Small RNA FRSR and detection method and application thereof

Abstract

The invention relates to the field of cell biology and biological medicine, and discloses application of FRSR (Ferroptosis Related Small RNA) and FRSR mimics related to ferroptosis and a product of the FRSR mimics related to ferroptosis, which have important application in detection, diagnosis and treatment of ferroptosis and high glucose damage. Wherein the nucleotide sequence of the small non-coding FRSR is as shown in SEQ ID NO. 1. According to the invention, overexpression of FRSR in the heart is realized by constructing a high-glucose injury model, utilizing an in-vitro experimental model and using FRSR mimics, so that it is explained that overexpression of FRSR can alleviate ferroptosis and improve the heart function after high-glucose injury, and a new idea and basis are provided for alleviating diabetic heart disease and researching and developing targeted drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a Ferroptosis related small RNA (FRSR) for detecting high glucose injury, and a detection method and application thereof. BACKGROUND

[0002] Cardiovascular disease is a major cause of death worldwide, and its incidence is rising due to population aging and lifestyle factors. The most common cardiovascular diseases are myocardial injury (such as myocardial ischemia, myocardial infarction), atrial fibrillation (AF), heart failure (HF) and diabetic cardiomyopathy (DCM). According to the data of American Heart Association (AHA)

[99] , about 1.4 million people died of cardiovascular diseases worldwide in 2021, and the prevalence of heart disease in young people is increasing over time. This emphasizes the urgent need to detect and properly treat cardiovascular diseases as early as possible.

[0003] DCM is a cardiovascular complication caused by high blood sugar in diabetic patients. The clinical manifestations of high glucose injury are abnormal myocardial cell contraction and relaxation, hypertension, and heart failure of coronary artery disease. In recent years, with the increasing incidence of diabetes, the number of patients with diabetic cardiomyopathy has also been increasing year by year, which brings great pain and economic pressure to patients and society. Therefore, it is of great significance to find more safe and effective drugs. TsRNA is generated by specific ribonuclease endonuclease cleavage of precursor tRNA or mature tRNA at different positions. Therefore, his biogenesis is regulated by tRNA modification. According to the cleavage of the leader sequence and the tail sequence generated by different modification sites, the nucleic acid encoded tsRNA can be roughly divided into two categories: tRNA-derived stress-induced RNA (tiRNA or tRH) and tRNA-derived fragments (tRF). At the same time, tsRNA can find RNA targets by binding to AGO2 protein and using miRNA-like base pairing to directly cleave nascent RNA in a sequence-specific manner; or directly bind to RNA binding protein (RBP), separate RBP from target RNA, and thus affect RNA stability and participate in gene expression regulation. If the effective silencing of pathogenic factors or death-related proteins can reverse the occurrence and development of diseases.

[0004] Ferroptosis is a form of iron-dependent programmed cell death characterized by excessive lipid peroxidation and disruption of cellular metabolism and intracellular redox homeostasis. The regulation of ferroptosis involves multiple molecules and signaling pathways, including negative regulators such as glutathione peroxidase 4 (GPX4), heat shock protein beta-1 (HSPB1) and nuclear factor erythroid 2-related factor 2 (NRF2), and positive regulators such as NADPH oxidase and p53. These regulators regulate ferroptosis by limiting the generation of reactive oxygen species (ROS), reducing cellular iron uptake, or by promoting ROS production and inhibiting the expression of SLC7A11. In addition, ferroptosis plays a regulatory role in DCM, so inhibiting ferroptosis can prevent myocardial cell death and be an effective treatment strategy for cardiovascular diseases, especially DCM. Some non-coding RNAs (ncRNAs) also play an important role in regulating cellular ferroptosis in cardiovascular diseases. Studies have found that ncRNAs can affect cellular ferroptosis by regulating related signaling pathways, thereby playing a key role in the development of cardiovascular diseases. Small RNAs play an important role in the treatment of cardiovascular diseases by regulating signaling pathways. This type of non-coding RNA has a unique "one-to-many" regulatory property, which can simultaneously act on multiple target proteins or target genes, thereby forming a complex regulatory network. Further research into its potential mechanisms will provide new targets for the treatment of cardiovascular diseases. SUMMARY

[0005] The present application proposes that when diabetic cardiomyopathy occurs, the subsequent high glucose injury leads to myocardial cell death and fibrosis, which in turn affects heart function. At the same time, the expression level of FRSR is reduced and shows a significant correlation with the process of myocardial cell ferroptosis; overexpression of FRSR can significantly inhibit the occurrence of ferroptosis and inhibit the expression of ferroptosis-related proteins such as GPX4, and plays an important role in the regulation of myocardial cell death. The treatment methods and treatment effects of diabetic cardiomyopathy are limited, therefore, the performance of FRSR in the regulation of myocardial cell death makes us believe that it can be used as a therapeutic target for diabetic cardiomyopathy and has the ability to improve the clinical manifestations of diabetic heart disease.

[0006] The present application adopts the following technical solutions: The first object of the present application is to provide a marker for high glucose injury, which is small RNA FRSR, and the nucleic acid sequence of FRSR is shown as SEQ ID NO. 1.

[0007] The present application also provides a kit for detecting high glucose injury, which comprises primer sequences for detecting FRSR, and the primer sequences are shown as SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.

[0008] The second object of the present application is to provide the application of FRSR in the product for preventing and / or treating myocardial cell ferroptosis.

[0009] The third object of the present application is to provide the application of FRSR as a specific biomarker in the preparation of a diagnostic kit for evaluating ferroptosis and diabetic cardiomyopathy.

[0010] The fourth object of the present application is to provide the use of a reagent for detecting a specific molecular marker comprising the nucleic acid sequence of FRSR in an in vitro diagnostic device for ferroptosis and diabetic cardiomyopathy.

[0011] The fifth object of the present application discloses an in vitro diagnostic system for clinically detecting ferroptosis and diabetic cardiomyopathy, characterized in that it comprises a nucleic acid sequence capable of specifically recognizing FRSR.

[0012] In some embodiments, the FRSR sequence is derived from mice, humans or other mammals.

[0013] In some embodiments, the reagent for detecting a specific molecular marker comprises primers for amplifying FRSR transcriptional RNA; preferably, the nucleotide sequences of the RT primers and the upstream and downstream primers in the primers are as shown in SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4.

[0014] The fifth object of the present application also provides a FRSR overexpression adenovirus having a nucleotide sequence as shown in SEQ ID NO. 5.

[0015] Further, the FRSR overexpression can be used to prepare a drug for treating myocardial cell ferroptosis and diabetic cardiomyopathy. Studies have shown that FRSR overexpression can significantly inhibit the occurrence of myocardial cell ferroptosis and improve cardiac dysfunction related to diabetic cardiomyopathy.

[0016] The sixth aspect of the present application also provides a drug for preventing and / or treating diabetic cardiomyopathy, which comprises a FRSR overexpression adenovirus. The preferred treatment method is intravenous injection, which targets the heart for precise treatment.

[0017] Compared with the prior art, the present application has at least the following beneficial effects: The present application first discovers that the expression of FRSR is reduced when diabetic cardiomyopathy occurs, and is highly related to the occurrence of myocardial cell ferroptosis, which provides a new idea for subsequent treatment. In addition, the present application realizes effective drug delivery of diabetic heart disease gene therapy by constructing an adenovirus vector. The treatment method of overexpressing FRSR wrapped by adenovirus as a gene delivery vector has the advantage of high-efficiency transduction. This drug delivery method with the highest biological safety and the least invasiveness will become a new gene therapy method. BRIEF DESCRIPTION OF DRAWINGS

[0018] To clearly explain the implementation path and innovation points of the technical solutions of the present application, the drawings involved in the embodiments will be described as follows: It should be particularly noted that the drawings in this part are only used to illustrate the core inventive concept, and do not limit the protection scope. Based on the illustrated principles, those skilled in the art can make reasonable extensions in accordance with the technical route of the present application.

[0019] Figure 1 Expression level of FRSR in a high-sugar injury model of mice; Figure 2 Expression level of FRSR in a diabetic cardiomyopathy model of mice; Figure 3 Expression level of X-CT ferroptosis protein after overexpression of FRSR after high-sugar injury Figure 4 Lipid peroxidation (MDA) level after overexpression of FRSR after high-sugar injury; Figure 5 Expression level of iron death marker gene Ptgs2 mRNA after overexpression of FRSR after high-sugar injury; Figure 6 Survival rate of myocardial cells after overexpression of FRSR after high-sugar injury; Figure 7 Fe level of myocardial cells after overexpression of FRSR after high-sugar injury 2+ level. DETAILED DESCRIPTION

[0020] The present application will be further described below in combination with specific embodiments. The examples are only limited to illustrate the present application, but not to limit the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application. C57 mice are used, and the Latin name of C57 mice is Mus musculus .

[0021] Example 1: Expression of FRSR in high-sugar injury of myocardial cells of mice The technical method involved in this embodiment is as follows: (1) Construct a high glucose injury model (High glucose HG): Take 1-2 day old neonatal mice (C57BL / 6) to isolate myocardial cells. Clean and disinfect the mouse by spraying 75% alcohol, use ophthalmic scissors to cut open the mouse chest to take out the heart, and wash it three times in pre-cooled PBS in a sterile environment. Cut the heart tissue with ophthalmic scissors, transfer the heart tissue to a conical flask containing 10 mL of digestion solution (trypsin 1.2 mg / mL, collagenase II 0.14 mg / mL), and shake gently in a 37°C water bath for 7 min. After each digestion, the supernatant obtained by digestion is transferred to a centrifuge tube containing serum on ice for storage. After removing the supernatant, add new digestion solution and repeat about ten times until the heart tissue disappears. Centrifuge the centrifuge tube containing the collected supernatant at 1000 rpm / min for 10 min, resuspend the precipitate with F12 / DMEM containing 10% serum, centrifuge again at 1000 rpm / min for 10 min, take the supernatant again, filter it with a 70-mesh cell sieve, and place the filtered myocardial cells in a 10 cm culture dish. Incubate in a 5% CO2 humidified incubator at 37°C for 1.5 h until the fibroblasts adhere. After the fibroblasts adhere, centrifuge the centrifuge tube containing the myocardial cells in the culture dish at 1000 rpm / min for 10 min. Discard the supernatant and resuspend it with new medium containing serum. Add 0.1 mM bromodeoxyuridine (BrdU) to inhibit the growth of non-myocardial cells. According to the experimental requirements, add high glucose F12 / DMEM medium (25-30 mM D-glucose as the high glucose treatment concentration) the next day, and then proceed with the subsequent experiments.

[0022] (2) Construct a diabetic cardiomyopathy model: Select 6 to 8 week old mice (C57BL / 6) for the experiment. They were raised under controlled temperature conditions (18-24°C) and reversed 12-hour light-dark cycles, and were allowed to eat and drink freely for one week. After feeding with 60% fat energy high-fat diet for one week, the model group mice were fasted for 12 hours every night, and continuously injected with streptozotocin (STZ, 35 mg / kg, dissolved in 0.1 mM citrate buffer, pH 4.4) for 5 days to induce insulin deficiency in mice. On days 7, 10, and 14 after STZ injection, blood samples were collected from the tail vein of the mice, and the fasting blood glucose level of the mice was measured using a blood glucose meter. Only mice with a blood glucose level of more than 16.7 mmol / l for three consecutive times were considered to be successfully modeled, and then fed with high-fat diet for 3 months before proceeding with the subsequent experiments.

[0023] (3) Real-time quantitative PCR (RT-qPCR): This implementation involves cell level and animal level. The cell level is divided into control group (CON) and high glucose injury group (HG), and the animal level is divided into control group (Sham) and severe diabetic cardiomyopathy group (DCM). The heart sample processing procedure is as follows: after collecting the heart tissue of the Sham group and the DCM group, about 1 / 3 of the tissue sample is accurately cut and placed in a sterile grinding tube. Add pre-cooled Trizol Reagent (1 mL, 4°C) and 4 mm zirconium oxide grinding beads to each centrifuge tube, and use a tissue homogenizer to perform three-stage homogenization treatment (10 s / time, interval 30 s) until a homogeneous suspension is obtained. After centrifugation to remove the culture solution, the Con group and the HG group cell samples are synchronized to complete RNA extraction according to the Trizol method. The RNA precipitate of all samples is dissolved with DEPC water, and the final concentration is adjusted to 1000 µg / mL after detection by a spectrophotometer. AGEvo M-MLV RT kit is used to synthesize cDNA. The SYBR Green fluorescent quantitative detection system is used for amplification reaction on the Thermo Fisher quantitative PCR instrument. The difference in gene expression is quantitatively evaluated by the 2-ΔΔCt method, and the relative expression is obtained after the experimental data are calibrated by the system reference.

[0024] Results: In order to study the expression of FRSR in high glucose injury of mouse myocardial cells and diabetic cardiomyopathy, high glucose injury samples (HG) and mouse diabetic cardiomyopathy samples (DCM) were collected. The expression level of FRSR was detected by RT-qPCR (as shown in Table 2), and the results showed that the expression of FRSR was reduced in high glucose injury and diabetic cardiomyopathy. Therefore, FRSR may be involved in the regulation of heart diseases such as diabetic cardiomyopathy. Figure 1 .2

[0025] Example 2: Inhibition of ferroptosis of FRSR in high glucose injury of mouse myocardial cells (1) Lipofectamine® 3000-mediated cell transfection: After treating the isolated primary myocardial cells of the mouse with F12 / DMEM medium for 24 h, take one 1.5 mL EP tube, and add 400 μL of DMEM high glucose medium to the 1.5 mL EP tube, add 5 μL of FRSR mimics, add 2.5 μL of Lipofectamine® 3000, and mix gently. Put it at room temperature for 20 min. Then add the prepared transfection reagent to the culture dish to be transfected. After 6 h of culture, discard the original culture medium, and add a newly prepared high glucose medium (25-30 mM D-glucose) to the HG+FRSR mimics group.

[0026] (2) Western blotting: total protein was extracted from cells using RIPA and PMSF, and the protein concentration was determined using a BCA kit. The OD value was determined by a microplate reader to determine the protein loading amount. The protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane, and X-CT antibody was used to detect protein expression. (3) Detection of lipid peroxidation (MDA) in cardiomyocytes: a malondialdehyde content detection kit was used to detect the malondialdehyde content in the cell sample. Malondialdehyde in the sample can condense with thiobarbituric acid (TBA) under acidic and high temperature conditions to generate a brown red 3,5,5-trimethyl oxazole-2,4-dione, which has a maximum absorption wavelength at 532 nm. After colorimetry, the content of peroxidized lipids in the sample can be estimated.

[0027] (4) Detection of iron death marker genes in cardiomyocytes Ptgs2 mRNA expression: total RNA was extracted from each cell sample using the Trizol method. The RNA precipitate was dissolved in deionized water to a concentration of about 1000 μg / mL; the RT Primer Mix (containing Oligo dT Primer and Random 6 mers Primer) of the reverse transcription kit was used to reverse the Ptgs2 and the reference gene, and mouse U6 was selected as the reference gene, and the amplification conditions were according to the instructions. The cDNA obtained after reverse transcription was quantitatively detected by real-time fluorescence quantitative PCR (Thermo Fisher) according to the 2^ (-ΔΔCt) method.

[0028] (5) Detection of Fe 2+ level: using ferrous ion colorimetric test kit (Elabscience), the content of ferrous ion in the cell sample was detected. The ferrous ion in the sample combined with the probe to generate a substance with a strong absorption peak at 593 nm wavelength, and within a certain range, its optical density value was linearly related to the concentration of ferrous ion.

[0029] Results: To further explore the role of FRSR in cell iron death, we established Control group, HG group, HG+NC group, and HG+FRSR mimics group at the cellular level. Western blotting results showed that under high glucose damage conditions, the expression of pyroptosis-related proteins was increased; but in the HG+FRSR mimics group, overexpression of FRSR reduced the expression of pyroptosis-related proteins. After iron death occurred, the lipid peroxidation (MDA) level and Ptgs2mRNA expression will increase. Therefore, the results show that under HG conditions, the indicators of ferroptosis are greatly increased; CCK-8 is an experiment for detecting cell proliferation and cell activity, and the CCK-8 results show that overexpression of FRSR alleviates the damage caused by ferroptosis. The results are shown in FIG. Figures 3-7 )Overexpression of FRSR reverses ferroptosis and alleviates the damage caused by diabetic cardiomyopathy.

[0030] In summary, FRSR plays an important role in the regulation of ferroptosis in myocardial cells and exhibits significant potential in regulating diabetic cardiomyopathy. This finding indicates that FRSR has multiple application values: first, it can be used as a biomarker for high glucose damage for clinical detection and diagnosis; second, it can be used as an active ingredient of therapeutic drugs; third, it can achieve precise targeted drug delivery. These characteristics provide a new idea for the treatment of cardiovascular diseases, that is, by enhancing the expression level of FRSR in heart tissue to inhibit cell ferroptosis, thereby effectively improving diabetic cardiomyopathy, which is expected to become an important breakthrough direction in the field of cardiovascular disease treatment.

Claims

1. A marker of high glyco-cariage, said marker being the small RNA FRSR, characterized in that: The nucleic acid sequence of the FRSR is shown as SEQ ID NO.

1.

2. A kit for detecting high glucose damage, characterized by: The kit is the primer sequence for the detection of the FRSR of claim 1, and the primer sequence is shown as SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO.

4.

3. The FRSR of claim 1 is used in the product for preventing and / or treating myocardial cell ferroptosis.

4. The FRSR of claim 1 is used as a specific biomarker in the preparation of a diagnostic kit for evaluating ferroptosis and diabetic cardiomyopathy.

5. Use of a reagent for detecting the FRSR nucleic acid sequence marker of claim 1 in an in vitro diagnostic device for ferroptosis and diabetic cardiomyopathy.

6. An in vitro diagnostic system for the clinical detection of ferroptosis and diabetic cardiomyopathy, characterized by: It comprises a nucleic acid sequence capable of specifically recognizing the FRSR of claim 1.

7. An FRSR overexpressing adenovirus, characterized in that: The FRSR overexpression adenovirus has a nucleotide sequence shown as SEQ ID NO.

5.

8. Use of the FRSR overexpression adenovirus of claim 7 in the preparation of a drug for treating myocardial cell ferroptosis and diabetic cardiomyopathy.

9. A drug for preventing and / or treating diabetic cardiomyopathy, comprising the FRSR overexpression adenovirus of claim 7.

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