Pyroptosis-related circular RNA (Ribonucleic Acid) for detecting myocardial ischemia-reperfusion injury as well as detection method and application thereof

Abstract

The invention belongs to the field of biomedical treatment, and particularly relates to pyroptosis related circular RNA for detecting myocardial ischemia reperfusion injury and a detection method and application thereof, the small RNA is named as pyroptosis related circular RNA (PYRCR), and the nucleotide sequence is shown as SEQ ID NO: 1. According to the invention, a myocardial ischemia reperfusion injury model is constructed, an in-vitro experimental model is utilized, and continuous and potent protein expression can be generated in a target tissue through adenovirus wrapping overexpression PYRCR. Therefore, the overexpressed PYRCY can relieve pyroptosis and improve the cardiac function after myocardial ischemia-reperfusion injury, and a new thought and basis are provided for relieving myocardial ischemia-reperfusion injury and researching and developing targeted drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to pyroptosis-related circular RNA for detecting myocardial ischemia-reperfusion injury, and a detection method and application thereof. Background Art

[0002] Cardiovascular disease risk factors are increasingly impacting public health. Among the many treatment modalities, gene therapy remains a research hotspot. Gene therapy uses gene-editing techniques, such as overexpressing or knocking out target genes, to influence disease progression. Historically, research has focused on protein-coding genes, a relatively small fraction of the genome. However, the Encyclopedia of DNA Elements (ENCODE) project clearly demonstrates that targeting only protein-coding genes is insufficient. ENCODE has revealed tens of thousands of non-coding RNAs with diverse roles. These non-coding RNAs not only participate in fundamental biological processes regulating growth and development but also play a crucial role in regulating gene expression. In recent years, an increasing number of non-coding genes have been shown to serve as biomarkers for various diseases, with circular RNAs (circRNAs) gradually coming into our view.

[0003] Unlike traditional linear RNA (linear RNA, with 5' and 3' ends), circRNAs exhibit a closed circular structure, making them impervious to RNA exonucleases, more stable in expression, and less susceptible to degradation. Functionally, circRNA molecules are rich in miRNA binding sites, acting as miRNA sponges in cells, thereby relieving the inhibitory effects of miRNAs on their target genes and increasing their expression levels. This mechanism of action is known as the competing endogenous RNA (ceRNA) mechanism. By interacting with disease-associated miRNAs, circRNAs play a key role in disease.

[0004] Among the many cardiovascular diseases, ischemic heart disease and myocardial infarction are the leading causes of death in patients with cardiovascular disease. Cardiomyocyte death is one of the main mechanisms leading to myocardial infarction and myocardial ischemia-reperfusion injury. On the one hand, cell death ensures normal host development by removing old, damaged, and useless cells. On the other hand, when cells are unable to maintain basic life functions, abnormal cell death can disrupt organ function and cause inflammation. However, due to the limited regenerative capacity of cardiomyocytes, effectively regulating cardiomyocyte death is a good therapeutic strategy for controlling the occurrence and development of cardiovascular diseases.

[0005] Pyroptosis is a common form of programmed cardiomyocyte death. Unlike other programmed cell death pathways, pyroptosis is primarily initiated by caspase-1 / 4 / 5 / 11. Pyroptosis can also mediate the cleavage of IL-1β, IL-18, and GSDMD. Caspase-mediated cleavage of GSDM family members is a necessary step in triggering pyroptosis. In addition to the release of two specific proinflammatory cytokines, IL-1β and IL-18, other inflammatory mediators are also released, including DNA fragmentation and high-mobility group protein B1. Pyroptosis has been implicated in the pathogenesis of many cardiovascular diseases. Pyroptosis of endothelial cells, macrophages, and smooth muscle cells is closely associated with the onset and progression of myocardial ischemia-reperfusion injury, myocardial infarction, and atherosclerosis. Therefore, effectively controlling and reversing pyroptosis is a promising therapeutic approach for myocardial ischemia-reperfusion injury. Among the many mechanisms regulating pyroptosis, the previously mentioned circRNAs can participate in multiple signaling pathways and influence the expression of inflammatory proteins. This has formed a new therapeutic approach, using tsRNA to intervene in the expression of inflammatory proteins, alleviate and reverse pyroptosis, and thus control the occurrence and development of myocardial ischemia-reperfusion injury. The regulatory pathways and molecular mechanisms involved can be further studied and applied, so circRNA intervention in myocardial ischemia-reperfusion injury is an application with outstanding potential.

[0006] Traditional clinical drugs for the treatment of myocardial ischemia-reperfusion injury may suffer from poor solubility, low drug selectivity, inability to achieve effective concentrations in target organs, or excessive release, resulting in limited therapeutic efficacy. Using adenovirus as a vector to encapsulate drugs is a common intervention approach, and intravenous injection can improve bioavailability, reduce toxic side effects, and maximize therapeutic efficiency. Summary of the Invention

[0007] To address the problems of the above-mentioned prior art, the present invention proposes a pyroptosis-related small RNA for detecting myocardial ischemia-reperfusion injury, as well as a detection method and application thereof. CircRNA treats myocardial ischemia-reperfusion injury by participating in the pyroptosis signaling pathway. This small RNA has the ability to regulate pyroptosis, so it is named pyroptosis-related circular RNA (PYRCR).

[0008] The first object of the present invention is to provide a marker for myocardial ischemia-reperfusion injury.

[0009] A second object of the present invention is to provide a marker for detecting pyroptosis.

[0010] The third object of the present invention is to provide a kit for detecting myocardial ischemia-reperfusion injury.

[0011] The fourth object of the present invention is to provide a use of a PYRCR drug for preventing and / or alleviating myocardial ischemia-reperfusion injury.

[0012] The present invention adopts the following technical solutions: A marker for myocardial ischemia-reperfusion injury is provided, wherein the marker is PYRCR, and the nucleic acid sequence of the PYRCR is shown in SEQ ID NO.1.

[0013] The present invention also provides a kit for detecting myocardial ischemia-reperfusion injury, which includes primers for PYRCR detection: PYRCR-F and PYRCR-R, whose nucleic acid sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0014] The present invention also provides the use of PYRCR in preparing gene medicines for myocardial cell pyroptosis and myocardial ischemia-reperfusion injury.

[0015] The gene medicine component for myocardial cell pyroptosis and myocardial ischemia-reperfusion injury is a PYRCY overexpression adenovirus, and the overexpression PYRCY nucleic acid sequence carried by the adenovirus is shown in SEQ ID NO: 4.

[0016] The PYRCR overexpression adenovirus is preferably administered by intravenous injection, so as to be delivered to the heart in a targeted manner to achieve precise treatment.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention utilizes adenovirus to achieve effective drug delivery for gene therapy of myocardial ischemia-reperfusion injury. Using adenovirus as a gene delivery vector offers the advantage of efficient transduction. Overexpression of PYRCR via adenovirus packaging can result in sustained and robust protein expression within target tissues. This convenient intravenous injection alleviates myocardial ischemia-reperfusion injury. This highly biosafe and minimally invasive administration method will become a novel gene therapy approach. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the expression level of PYRCR in the mouse myocardial ischemia-reperfusion model; Figure 2 is the expression level of PYRCR in the mouse cardiomyocyte hypoxia / reoxygenation model; Figure 3 is the expression level of PYRCR in human heart failure; Figure 4 is the expression level of pyroptosis-related proteins after overexpression of PYRCR after hypoxia; Figure 5is the level of lactate dehydrogenase (LDH) after overexpression of PYRCR after hypoxia; Figure 6 is the survival rate of cardiomyocytes after overexpressing PYRCR after hypoxia; Figure 7 is the interleukin-18 (IL-18) level after overexpression of PYRCR after hypoxia; Figure 8 After establishing a myocardial ischemia-reperfusion model in adult mice, the expression levels of pyroptosis-related proteins after overexpression of PYRCR were analyzed; Figure 9 After establishing a myocardial ischemia-reperfusion model in adult mice, the ratio of left ventricular systolic diameter to diastolic diameter (FS) after overexpression of PYRCR was measured. DETAILED DESCRIPTION

[0019] The present invention will be further described below with reference to specific embodiments. These examples are intended to illustrate the present invention, not to limit the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0020] Example 1 Expression of PYRCR in Mouse Cardiomyocytes with Hypoxia and Human Heart Diseases (1) Establishment of myocardial ischemia reperfusion injury model: The myocardial ischemia reperfusion injury model was established in adult (6-8 weeks) mice by ligating the left anterior descending coronary artery (LAD). Mice were anesthetized by intraperitoneal injection of 5% chloral hydrate and fixed on a warming board when they were unable to resist. The hair on the trachea and chest of the mice was removed with surgical scissors (to fully expose the surgical area), and the surgical area was disinfected with 75% alcohol. The ventilator was turned on, and after adjusting the various parameters (respiratory rate 110 bpm), the trachea was cut open and assisted breathing was performed using an animal ventilator. The chest cavity was cut open at the appropriate position, and a chest expander was directly inserted between the third and fourth ribs to expose most of the mouse heart. The LAD was ligated 2.5 mm distal to the ascending aorta with nylon suture (6-0, Ningbo Medical Suture Needle Co., Ltd., China) to establish the myocardial I / R injury model in adult mice. After LAD surgery, the muscles and chest incisions of the mice were sutured with 4-0 nylon sutures. The mice were then unfixed and placed on a warming board until they could regain voluntary movement. Mice in the sham group did not undergo LAD ligation; instead, they were anesthetized, had their chests opened, and were sutured.

[0021] (2) Construction of cardiomyocyte hypoxia / reoxygenation model (H / R): Take 1-3 days old suckling mice (C57BL / 6), soak them in 75% ethanol for disinfection, open the chest and remove the heart, quickly transfer it to pre-cooled sterile PBS and rinse it three times to remove blood cells and connective tissue. Cut the heart tissue into pieces and transfer it to 10 mL digestion solution (trypsin 1.2 mg / mL, collagenase II 0.14 mg / mL), and shake it gently in a 37℃ water bath for 6 minutes. After each digestion, transfer the supernatant to a centrifuge tube containing serum, add digestion solution to the heart tissue again and continue to shake it gently until the large piece of heart is completely digested. After centrifugation at 1000 rpm / min for 10 minutes, remove the supernatant and resuspend the pellet in F12 / DMEM containing 10% serum. Centrifuge again and remove the supernatant. Then filter the supernatant through a 70-mesh cell sieve into a 10 cm dish. Culture in a 37°C, 5% CO2 incubator for 1.5 hours until fibroblasts adhere. Remove the culture medium and transfer it to a new centrifuge tube, centrifuging at 1000 rpm / min for 10 minutes. Discard the supernatant, resuspend the cells in fresh culture medium, and add 0.1 mM bromodeoxyuridine (BrdU). Incubate in a 37°C, 5% CO2 incubator. The next day, change the medium and continue incubation overnight. Subsequently, place the culture dish in a hypoxic incubator for 12 hours overnight, followed by 6 hours in a normal incubator.

[0022] (3) Real-time fluorescence quantitative PCR (RT-qPCR): The surgical subjects were adult C57BL / 6J mice aged about 8 weeks. The complete heart tissue was dissected out from the mice in the Sham group and the I / R group, and one-third of the heart tissue was divided and transferred to a 2 ml clean grinding tube. 1 ml of 4°C pre-cooled Trizol Reagent and 4 mm zirconium oxide grinding beads were added to each tube. Then, a high-throughput tissue grinder was used to grind for ten seconds each time, and repeated three times until the heart tissue became a homogenate; the supernatant of the cells in the Con group and the H / R group was discarded, and the Trizol method was used to extract the RNA in the same way as the tissue RNA. After RNA extraction, DEPC water was added to dissolve the RNA precipitate. The extracted RNA concentration was measured and continued to be diluted with DEPC water until the concentration was about 1000 μg / mL. The total RNA was converted into cDNA using the AG Evo M-MLVRT Kit reverse transcription kit. Fluorescence quantitative PCR was performed using the SYBR Green Kit. The RT-qPCR was detected using the Thermo Fisher real-time fluorescence quantitative PCR detection system. The experimental results were analyzed using 2 -△△Ct The relative expression levels were qualitatively analyzed by qPCR. The qPCR primer sequences were as follows: PYRCR-F SEQ ID NO.2: 5'--CTAGCTGAGCTGGGAAGTAACA --3' PYRCR-R SEQ ID NO.3 5'--AGCAAGGTGTATTCTGGGGAG--3'.

[0023] Results: To investigate the role of PYRCR in cardiovascular disease, myocardial hypoxia / reoxygenation samples (H / R), mouse ischemia-reperfusion injury samples (I / R), and human heart failure samples (HF) were collected and the expression level of PYRCR was detected by RT-qPCR (e.g. Figure 1-3 The results showed that PYRCR expression was decreased in the hearts of mice with hypoxia / reoxygenation and myocardial ischemia-reperfusion injury, and PYRCR expression was decreased in human heart failure. These results indicate that PYRCR may be involved in the regulation of myocardial ischemia-reperfusion injury.

[0024] Example 2: Inhibitory Effect of PYRCR on Pyroptosis in Mouse Cardiomyocytes During Hypoxia (1) Adenovirus-mediated cell infection: This experiment involved a control group (Control group), a hypoxia / reoxygenation group (H / R group), a hypoxia / reoxygenation control group (H / R+NC group), and a hypoxia / reoxygenation + PYRCR overexpression group. Mouse primary cardiomyocytes were cultured in DMEM / F12 medium containing 10% FBS. After the cells grew to 60% confluence, complete medium containing virus (virus: medium = 1:1000) was added and cultured for 24 hours before subsequent experiments.

[0025] (2) Immunoblotting: The cells must first be freed of culture medium, washed twice with PBS, and then collected into a 1.5 ml centrifuge tube using a cell scraper. After centrifugation, the supernatant was discarded and 100 μl of the prepared lysis buffer (PMSF:RIPA = 1:100) was added. Lysis was continued on ice for 30 minutes, and the supernatant was collected by centrifugation. The supernatant was added and boiled for 10 minutes to obtain the protein sample. The protein sample was subjected to SDS-PAGE electrophoresis using a 12% separation gel and a stacking gel, and then transferred to a PVDF membrane. Specific antibodies were used for expression. Antibodies used included NLRP3, Caspase-1, and GSDMD-N.

[0026] Results: To further explore the role of PYRCR in pyroptosis, we established control, H / R, H / R + NC, and H / R + PYRCR overexpression groups. Immunoblotting results showed that hypoxia / reoxygenation conditions enhanced the expression of pyroptosis-related proteins, while overexpression of PYRCR reduced their expression. Following pyroptosis, lactate dehydrogenase (LDH) is released. LDH assays showed increased LDH release under hypoxia / reoxygenation. CCK-8 assays, which measure cell proliferation and viability, demonstrated that PYRCR overexpression alleviated pyroptosis-induced cell death. Interleukin-18 (IL-18), a proinflammatory cytokine that indicates the severity of cell damage, was found to be decreased in the hypoxia / reoxygenation + PYRCR overexpression group. These results, as shown in Figures 4-7, indicate that PYRCR overexpression alleviates pyroptosis-induced damage.

[0027] Example 3 The therapeutic effect of PYRCR in myocardial ischemia-reperfusion injury The PYRCR overexpression nucleic acid sequence using adenovirus as a vector is shown in SEQ ID NO: 4 and was synthesized by Hanheng Biotechnology.

[0028] (1) Six to eight-week-old mice (C57BL / 6) were selected and injected into their tail veins to establish the myocardial ischemia-reperfusion model described in Example 1. During this period, the PYRCR overexpression virus was injected once every five days to maintain its expression. The expression of pyroptosis-related proteins was detected by immunoblotting according to the method described in Example 2.

[0029] (2) Left ventricular FS by echocardiography: FS refers to the ratio of the left ventricular systolic diameter to the left ventricular diastolic diameter. FS is an important indicator for evaluating cardiac function and describes cardiac function from the perspective of cardiac muscle strength. The specific implementation method is to inject 200 μL of 5% chloral hydrate into the mouse 24 hours after the establishment of a myocardial ischemia-reperfusion model for anesthesia and fix it on a warming board. Transthoracic echocardiography is performed using a 6 LAB portable small animal ultrasound imaging system (VINNO) equipped with a probe and coupling agent. Based on the received images, the established standard formula is used to calculate the FS of the mouse left ventricle echocardiography.

[0030] Results: To further test the function of PYRTS in mice, we used a commercially customized PYRCR overexpression adenovirus vector from Hanbio. We selected 6- to 8-week-old mice (C57BL / 6) and injected them into their tail veins. The myocardial ischemia-reperfusion model described in Example 1 was established. During this period, the PYRCR overexpression virus was injected once every five days to maintain its expression. The experimental groups were divided into the control group (Sham group), myocardial ischemia-reperfusion injury (I / R group), myocardial ischemia-reperfusion injury + control group (I / R+NC group), and myocardial ischemia-reperfusion injury + PYRCY overexpression group. The results showed that ( Figure 8-9 As shown in Figure 3 , the expression of pyroptosis-related proteins was significantly increased in the I / R group, but pyroptosis was alleviated in the myocardial ischemia-reperfusion injury + PYRCY overexpression group. Combined with FS indicators, it can be seen that overexpression of PYRCR can alleviate the damage caused by pyroptosis.

[0031] In summary, PYRCR is involved in regulating cardiomyocyte pyroptosis and has the potential to modulate myocardial ischemia-reperfusion injury. Therefore, the PYRCR provided by the present invention can be used as a marker for detecting, diagnosing, and treating myocardial ischemia-reperfusion injury; it can be used as an active ingredient in drugs treating myocardial ischemia-reperfusion injury; and it can also achieve targeted drug delivery. Therefore, increasing PYRCR expression in the heart to reverse pyroptosis and thereby alleviate myocardial ischemia-reperfusion injury is an emerging approach for treating cardiovascular diseases.

Claims

1. A marker for myocardial ischemia-reperfusion injury, characterized in that: The marker is circular RNA-PYRCR, and the nucleic acid sequence of PYRCR is shown in SEQ ID NO.

1.

2. A kit for detecting myocardial ischemia-reperfusion injury, comprising primers for PYRCR detection: PYRCR-F and PYRCR-R, whose nucleic acid sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

3. Use of the marker PYRCR as claimed in claim 1 in the preparation of gene drugs for cardiomyocyte pyroptosis and myocardial ischemia-reperfusion injury.

4. The gene medicine component for myocardial cell pyroptosis and myocardial ischemia-reperfusion injury according to claim 3 is a PYRCY overexpressing adenovirus, and the overexpressed PYRCY nucleic acid sequence carried by the adenovirus is shown in SEQ ID NO:

4.

5. The PYRCR overexpression adenovirus according to claim 4, characterized in that The preferred route of administration is intravenous injection.

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