Circular RNA PYRCR and detection method and application thereof

Abstract

The invention relates to the field of biomedical treatment, in particular to circular RNA PYRCR and a detection method and application thereof. The nucleic acid sequence of the PYRCR is as shown in SEQ ID NO. 1. Effective drug delivery of myocardial ischemia reperfusion injury gene therapy can be realized by selecting adenovirus. The adenovirus serving as a gene delivery vector has the advantage of efficient transduction. The overexpressed PYRCR is wrapped by the adenovirus, so that continuous and potent protein expression can be generated in a target tissue. The injury caused by myocardial ischemia reperfusion is relieved in a simple and convenient mode of intravenous injection. The administration mode with the maximum biological safety and the minimum invasion will become a novel gene therapy means.

Description

Technical Field

[0001] The present invention relates to the field of biomedical science, and particularly to circular RNA PYRCR, its detection method and application. Background Art

[0002] With the development of social economy and the change of national economy, the impact of cardiovascular disease risk factors on the health of residents has become increasingly significant. Among various treatment modalities, gene therapy has always been a research hotspot. Gene therapy affects the occurrence and development of diseases through gene editing techniques such as overexpression or knockout of target genes. All along, the research focus has been on the genes encoding proteins that account for a very small proportion in the genome. However, it can be clearly seen from the Encyclopedia of DNA Elements (ENCODE) project that only targeting the "effective genes" encoding proteins is far from enough. ENCODE reveals that tens of thousands of non-coding RNAs have different functions. These non-coding RNAs not only participate in the basic biological processes regulating growth and development, but also play an important role in gene expression regulation. In recent years, more and more non-coding genes have been proven to be biomarkers for various diseases. Among them, circular RNA (circRNA) has gradually come into our view.

[0003] Different from traditional linear RNA (linear RNA, containing 5' and 3' ends), circRNA has a closed circular structure, is not affected by RNA exonucleases, and is more stable and not easily degraded in terms of expression. Functionally, circRNA molecules are rich in miRNA binding sites and play the role of miRNA sponge in cells, thereby relieving the inhibition of miRNA on its target genes and increasing the expression level of target genes; this mechanism of action is called the competing endogenous RNA (ceRNA) mechanism. By interacting with miRNAs associated with diseases, circRNA plays a key role in diseases.

[0004] Among various cardiovascular diseases, ischemic heart disease and myocardial infarction are the main causes of death for cardiovascular disease patients. Myocardial cell death is one of the main mechanisms leading to myocardial infarction and myocardial ischemia-reperfusion injury. On the one hand, cell death ensures the normal development of the host by removing old, damaged and useless cells. On the other hand, when cells are unable to maintain basic life functions, abnormal cell death will damage organ functions and cause inflammation. However, due to the limited regenerative ability of myocardial cells, effective regulation of myocardial cell death is a good treatment strategy for controlling the occurrence and development of cardiovascular diseases.

[0005] Pyroptosis is a common form of programmed cardiomyocyte death. Different from other programmed cell deaths, the main executors of death are caspase-1 / 4 / 5 / 11. Pyroptosis can also mediate the cleavage of IL-1β, IL-18, and GSDMD. The cleavage of GSDM family members mediated by caspases is a necessary step to trigger pyroptosis. In addition to releasing two specific pro-inflammatory cytokines, IL-1β and IL-18, it also includes other inflammatory mediators such as DNA fragments and high-mobility group protein B1. Pyroptosis is related to the pathogenesis of many cardiovascular diseases. Endothelial cell pyroptosis, macrophage pyroptosis, and smooth muscle cell pyroptosis are closely related to the occurrence and development of myocardial ischemia-reperfusion injury, myocardial infarction, and atherosclerosis. Therefore, effectively controlling and reversing pyroptosis is a good treatment method for myocardial ischemia-reperfusion injury. Among the many ways to regulate pyroptosis, the previously mentioned CircRNA can participate in multiple signaling pathways and affect the expression of inflammatory proteins. This forms a new treatment route, where tsRNA intervenes in the expression of inflammatory proteins, alleviates and reverses pyroptosis, thereby controlling the occurrence and development of myocardial ischemia-reperfusion injury. The regulatory route and molecular mechanism can be further studied or 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 have problems such as poor solubility, low drug selectivity, inability to reach effective concentrations in target organs, or rapid release, so the therapeutic effect is limited. Using adenovirus as a vector to encapsulate drugs is a common intervention method, which can achieve the purpose of improving bioavailability, reducing toxic and side effects, and maximizing the therapeutic efficiency through intravenous injection. Summary of the Invention

[0007] To solve the problems existing in the above-mentioned prior art, the present invention provides a pyroptosis-related circular RNA for detecting myocardial ischemia-reperfusion injury, its detection method and application. CircRNA participates in the pyroptosis signaling pathway to treat myocardial ischemia-reperfusion injury. 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 circular RNA PYRCR, and the nucleic acid sequence of the PYRCR is shown in SEQ ID NO.1.

[0009] The second object of the present invention is to provide a biomarker for myocardial ischemia-reperfusion injury, and the biomarker is the circular RNA PYRCR.

[0010] The present invention also provides a kit for detecting myocardial ischemia-reperfusion injury, the kit comprising primer sequences for detecting PYRCR, and the primer sequences are as shown in SEQ ID NO.2 and SEQ ID NO.3:

[0011] A third object of the present invention is to provide the application of the PYRCR sequence as a specific biomarker in the preparation of products for detecting pyroptosis of cardiomyocytes and myocardial ischemia-reperfusion injury.

[0012] Further, the product is a kit.

[0013] The present invention also includes the application of a PYRCR-specific detection reagent in the preparation of the above-mentioned pathological state diagnosis products, and the detection reagent is the PYRCR sequence.

[0014] A third object of the present invention also provides the application of PYRCR in a drug for preventing and / or treating myocardial ischemia-reperfusion injury.

[0015] A fourth object of the present invention is to provide an adenovirus overexpressing PYRCY, and the adenovirus overexpressing PYRCY has a nucleotide sequence as shown in SEQ ID NO.4.

[0016] Further, the application of the adenovirus overexpressing PYRCY as a therapeutic drug for pyroptosis of cardiomyocytes and myocardial ischemia-reperfusion injury, overexpressing PYRCY can significantly alleviate pyroptosis and improve cardiac function after myocardial ischemia-reperfusion injury.

[0017] A fifth object of the present invention is to provide a drug, the drug comprising an adenovirus overexpressing PYRCR, the drug is completed based on the adenovirus overexpressing PYRCR, and the administration method preferably selects intravenous injection to target delivery to the heart to achieve precise treatment.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention can select an adenovirus to achieve effective drug delivery for gene therapy of myocardial ischemia-reperfusion injury. Using an adenovirus as a gene delivery vector has the advantage of efficient transduction. By wrapping an adenovirus overexpressing PYRCR, continuous and potent protein expression can be generated in the target tissue. The injury of myocardial ischemia-reperfusion can be alleviated by this simple method of intravenous injection. This administration method with the greatest biosafety and the least invasiveness will become a new gene therapy means. Description of the Drawings

[0020] Figure 1 is the expression level of PYRCR in a mouse model of myocardial ischemia-reperfusion;

[0021] Figure 2Expression level of PYRCR in the hypoxia / reoxygenation model of mouse cardiomyocytes;

[0022] Figure 3 Expression level of PYRCR in human heart failure;

[0023] Figure 4 Expression level of pyroptosis-related proteins after overexpressing PYRCR after hypoxia;

[0024] Figure 5 Level of lactate dehydrogenase (LDH) after overexpressing PYRCR after hypoxia;

[0025] Figure 6 Survival rate of cardiomyocytes after overexpressing PYRCR after hypoxia;

[0026] Figure 7 Level of interleukin 18 (IL-18) after overexpressing PYRCR after hypoxia;

[0027] Figure 8 Expression level of pyroptosis-related proteins after overexpressing PYRCR after establishing a myocardial ischemia-reperfusion model in adult mice;

[0028] Figure 9 Ratio of left ventricular systolic diameter to diastolic diameter (FS) after overexpressing PYRCR after establishing a myocardial ischemia-reperfusion model in adult mice. Detailed implementation manners

[0029] The present invention will be further described below in conjunction with specific embodiments. The embodiments are only for illustrating the present invention and not for limiting the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention. The biological source of PYRCR is a mouse.

[0030] Example 1 Expression of PYRCR in Hypoxia of Mouse Cardiomyocytes and Human Heart Diseases

[0031] The technical methods involved in this example are as follows:

[0032] (1) Establish a myocardial ischemia-reperfusion injury model (Ischemia Reperfusion I / R): An adult (6-8 weeks) mouse myocardial ischemia-reperfusion injury model was established by ligating the left anterior descending coronary artery (LAD) of the mouse. The mouse was anesthetized by intraperitoneal injection of 5% chloral hydrate. When the mouse had no resistance, it was fixed on a warming plate. The hair on the trachea and chest of the mouse was removed with surgical scissors (fully exposing the surgical area), and the surgical area was disinfected with 75% alcohol. The ventilator was turned on, and after adjusting the parameters (respiratory rate 110 bpm), the trachea was cut open, and the animal ventilator was used to assist breathing. The chest cavity was cut open at an appropriate position, and a rib spreader was inserted directly between the third and fourth ribs to expose most of the mouse's heart. The LAD was ligated with a nylon suture (6-0, Ningbo Medical Suture Needle Co., Ltd., China) 2.5 mm distal to the ascending aorta to establish an adult mouse myocardial I / R injury model. After the LAD surgery, the mouse's muscle and chest incision were sutured with a 4-0 nylon suture, and the mouse was removed from the fixation and placed on the warming plate to recover until it had the ability of autonomous movement. The mice in the Sham group did not undergo the LAD ligation step, but only received anesthesia, thoracotomy, and suture.

[0033] (2) Establish a myocardial cell hypoxia / reoxygenation model (H / R): Newborn (1-3 days old) suckling mice (C57BL / 6) were taken. After disinfection with 75% ethanol immersion, the chest was opened to remove the heart, which was quickly transferred to pre-cooled sterile PBS and rinsed 3 times to remove blood cells and connective tissues. The heart tissue was minced and transferred to 10 mL of digestive solution (trypsin 1.2 mg / mL, collagenase II 0.14 mg / mL), and gently shaken in a 37°C water bath for 6 min. After each digestion, the supernatant was transferred to a centrifuge tube containing serum, and digestive solution was added to the heart tissue again and gently shaken until the large pieces of the heart were completely digested. After centrifugation at 1000 rpm / min for 10 min, the supernatant was taken and the precipitate was resuspended with F12 / DMEM containing 10% serum. After centrifugation again and taking the supernatant, the supernatant was filtered through a 70-mesh cell sieve into a 10-cm dish. The cells were cultured in a 37°C 5% CO2 incubator for 1.5 h until the fibroblasts adhered to the wall. The culture medium was taken and centrifuged at 1000 rpm / min for 10 min in a new centrifuge tube. The supernatant was discarded, and the cells were resuspended with a new culture medium and 0.1 mM bromodeoxyuridine (BrdU) was added. The cells were cultured in a 37°C 5% CO2 incubator. The culture medium was changed the next day and continued to be cultured until night. Subsequently, the culture dish was placed in an anaerobic incubator and cultured for 12 h overnight, and then cultured in a normal incubator for 6 h.

[0034] (3) Real-time fluorescence quantitative PCR (RT-qPCR): The surgical subjects were adult C57BL / 6J mice at about 8 weeks of age. Intact heart tissues were dissected from the mice in the Sham group and the I / R group respectively, and one-third of the heart tissues were separated and transferred into 2 ml clean grinding tubes. 1 ml of Trizol Reagent pre-cooled at 4°C and 4 mm zirconia grinding beads were added to each tube. Subsequently, a high-throughput tissue grinder was used for grinding, grinding for 10 seconds each time, and repeating three times until the heart tissue became homogenate; for the cells in the Con group and the H / R group, the supernatant was discarded, and the RNA was extracted by the Trizol method in the same way as the tissue RNA. After extracting the RNA, DEPC water was added to dissolve the RNA precipitate. The concentration of the extracted RNA was measured and continued to be diluted with DEPC water until the concentration was about 1000 μg / mL. The AG Evo M-MLV RT Kit reverse transcription kit was used to convert the total RNA into cDNA. SYBR Green Kit was used for fluorescence quantitative PCR. RT-qPCR was performed using the Thermo Fisher real-time fluorescence quantitative PCR detection system. The experimental results were qualitatively analyzed by the 2-△△Ct method.

[0035] Results:

[0036] To study the role of PYRCR in cardiovascular diseases, myocardial cell 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 (as Figures 1-3 shown). The results showed that the expression of PYRCR was decreased in the hearts of mice with hypoxia / reoxygenation and myocardial ischemia-reperfusion injury, and the expression of PYRCR was decreased in human heart failure. It can be seen that PYRCR may be involved in the regulation of myocardial ischemia-reperfusion injury.

[0037] Example 2 Inhibitory effect of PYRCR on pyroptosis in mouse cardiomyocytes under hypoxia

[0038] (1) Adenovirus-mediated cell infection: This implementation involves 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 + overexpression of PYRCR group. Mouse primary cardiomyocytes were cultured in DMEM / F12 medium containing 10% FBS. After the cells grew to 60%, complete medium containing virus (virus: medium = 1:1000) was added and cultured for 24 h before subsequent experiments could be carried out.

[0039] (2) Western blot assay: The cells were first discarded the culture medium, washed twice with PBS, and then collected into a 1.5 ml centrifuge tube with a cell scraper. After centrifugation, the supernatant was discarded, 100 μl of the prepared lysis buffer (PMSF: RIPA = 1:100) was added, and the cells were lysed on ice for 30 minutes, then centrifuged to collect the supernatant, and loading buffer was added and boiled for 10 minutes to obtain the protein sample. The protein sample was loaded onto a 12% separating gel and a stacking gel for SDS-PAGE electrophoresis, and then transferred to a PVDF membrane for expression detection using specific antibodies. The antibodies used included NLRP3, Caspase-1, and GSDMD-N.

[0040] Results:

[0041] To further explore the role of PYRCR in pyroptosis, at the cellular level, we established a Control group, an H / R group, an H / R + NC group, and an H / R + overexpression of PYRCR group. Western blot results showed that under hypoxia / reoxygenation conditions, the expression of pyroptosis-related proteins was enhanced; however, after overexpression of PYRCY, the expression of pyroptosis-related proteins was decreased. After pyroptosis occurred, lactate dehydrogenase (LDH) was released. Therefore, the LDH results showed that under hypoxia / reoxygenation conditions, the release of LDH increased; CCK-8 is an experiment to detect cell proliferation and cell viability, and the CCK-8 results showed that overexpression of PYRCR alleviated cell death caused by pyroptosis. Interleukin-18 (IL-18) is a pro-inflammatory cytokine, and its results can show the severity of cell damage. After verification, IL-18 decreased in the hypoxia / reoxygenation + overexpression of PYRCR group. All the above results are shown in Figures (4-7), and overexpression of PYRCR alleviated the damage caused by pyroptosis.

[0042] Example 3 Therapeutic effect of PYRCR on myocardial ischemia-reperfusion injury

[0043] In this example, Hanheng Biotech was used to commercially customize the PYRCR overexpression adenovirus vector, and the sequence is shown in SEQ ID NO.4.

[0044] (1) Six- to eight-week-old mice (C57BL / 6) were selected for tail vein injection, and the myocardial ischemia-reperfusion model in Example 1 was constructed. 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 according to the Western blot method mentioned in Example 2.

[0045] (2) Echocardiography was used to detect left ventricular FS: FS refers to the ratio of the left ventricular internal diameter in systole to that in diastole. FS is an important indicator for evaluating cardiac function, which describes cardiac function from the perspective of the muscle strength of the heart. The specific implementation method was as follows: 24 hours after establishing the myocardial ischemia-reperfusion model in mice, 200 μL of 5% chloral hydrate was injected into the mice for anesthesia and the mice were fixed on a warming plate. A 6LAB portable small animal ultrasonic imaging system (VINNO), equipped with a probe and coupling agent, was used for transthoracic echocardiography. According to the received images, the FS of the left ventricular echocardiogram of the mice was calculated using a predefined standard formula.

[0046] Results:

[0047] To further detect the function of PYRTS in mice, a commercially customized PYRCR overexpression adenovirus vector from Hanheng Biotechnology was used. Mice (C57BL / 6) aged 6 to 8 weeks were selected and injected via the tail vein, and the myocardial ischemia-reperfusion model 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 a control group (Sham group), a myocardial ischemia-reperfusion injury group (I / R group), a myocardial ischemia-reperfusion injury + control group (I / R + NC group), and a myocardial ischemia-reperfusion injury + PYRCR overexpression group. The results showed that as shown in ( Figures 8-9 ), the expression of pyroptosis-related proteins was significantly increased in the I / R group, but pyroptosis was alleviated in the myocardial ischemia-reperfusion injury + PYRCR overexpression group. Combining with the FS index, it can be seen that overexpression of PYRCR can alleviate the damage caused by pyroptosis.

[0048] In summary, PRYCR is involved in regulating myocardial cell pyroptosis and has the potential to regulate myocardial ischemia-reperfusion injury. Therefore, 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 of a drug for treating myocardial ischemia-reperfusion injury; and it can achieve targeted drug delivery. Therefore, reversing pyroptosis by increasing the expression of PYRCR in the heart and thus alleviating myocardial ischemia-reperfusion injury will be a new approach for treating cardiovascular diseases.

Claims

1. A circular RNA PYRCR, characterized in that: The nucleic acid sequence of the PYRCR is shown in SEQ ID NO.

1.

2. A marker for myocardial ischemia-reperfusion injury, characterized in that: The biomarker is the circular RNA PYRCR described in claim 1.

3. A kit for detecting myocardial ischemia-reperfusion injury, characterized in that: The kit includes primer sequences for detecting the PYRCR described in claim 1, and the primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.

3.

4. Use of the PYRCR sequence described in claim 1 in the preparation of a detection product for pyroptosis of cardiomyocytes and myocardial ischemia-reperfusion injury by a specific biomarker.

5. The application according to claim 4, wherein: The product is a kit.

6. Use of the PYRCR described in claim 1 in a medicament for preventing and / or treating myocardial ischemia-reperfusion injury.

7. An adenovirus with overexpression of PYRCY according to claim 1, characterized in that: The overexpressing adenovirus of the PYRCY has a nucleotide sequence shown in SEQ ID NO.

4.

8. Use of the adenovirus expressing the PYRCY described in claim 7 as a therapeutic medicament for pyroptosis of cardiomyocytes and myocardial ischemia-reperfusion injury.

9. A drug, characterized in that: The medicament includes the overexpressing adenovirus of the PYRCR described in claim 7, and the medicament is completed based on the overexpressing adenovirus of the PYRCR.

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