treated group refers to mice injected intraperitoneally with AngⅡ;

[0029] AngⅡ+CTRL: AngⅡ-treated group after tail vein injection of control adenovirus;

[0030] AngⅡ+Psmg4-FL: AngⅡ-treated group after tail vein injection of adenovirus targeting the amplification of the Psmg4 gene;

[0031] Heart / Body weight (mg / g): ratio of mouse heart to body weight;

[0032] Cross-sectional area (μm 2 ): myocardial cell cross-sectional area;

[0033] BNP mRNA levels: relative expression levels of BNP mRNA;

[0034] LV collagen volume fraction (%): left ventricular collagen volume fraction;

[0035] Ejection Fraction (%): left ventricular ejection fraction;

[0036] Control: normal control group, which served as the control for the AngⅡ group;

[0037] AngⅡ: AngⅡ-treated group, refers to the mouse cardiomyocytes induced by AngⅡ;

[0038] AngⅡ+CTRL: group in which mouse cardiomyocytes were treated with AngⅡ after being transfected with control adenovirus;

[0039] AngⅡ+Psmg4-FL: mouse cardiomyocytes were treated with AngⅡ after transfection with adenovirus targeting the amplification of the Psmg4 gene;

[0040] Percent spliced ​​in (%): percentage of splicing of Psmg4 gene;

[0041] %Conversion of protein (Psmg4-FL / total Psmg4): percentage of Psmg4-FL protein to total Psmg4 protein;

[0042] Cell area (fold of control): cardiomyocyte surface area relative to that of the control group.

[0043] Example 1

[0044] This example provides subsequent experimental animals and constructs an Ang II-induced cardiac hypertrophy model, specifically:

[0045] 1.1 Experimental Animals: C57BL / 6 male mice, 8–10 weeks old, were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. All mice were housed in a temperature-controlled, normal light / dark cycle at the Qingdao University Animal Center and had free access to food and water throughout the experiment. All animal studies were conducted in accordance with protocols approved by the Institutional Animal Care Committee and by governmental authorities.

[0046] 1.2 Ang II-induced cardiac hypertrophy: Mice were intraperitoneally injected with Ang II for 4 consecutive weeks to induce cardiac hypertrophy. The control group of mice was intraperitoneally injected with normal saline for 4 consecutive weeks as a control. For the purpose of achieving Psmg4 gene overexpression in vivo, an adenovirus-based Psmg4 gene overexpression vector was constructed. This was usually injected into the tail vein of the mouse 5 days before Ang II injection, and the adenovirus vector was injected again 2 weeks after Ang II injection. The viral infection titer was 1×10 10～ 14 PFU, preferably 1 × 10 12 PFU.

[0047] The original plasmid vector used in the construction of the Psmg4 gene overexpression vector using adenovirus is ADV6, and the plasmid map is shown in FIG. Figure 3 As shown, the Psmg4 CDS region sequence (SEQ ID NO: 7) was inserted between the EcoRI and BamHI elements of the original plasmid vector to construct a recombinant viral vector that overexpresses the target gene Psmg4. The inserted nucleotide sequence of the recombinant viral vector is shown below:

[0048]

[0049] Example 2

[0050] In this example, cardiomyocytes were prepared for experiment and treated with angiotensin II (Ang II), specifically:

[0051] 2.1 Cardiomyocytes: The cardiomyocytes used in this experiment are primary mouse cardiomyocytes. The hearts of 1-2 day old newborn mice were taken and rinsed 3-4 times with pre-cooled PBS. The ventricular region was then separated and minced in HEPES buffered saline. The tissue was dispersed in a digestive solution containing pancreatin and collagenase II at 37°C, and a series of repeated digestion treatments were performed to collect the first type of cardiomyocytes. Based on the first type of cardiomyocytes, adenovirus was used to construct a Psmg4 gene overexpression vector in the cells to achieve the purpose of overexpressing the Psmg4 gene. Adenovirus is usually added to cells in good growth condition, and the infection titer is 1×10 10 ～12 PFU, preferably 1×10 10 PFU, and obtain the second type of cardiomyocytes.

[0052] 2.2 AngⅡ treatment: The second type of cardiomyocytes were added to DMEM / F12 medium containing 5% serum containing AngⅡ. The control group was treated with DMEM / F12 medium containing 5% serum containing 5% of the first type of cardiomyocytes. The cells were placed in an incubator at 37°C, 5% CO2, and 95% O2 for reoxygenation for 24 hours, and the cells were collected for subsequent experiments.

[0053] Example 3

[0054] In this example, RNA extraction, RT-qPCR, PCR amplification, and agarose gel electrophoresis were performed on the myocardial tissue of the animal model of Example 1 and the myocardial cells treated in Example 2, specifically:

[0055] 3.1 RNA extraction: Total RNA was isolated from cardiomyocytes and tissue samples using Trizol reagent.

[0056] Specifically, after mixing the cells or tissues with Trizol, add chloroform in a volume ratio of 1:0.2, shake vigorously, let it stand for 5 minutes, and use a 4°C centrifuge at 12000 rpm / min for 15 minutes to obtain the supernatant; then add isopropanol in a volume ratio of 1:1, let it stand for 30-60 minutes, and centrifuge at 12000 rpm / min for 15 minutes to separate and precipitate RNA; add 75% ethanol prepared with pre-cooled DEPC water, wash and dry to obtain total RNA.

[0057] 3.2 RT-qPCR: Use a reverse transcription kit according to the manufacturer's instructions to remove genomic DNA and reverse transcribe cDNA. Then, perform qRT-PCR using a fluorescent quantitative qPCR instrument using quantitative reagents. Normalize the results using GAPDH. The primer sequences used in this experiment are listed in the table below.

[0058]

[0059] 3.3 PCR Amplification: The cDNA obtained in 3.2 was amplified using PCR Mix in a PCR instrument. The primer sequences involved in this experiment are shown in the table below.

[0060]

[0061]

[0062] 3.4 Agarose gel electrophoresis: The PCR product obtained in 3.3 was electrophoresed in a 1-2% agarose gel, and the band distribution was observed in a developer, and quantitative analysis was performed using imageJ image analysis software.

[0063] Example 4

[0064] In this example, the heart treated in Example 1 was subjected to frozen sectioning, WGA staining, and quantitative analysis of the cross-sectional area of ​​myocardial cells, specifically:

[0065] 4.1 Heart frozen sections: Remove the mouse heart, wash it with PBS, embed it with OTC embedding medium, and slice it using a freezing microtome; the slices should be 5-7 μm thick and stored in a refrigerator.

[0066] 4.2 WGA staining: After washing the frozen sections with PBS at room temperature, fix them with 4% paraformaldehyde at room temperature for 15-25 minutes; wash them with PBS three times, stain them with WGA dye for 30 minutes at 37°C in the dark; wash them with PBS three times, and then stain and seal the sections with DAPI solution.

[0067] 4.3 Quantitative analysis: The WGA-stained slides were observed and photographed under a confocal microscope; the length of the cardiomyocytes was measured along their long axis, and the width of the cells was measured along the midpoint cross-section.

[0068] Example 5

[0069] In this example, the mice and their hearts treated in Example 1 were weighed and quantitatively analyzed, specifically:

[0070] First weigh the mouse, then remove the mouse heart and weigh the heart mass; calculate the heart-to-weight ratio based on the mouse heart mass / body weight.

[0071] Example 6

[0072] In this example, the heart treated in Example 1 was subjected to paraffin sectioning, Masson staining, and quantitative analysis of myocardial fibrosis area, specifically:

[0073] 6.1 Heart paraffin sectioning: The mouse heart was removed and washed with PBS, fixed with 4% paraformaldehyde at room temperature, and embedded in paraffin; the heart was sliced ​​using a microtome with a thickness of 5-7 μm.

[0074] 6.2 Masson staining: After dewaxing and rehydrating the paraffin sections, stain them using the Masson kit according to the instructions; then seal the sections with glycerol gelatin and air dry.

[0075] 6.3 Quantitative analysis of fibrosis: Masson-stained slides were observed and photographed under a microscope; imageJ image analysis software was used to calculate the area of ​​myocardial fibrosis.

[0076] Example 7

[0077] This example performs cardiac ultrasound testing on the mouse model constructed in Example 1, specifically:

[0078] After AngⅡ or Saline injection, the mice were subjected to M-mode echocardiography using Vevo2100 (Visual Sonic) imaging system to evaluate cardiac function, and the system was used to calculate the cardiac ejection fraction (EF%).

[0079] Example 8

[0080] In this example, protein extraction, Western blotting (WB), and quantitative analysis were performed on the myocardial tissue of the animal model of Example 1 and the myocardial cells treated in Example 2, specifically:

[0081] 8.1 Protein extraction: Isolate total protein from cardiomyocytes or myocardial tissue samples using RIPA lysis buffer supplemented with protease inhibitors at low temperature.

[0082] Specifically, add RIPA lysis buffer containing protease inhibitors to the disrupted tissue homogenate or cardiomyocytes, and lyse on ice for 30-60 minutes; then use a 4°C centrifuge at 12,000 rpm / min for 15 minutes, and transfer the supernatant; add 5× protein loading buffer to the supernatant at a volume ratio of 4:1, and use a metal bath to boil at 99°C for 10 minutes.

[0083] 8.2 Western blotting: Separate the collected protein samples by SDS-PAGE gel electrophoresis and transfer them to a PVDF membrane. Block the membrane with 5% skim milk powder in PBS at room temperature for 1 hour. Then, incubate with primary antibodies against PSMG4 and GAPDH for 2-3 hours at room temperature, and with secondary antibodies for 1 hour at room temperature. GAPDH was used as an internal control. Signals were detected using an ECL chemiluminescence detection system.

[0084] 8.3 Quantitative analysis: The images obtained in 8.2 were used to calculate the protein signal intensity using imageJ image analysis software.

[0085] Example 9

[0086] In this example, the cardiomyocytes treated in Example 2 were stained with phalloidin and quantitatively analyzed, specifically:

[0087] 9.1 Phalloidin staining: The cardiomyocytes treated in Example 2 were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15-25 min. After washing three times with PBS, the cells were stained with phalloidin probe dye for 30 min at room temperature in the dark. The cells were washed three times with PBS and then stained with DAPI solution and mounted.

[0088] 9.2 Quantitative Analysis: Observe the staining results of the slides using a confocal fluorescence microscope; the phalloidin dye will mark the cytoskeleton in red, and DAPI will mark all cell nuclei in blue; use imageJ image analysis software to measure the surface area of ​​cardiomyocytes.

[0089] The experimental data obtained from the above examples were statistically analyzed. All data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using Graphpad Prism. Multiple group comparisons were performed using one-way ANOVA followed by Tukey's multiple comparisons. Two independent samples were compared using the t-test. Experiments were repeated at least three times, and similar results were obtained. A p value of < 0.05 was considered statistically significant.

[0090] The experimental results and analysis of the above embodiments are as follows:

[0091] Experimental results related to mouse models are as follows Figure 1 The results of cardiomyocyte-related experiments are shown in Figure 2 As shown in the figure, it can be seen that:

[0092] In vivo, after AngⅡ injection, the heart of mice became larger and the heart-to-body weight ratio increased ( Figure 1 A in the figure), the cross-sectional area of ​​myocardial cells increases ( Figure 1 B), this situation was alleviated to some extent after overexpression of Psmg4; overexpression of Psmg4 gene alleviated the increase of BNP mRNA, a hypertrophy marker, in myocardial tissue induced by AngⅡ ( Figure 1 C in the figure), reduced the area of ​​cardiac fibrosis ( Figure 1 D) and improved ventricular function ( Figure 1 E).

[0093] In vitro, the Psmg4 gene in cardiomyocytes treated with AngⅡ was spliced, resulting in long and short fragments, with the long fragment reduced ( Figure 2 A in the figure); protein level detection of PSMG4 expression in cardiomyocytes treated with AngⅡ, the short fragment protein increased ( Figure 2 Overexpression of Psmg4 blocked the increase in cardiomyocyte surface area induced by AngⅡ ( Figure 2 C) and the increase of hypertrophy marker BNP mRNA ( Figure 2 D) in.

[0094] This indicates that in damaged cardiomyocytes and the heart, the Psmg4 gene undergoes splicing, with an increase in short fragments and a decrease in long fragments. Overexpression of the Psmg4 gene can effectively reduce myocardial hypertrophy and fibrosis, improving cardiac function. Therefore, the Psmg4 gene can be used as a biomarker in the development of diagnostic products for related heart diseases. Furthermore, targeting the Psmg4 gene for the development of therapeutic drugs for related heart diseases holds broad application prospects.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of the Psmg4 gene for preparing a drug for preventing or treating myocardial hypertrophy, characterized in that: The drug comprises a viral vector that overexpresses the Psmg4 gene, and the viral vector is selected from an adenoviral vector, an adeno-associated viral vector, a lentiviral vector or a retroviral vector.

2. The use according to claim 1, characterized in that The viral vector is an adenoviral vector.

3. The use according to claim 1 or 2, characterized in that The drug further comprises a drug delivery preparation, and / or an excipient; the drug delivery preparation is a liposome; and the excipient is selected from one or more of phosphate buffer, mannitol or physiological saline.

4. The use according to claim 3, characterized in that The auxiliary material is phosphate buffer.

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