Application of PYRTS and simulant thereof in myocardial infarction treatment or detection products

Abstract

The invention belongs to the field of gene drugs, and particularly relates to application of PYRTS and simulants thereof in myocardial infarction treatment or detection products. The nucleotide sequence of the PYRTS is as shown in SEQ ID NO. 1. When myocardial infarction occurs, the expression activity of the tsRNA is reduced, so that the application of the tsRNA as a biomarker in preparation of detection products is provided. According to the characteristics of tsRNA, the tsRNA is named as PYRTS (tsRNA associated with pyroptosis), overexpression of the PYRTS significantly inhibits occurrence of pyroptosis of myocardial cells, a PYRTS simulant and application of the PYRTS simulant in preparation of drugs for prevention and / or treatment of heart diseases are provided, the PYRTS simulant is preferably PYRTS-Agomir, and increase of PYRTS expression effectively alleviates the problems of poor prognosis of myocardial infarction patients and treatment of heart diseases.

Description

Technical Field

[0001] The present invention belongs to the field of gene drugs, and particularly relates to the application of PYRTS and its mimetics in the treatment or detection products of myocardial infarction. Background Art

[0002] With the acceleration of the urbanization process and the change of lifestyle, the incidence of cardiovascular diseases has been increasing year by year, bringing great pressure to the medical system. Myocardial infarction deserves attention and in-depth research, so it is crucial to explore the early diagnosis and treatment of myocardial infarction. Myocardial infarction (MI) is a serious cardiovascular disease, usually caused by the acute obstruction of the coronary artery, leading to myocardial ischemia and necrosis. In recent years, transfer RNA-derived small RNAs (tsRNAs), as a novel type of non-coding RNA, have played important roles in various physiological and pathological processes, including pathological changes such as myocardial fibrosis after myocardial infarction. Studies have shown that the expression pattern of tsRNAs changes significantly after myocardial infarction, which may be closely related to its role in regulating gene expression, apoptosis, and inflammatory responses. For example, a study identified sncRNAs related to myocardial fibrosis through PANDORA sequencing technology and found that N-acetyltransferase 10 (NAT10) was highly expressed in ischemic myocardial tissue, and it participated in the occurrence of myocardial fibrosis by regulating the mRNA acetylation of early growth response 3 (EGR3). In addition, studies have also found that specific tsRNAs, such as tsr007330, are down-regulated in MI tissues, and their overexpression can improve cardiac function after MI and reduce myocardial fibrosis.

[0003] Necroptosis is a form of programmed cell death that has attracted extensive attention in the study of cardiovascular diseases (CVDs) in recent years. The pathogenesis of cardiovascular diseases is complex, and necroptosis is considered to be one of the important factors leading to cardiac dysfunction and tissue damage. There is a close interaction between necroptosis and other forms of programmed cell death (such as apoptosis, inflammatory necrosis, etc.), and these processes jointly affect the homeostasis and pathological changes of the cardiovascular system.

[0004] In cardiovascular diseases, pyroptosis participates in the pathological process by regulating inflammatory responses and cell damage. For example, studies have shown that under conditions such as atherosclerosis and myocardial infarction, the activation of pyroptosis exacerbates local inflammation and promotes the development of lesions. In addition, mitochondrial dysfunction is closely related to pyroptosis, and mitochondrial dysregulation can lead to the overproduction of reactive oxygen species (ROS), thereby inducing pyroptosis and other types of programmed cell death. Non-coding RNA (ncRNA) also plays an important role in regulating pyroptosis in cardiovascular diseases. Studies have found that ncRNA can affect pyroptosis by regulating related signaling pathways, thus playing a key role in the development of cardiovascular diseases. For example, some ncRNAs have been shown to inhibit or promote pyroptosis, which provides new potential targets for the treatment of cardiovascular diseases.

[0005] Agomir is a double-stranded small RNA with special labeling and chemical modification, which regulates the biological functions of target genes by mimicking endogenous tsRNA and is expected to become a basic method for treating various infectious diseases, hematological tumors, cardiovascular and neurodegenerative diseases. Agomir is a double-stranded small RNA molecule designed according to the mature tsRNA sequence to mimic the endogenous mature tsRNA sequence. Agomir includes a sequence identical to the target tsRNA mature sequence and a sequence complementary to the tsRNA mature sequence. Specific Agomir can be introduced into cells expressing the corresponding tsRNA to mimic the role of tsRNA. As a tsRNA mimic, Agomir has become an important tool in molecular biology research and potential treatment due to its advantages such as high efficiency, stability, and strong targeting. Summary of the Invention

[0006] Based on the above background technical content, according to the characteristics of tsRNA, the present invention names this tsRNA PYRTS (tsRNA associated with pyroptosis). The present invention has found that during myocardial infarction, the expression level of PYRTS decreases and is highly correlated with cardiomyocyte pyroptosis; overexpression of PYRTS can significantly inhibit the area of myocardial infarction and plays an important role in the regulation of cardiomyocyte death. The current drug treatment for myocardial infarction has limited effects. Therefore, PYRTS plays an important role in the regulation of cardiomyocyte death and can regulate the treatment of cardiomyocyte death and ischemia-reperfusion injury by participating in the regulation of cardiomyocyte pyroptosis, including key events such as inhibiting the expression of pyroptosis-related protein GSDMD, thereby improving the clinical prognosis of ischemia-reperfusion injury.

[0007] In the first aspect of the present invention, there is provided the use of PYRTS in the treatment or detection of myocardial infarction products, and the nucleotide sequence of the PYRTS is as shown in SEQ ID NO: 1.

[0008] In the second aspect of the present invention, there is provided the use of PYRTS as a biomarker in the preparation of a product for detecting pyroptosis of cardiomyocytes or myocardial infarction.

[0009] In the third aspect of the present invention, there is provided a product for detecting pyroptosis of cardiomyocytes or myocardial infarction, comprising a reagent for detecting a biomarker, wherein the biomarker is a PYRTS sequence.

[0010] Further, the reagent for detecting the biomarker comprises a primer pair for amplifying RNA transcribed from PYRTS.

[0011] Preferably, in the primer pair, the nucleotide sequences of the RT primer, the upstream primer and the downstream primer are shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively, and U6 is selected as the internal reference gene, and the nucleotide sequences of the upstream and downstream primers of U6 are shown in SEQ ID NO.6 and SEQ ID NO.7 respectively.

[0012] The present invention also provides the use of the above product for detecting pyroptosis of cardiomyocytes or myocardial infarction in the preparation of a product for detecting pyroptosis of cardiomyocytes or myocardial infarction.

[0013] In some embodiments, the biomarker is selected from the PYRTS sequence. PYRTS is related to pyroptosis of cardiomyocytes, and the occurrence of pyroptosis of cardiomyocytes can be characterized by the RNA transcribed from the PYRTS sequence.

[0014] In some embodiments, the PYRTS sequence is derived from a mouse, a human or other mammals.

[0015] In some embodiments, the product for detecting pyroptosis of cardiomyocytes or myocardial infarction is a detection kit.

[0016] In the fourth aspect of the present invention, there is provided the use of a PYRTS mimetic as a therapeutic drug for pyroptosis of cardiomyocytes and myocardial infarction. Overexpression of PYRTS can significantly reduce cardiomyocyte death, activate the repair of damaged myocardial tissue, and overexpression of PYRTS effectively alleviates the problems of poor prognosis of patients with myocardial infarction and the treatment of heart diseases.

[0017] Further, the PYRTS mimetic is a PYRTS Agomir, which has a nucleotide sequence shown in SEQ ID NO.5.

[0018] The present invention also provides the use of the PYRTS mimetic in the preparation of a drug for preventing and / or treating heart diseases, wherein the heart diseases include myocardial infarction or pyroptosis of cardiomyocytes.

[0019] The fifth aspect of the present invention also provides a drug for preventing and / or treating heart diseases, said drug comprising a PYRTS mimetic, said PYRTS mimetic being PYRTS Agomir and having a nucleotide sequence as shown in SEQ ID NO.5.

[0020] The drug is based on the already mature Agomir; relying on Agomir, a PYRTS mimetic is constructed and delivered to the location of the heart disease lesion, and can be used as a drug for treating myocardial infarction to achieve effective treatment of the lesion.

[0021] In some embodiments, the heart disease is myocardial infarction or cardiomyocyte pyroptosis.

[0022] Preferably, the administration method of the drug is to directly inject the therapeutic drug intravenously into the body.

[0023] Compared with the prior art, the beneficial effects of the present invention include:

[0024] 1. The inventors first discovered that during the occurrence of myocardial infarction, the expression of PYRTS decreases, which is highly correlated with cardiomyocyte pyroptosis, and overexpression of PYRTS can significantly inhibit the area of myocardial infarction;

[0025] 2. The present invention provides the application of PYRTS as a biomarker according to the regulatory mechanism of PYRTS in cardiomyocyte death, and provides a product for detecting cardiomyocyte pyroptosis or myocardial infarction;

[0026] 3. The present invention relies on the PYRTS mimetic (PYRTS Agomir), constructs a PYRTS mimetic, delivers it to the location of the heart disease lesion, can be used as a drug for treating myocardial infarction, achieves effective treatment of the lesion, and provides a drug for preventing and / or treating heart diseases. Description of the Drawings

[0027] Figure 1 Shows the expression level of PYRTS in a mouse myocardial infarction model;

[0028] Figure 2 Shows the expression level of PYRTS in a mouse cardiomyocyte hypoxia model;

[0029] Figure 3 Shows the expression level of PYRTS in human dilated cardiomyopathy;

[0030] Figure 4 Shows the expression level of PYRTS in human heart failure;

[0031] Figure 5 Shows the expression level of pyroptosis-related proteins after overexpression of PYRTS after hypoxia;

[0032] Figure 6The level of lactate dehydrogenase (LDH) after overexpressing PYRTS following hypoxia;

[0033] Figure 7 The survival rate of cardiomyocytes after overexpressing PYRTS following hypoxia;

[0034] Figure 8 The level of lipid peroxidation after overexpressing PYRTS following hypoxia;

[0035] Figure 9 The expression level of pyroptosis-related proteins after overexpressing PYRTS in an adult mouse myocardial infarction model. Detailed implementation manners

[0036] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation manners. Examples of the embodiments are shown in the drawings. It should be understood that the specific embodiments described in the following implementation manners of the present invention are only illustrative examples of the specific implementation manners of the present invention, intended to explain the present invention, and not to limit the present invention. Those not specifying specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not indicating the manufacturer can be obtained as conventional products through commercial purchase. The source of PYRTS is a mouse, Mus musculus in Latin.

[0037] In the ranges disclosed herein, the endpoints and any value are not limited to the exact range or value, and these ranges or values should be understood to include those close to these ranges.

[0038] Example 1: Expression of PYRTS in hypoxic mouse cardiomyocytes and human heart diseases

[0039] 1) Hypoxia model of cardiomyocytes (Hypoxia)

[0040] Isolate cardiomyocytes from 1- to 2-day-old neonatal mice (C57BL / 6). Wash the neonatal mice with 75% alcohol, surgically excise the heart, place it in a pre-cooled PBS culture dish in a sterile environment, wash the heart 3 times with PBS, and cut the heart tissue into pieces with surgical scissors. Transfer the heart tissue to 10 mL of digestion solution (trypsin 1.2 mg / mL, collagenase II 0.14 mg / mL), and gently shake it in a 37°C water bath for 6 min. After each digestion, transfer the supernatant to a centrifuge tube containing serum, store it on ice, and add fresh digestion solution again until the heart tissue disappears. Centrifuge at 1000 rpm / min for 10 min, take the supernatant, resuspend it with F12 / DMEM containing 10% serum, centrifuge again at 1000 rpm / min for 10 min, take the supernatant, filter it through a 70-mesh cell sieve, and place the filtered cardiomyocytes in a 10-cm culture dish. Culture at 37°C in a 5% CO2 humidified incubator for 1.5 h. The fibroblasts adhere to the wall, collect the medium containing cardiomyocytes into a new centrifuge tube, centrifuge at 1000 rpm / min for 10 min, discard the supernatant, and resuspend it with a new medium containing serum. Add 0.1 mM bromodeoxyuridine (BrdU). According to the experimental design, culture in a 37°C 5% CO2 humidified incubator. The next day, change the medium in the culture dish and continue to grow for one day. According to the experimental grouping, place the cells that need to be hypoxic in a hypoxic incubator for 12 hours, and then perform subsequent experiments.

[0041] 2) Establish a mouse myocardial infarction model (MI)

[0042] Select 6- to 8-week-old mice (C57BL / 6) for the experiment. Anesthetize the mice by intraperitoneal injection of 4% chloral hydrate, remove the hair on the chest and armpits with surgical scissors (fully expose the surgical area), and disinfect the surgical area with 75% ethanol. Turn on the ventilator, set the parameters (respiratory rate 110 bpm), and intubate the mice tracheally. Use ophthalmic scissors to open the chest between the third and fourth intercostal spaces to fully expose the heart and fully expose the left anterior descending coronary artery (LAD) or the area where it is located. Hold a 6-0 suture with a needle holder, insert the needle 2 mm below the lower edge of the left atrial appendage, pass the suture through the LAD to completely block the blood flow of the LAD. After ligation, completely suture the thoracic cavity opening with a 4-0 suture (ensure no gaps and no misalignment), close the thoracic cavity, and suture each layer of muscle and skin layer by layer from the inside out.

[0043] 3) Real-time fluorescence quantitative PCR (RT-qPCR)

[0044] Total RNA was extracted from mouse cardiomyocytes or tissues using TRIzol reagent, and the concentration and purity of the total RNA were determined using a nanodrop One. The total RNA was converted into cDNA using an AG Evo M-MLV RT Kit reverse transcription kit. Fluorescent quantitative PCR was performed using a SYBR Green Kit. RT-qPCR was performed using a Thermo Fisher real-time fluorescent quantitative PCR detection system. Reaction conditions: 95°C for 10 min; 95°C for 30 s; 95°C for 10 s, 60°C for 30 s, for a total of 40 cycles. The experimental results were qualitatively analyzed for relative expression levels using the 2-ΔΔCt method.

[0045] Results:

[0046] To study the expression of PYRTS in mouse cardiomyocyte hypoxia and human heart diseases, cardiomyocyte hypoxia samples (Hypoxia), mouse myocardial infarction model samples (MI), human dilated cardiomyopathy samples (DCM), and human heart failure samples (HF) were collected. The expression level of PYRTS was detected by RT-qPCR (as Figure 1-4 shown). The results showed that the expression of PYRTS was decreased in the hearts of mice with hypoxia and myocardial infarction, and the expression of PYRTS was decreased in human dilated cardiomyopathy and heart failure. It can be seen that PYRTS may be involved in the regulation of heart diseases such as myocardial infarction.

[0047] Example 2: Inhibitory effect of PYRTS on pyroptosis in mouse cardiomyocyte hypoxia

[0048] 1) Cell transfection mediated by 3000

[0049] (1) Primary mouse neonatal cardiomyocytes were isolated and left for treatment after changing the medium.

[0050] (2) Take a 1.5 mL EP tube, make a mark, and pipette 250 μL of high-glucose DMEM medium into the 1.5 mL EP tube. Add 5 μL of PYRTS mimic (Agomir) and 3.75 μL 3000, gently pipette and mix well, and leave at room temperature.

[0051] (3) Take out the six-well plate from the cell culture incubator, discard the original medium, and rinse with PBS.

[0052] (4) Add the mixture from (3) to the corresponding six-well plate and place the six-well plate in the incubator for 6 h.

[0053] (5) After culturing for six hours, discard the original medium, add fresh medium, and place the six-well plate in the incubator for continued culture for 24 - 48 h.

[0054] 2) Immunoblotting assay

[0055] Total protein was extracted from mouse tissues using RIPA and PMSF. Total protein concentration was determined using a BCA kit and OD values were read using a microplate reader. Protein loading buffer was added to the protein samples according to their concentration and incubated at 95°C for 10 min. Protein samples were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes. Protein expression was detected using specific antibodies. The following antibodies were used: IL-1β (ABclonal, Cat: A22257, 1:1000); NLRP3 (ABclonal, Cat: A24294, 1:1000); IL-18 (ABclonal, Cat: A20473, 1:1000); GSDMD-N (ABclonal, Cat: A18281, 1:1000); Caspase 1 (Proteintech, Cat: 22915-1-AP, 1:1000); and GAPDH (ABclonal, Cat: A19056, 1:5000).

[0056] result:

[0057] To further elucidate the role of PYRTS in cardiomyocyte pyroptosis, hypoxia (hypoxia) was used to simulate myocardial infarction in adult mice. The experiments were divided into a control group (control), a hypoxia group (hypoxia), a hypoxia control group (hypoxia+NC), and a hypoxia experimental group (cells were hypoxic-treated and then transfected with PYRTS Agomir, and hypoxia+PYRTS Agomir). Immunoblotting results showed that after hypoxia, the expression of pyroptosis-related proteins increased. Lactate dehydrogenase (LDH) is released extracellularly upon cell damage, and the extent of cell damage was assessed by measuring LDH activity in the culture medium. LDH levels increased after hypoxia. The CCK-8 (Cell Counting Kit-8) assay is a commonly used method for detecting cell proliferation and cytotoxicity. It is based on the reduction of the WST-8 reagent to a water-soluble formazan dye during cellular metabolism, whose absorbance is proportional to the number of viable cells. CCK-8 assay results showed that hypoxia increased cardiomyocyte cell death, while overexpression of PYRTS alleviated this. The MDA (malondialdehyde) assay is a commonly used lipid peroxidation assay used to assess oxidative stress in cells or tissues. MDA is one of the end products of lipid peroxidation, and its content can reflect the extent of free radical damage to cell membranes. The results of the MDA assay were consistent with those of the CCK-8 assay. Figure 5-8 As shown, Figure 5 is the expression level of pyroptosis-related proteins after overexpression of PYRTS after hypoxia; Figure 6is the level of lactate dehydrogenase (LDH) after overexpression of PYRTS after hypoxia; Figure 7 is the survival rate of cardiomyocytes after overexpressing PYRTS after hypoxia; Figure 8 =The lipid peroxidation level after overexpression of PYRTS after hypoxia. In summary, overexpression of PYRTS effectively alleviates the occurrence of cardiomyocyte pyroptosis. Example 3: PYRTS as a therapeutic molecule for myocardial infarction

[0058] 1) Genetron Health commercially customized a PYRTS mimetic (Agomir), the amino acid sequence of which is shown in SEQ ID NO.5.

[0059] 2) 6- to 8-week-old mice (C57BL / 6) were selected for the experiment. Agomir was injected through the tail vein of the mice. According to the method for constructing a mouse myocardial infarction model in Example 1, Agomir was injected every 5 days to maintain the expression level of Agomir.

[0060] 3) According to the immunoblotting method in Example 1, the expression level of pyroptosis-related proteins was detected by Western Blot.

[0061] result:

[0062] In order to further detect the function of PYRTS in mice, we used commercially customized PYRTS mimics (Agomir) from GeneGene, and selected 6 to 8-week-old mice (C57BL / 6) for the experiment. Agomir was injected through the tail vein of the mice. According to the method of constructing a mouse myocardial infarction model in Example 1, Agomir was injected every 5 days to maintain the expression level of Agomir. The experiment was divided into a control blank group (Sham-NC), a control experimental group (Sham-Agomir), a myocardial infarction control group (MI-NC), and a myocardial infarction experimental group (after the mouse tail vein was injected with PYRTS Agomir, a myocardial infarction model was constructed, MI-PYRTSAgomir). The results of the immunoblotting experiment showed that according to Figure 9 The results showed that after overexpression of PYRTS, cardiac pyroptosis in mice was alleviated, indicating that the PYRTS Agomir of the present invention can be used to treat cardiomyocyte pyroptosis and myocardial infarction.

Claims

1. Application of PYRTS in products for the treatment or detection of myocardial infarction, wherein the nucleotide sequence of PYRTS is as shown in SEQ ID NO.

1.

2. Application of PYRTS as a biomarker in the preparation of products for detecting pyroptosis of cardiomyocytes or myocardial infarction.

3. A product for detecting pyroptosis of cardiomyocytes or myocardial infarction, characterized in that: The product is a reagent containing a biomarker for detection, and the biomarker is the PYRTS sequence.

4. The product for detecting pyroptosis or myocardial infarction of cardiomyocytes according to claim 3, characterized in that: The reagent for detecting the biomarker contains a primer pair for amplifying the RNA transcribed by PYRTS.

5. A product for detecting pyroptosis or myocardial infarction of cardiomyocytes according to claim 4, characterized in that: In the primer pair, the nucleotide sequences of the RT primer, the upstream primer, and the downstream primer are as shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively.

6. The application of PYRTS in the products for the treatment or detection of myocardial infarction according to claim 1, wherein: The PYRTS sequence is derived from mice, humans, or other mammals.

7. Application of PYRTS mimics as drugs for the treatment of pyroptosis of cardiomyocytes and myocardial infarction.

8. The application according to claim 8, characterized in that: The PYRTS mimic is PYRTS Agomir, which has a nucleotide sequence as shown in SEQ ID NO.

5.

9. A drug for preventing and / or treating heart diseases, characterized in that: The drug includes a PYRTS mimic, and the PYRTS mimic is PYRTS Agomir, which has a nucleotide sequence as shown in SEQ ID NO.

5.

10. The drug according to claim 9, wherein: The heart disease includes myocardial infarction or pyroptosis of cardiomyocytes.

Images