Ferroptosis-related small 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 cytobiology and biological medicine, and relates to ferroptosis-related small RNA for detecting myocardial ischemia-reperfusion injury and a detection method and application of the ferroptosis-related small RNA. The small RNA is named as ferroptosis related small RNA (FASR), and the nucleotide sequence of the small RNA is shown as SEQ ID NO: 1. The expression of the FASR in the heart suffering from ischemia-reperfusion injury is remarkably reduced. FASR mimics or FASR agomir is used for realizing overexpression of FASR in the heart, ferroptosis can be remarkably inhibited, left ventricular FS and EF after myocardial ischemia reperfusion injury can be remarkably improved, and a cardiac function protection effect is achieved. The medicine contains at least one of FASR, FASR mimics and FASR agomir, and the myocardial ischemia reperfusion injury can be well prevented and / or treated through overexpression of the FASR.

Description

Technical Field

[0001] The present invention belongs to the fields of cell biology and biomedicine, and relates to ferroptosis-related small RNAs for detecting myocardial ischemia-reperfusion injury, and detection methods and applications thereof. Background Art

[0002] For patients with cardiovascular diseases, ischemic heart diseases (such as angina pectoris and acute myocardial infarction), hemorrhagic stroke, and ischemic stroke are the three major causes of death. Strengthening basic research on cardiovascular diseases and exploring new strategies for effectively treating ischemic heart diseases contribute to ensuring the health of Chinese residents. Most cells maintain their functional homeostasis through continuous renewal. However, as terminally differentiated cells, cardiomyocytes can hardly repair themselves through self-renewal if a large number of cardiomyocytes die. Myocardial infarction, myocardial ischemia / reperfusion (I / R), atherosclerosis, and diabetic heart disease are all results of excessive cell death, which involves the control mechanism of cell death. Accidental cell death and regulated cell death are two different ways of cell death, and regulated cell death is particularly important for the development and control of heart diseases.

[0003] Ferroptosis, as a form of regulated cell death, has great potential in the treatment of cardiovascular diseases. It is a new form of programmed cell death that highly depends on ferrous ion (Fe 2+ ), rather than other divalent metals, and Fe 2+ is usually also used as a detection index for ferroptosis. The essence of ferroptosis is the reduction of the activity of glutathione peroxidase 4 (GPX4) and the depletion of glutathione, resulting in the inability of lipid peroxides to be normally metabolized by the glutathione reductase reaction catalyzed by GPX4. Intracellular Fe 2+ utilizes the Fenton reaction to catalyze the production of ROS and attack cells, causing cellular oxidative stress, accompanied by the increase of indicators such as cyclooxygenase 2 (Recombinant prostaglandin endoperoxide synthase 2, Ptgs2 ), lactic dehydrogenase (LDH), and malonic dialdehyde (MDA), and can be manifested as the rupture of the cell membrane phospholipid bilayer, mitochondrial damage, and cell dysfunction. By detecting the level of MDA, the degree of lipid oxidation in cells can be reflected. Therefore, the determination of MDA is widely used as an index of lipid oxidation. By detecting Fe 2+ , Ptgs2 the degree of ferroptosis can be reflected. Therefore, Ptgs2It is often used for the detection of ferroptosis in cells as a ferroptosis marker gene.

[0004] Biochemically, ferroptosis is mainly manifested as disorders in the metabolism of iron, amino acids, and lipids, leading to the accumulation of lipid peroxides in cell membranes. Molecularly, GPX4 is an enzyme considered to be a central regulator of ferroptosis, but inhibiting GPX4 alone cannot completely prevent ferroptosis, which reflects that there may be other GPX4-like pathways to resist ferroptosis. With further research, more ferroptosis-related diseases and their pathogenesis have been discovered. Maintaining iron homeostasis is crucial for the normal function of the heart, and ferroptosis has been found to play an important role in the pathophysiology of cardiovascular diseases such as atherosclerosis, myocardial infarction, myocardial ischemia-reperfusion, heart failure, and myocardial hypertrophy. In addition, ferroptosis plays a regulatory role in various ischemia-reperfusion diseases. Therefore, inhibiting ferroptosis to prevent cardiomyocyte death may be an effective treatment strategy for cardiovascular diseases, especially myocardial ischemia-reperfusion injury.

[0005] There are various regulatory ways for myocardial ischemia-reperfusion injury. Among them, the regulation of gene translation by small RNAs is an important regulatory way, and the regulatory pathway can involve multiple proteins or genes. Small RNAs are a class of short-chain non-coding RNAs with a length of about 24 to 31 nucleotides. For small RNAs, the characteristics of their regulatory ways determine that their target proteins or target genes are not just one, but in a one-to-many manner, which makes the functional research of small RNAs very rich. In addition, more and more studies have shown that small RNAs abnormally expressed in heart diseases may become novel biomarkers and therapeutic targets for the diagnosis and treatment of heart diseases, and their potential functions or mechanisms can be further studied or applied. Therefore, treating myocardial ischemia-reperfusion injury through small RNAs is a promising research direction. Summary of the Invention

[0006] To solve the problems existing in the above-mentioned prior art, the present invention provides a ferroptosis-related small RNA for detecting myocardial ischemia-reperfusion injury, its detection method and application. For a novel treatment method for myocardial ischemia-reperfusion injury, this small RNA is involved in a cardiomyocyte death mode - ferroptosis - triggered by myocardial ischemia-reperfusion injury. Therefore, this small RNA is named ferroptosis-associated small RNA (FASR).

[0007] The first object of the present invention is to provide a biomarker for myocardial ischemia-reperfusion injury.

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

[0009] The third object of the present invention is to provide the use of FASR, FASR mimics and FASR agomir in the preparation of drugs for preventing and / or treating myocardial ischemia-reperfusion injury.

[0010] The present invention adopts the following technical solutions: The present invention provides a biomarker for myocardial ischemia-reperfusion injury, and the biomarker is FASR. The nucleic acid sequence of the FASR is as shown in SEQ ID NO: 1: SEQ ID NO: 1 5'-UAGCUGCAUUCAAUAAGGCAGAAUUUUGUC-3' The present invention also provides a kit for detecting myocardial ischemia-reperfusion injury. The kit includes primers for FASR detection, FASR RT, FASR-F, and FASR-R. The nucleic acid sequences are as shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 respectively: SEQ ID NO: 2 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGACAAA-3' SEQ ID NO: 3 5'-GCTGCATTCAATAAGGCAGAAT-3' SEQ ID NO: 4 5'-AGTGCAGGGTCCGAGGTATT-3' The present invention also provides the use of FASR in the preparation of drugs for preventing and / or treating myocardial ischemia-reperfusion injury.

[0011] The component for preparing the drug for preventing and / or treating myocardial ischemia-reperfusion injury is any one of FASR, FASR mimics and FASR agomir.

[0012] The FASR mimics is a FASR mimic, and is transfected into cardiomyocytes using LipofectamineTM 8000 (Shanghai Beyotime Biotechnology) to achieve overexpression of FASR in cardiomyocytes. The FASR mimics sequence includes SEQ ID NO: 5 and SEQ ID NO: 6: 5'-UAGCUGCAUUCAAUAAGGCAGAAUUUUGUC-3', that is, SEQ ID NO: 5; 5'-CAAAAUUCUGCCUUAUUGAAUGCAGCUAUU-3', namely SEQ ID NO: 6.

[0013] The FASR agomir is an activator of FASR. After being diluted with enzyme-free water and directly injected into mice via the tail vein, it can achieve overexpression of FASR in mice. The FASR agomir sequence includes SEQ ID NO: 7 and SEQ ID NO: 8: 5'-UAGCUGCAUUCAAUAAGGCAGAAUUUUGUC-3', namely SEQ ID NO: 7; 5'-CAAAAUUCUGCCUUAUUGAAUGCAGCUAUU-3', namely SEQ ID NO: 8.

[0014] The role of FASR in myocardial ischemia-reperfusion injury and the application, regulation of its expression information further confirm that FASR has the role of predicting, diagnosing, and differentiating myocardial ischemia-reperfusion injury, providing a new drug action target for the treatment of myocardial ischemia-related heart diseases. Description of the Drawings

[0015] Figure 1 It shows that FASR is abnormally lowly expressed in myocardial ischemia-reperfusion injury; Figure 2 It shows the role of FASR mimics in inhibiting ferroptosis in myocardial ischemia-reperfusion injury; Figure 3 It shows the role of FASR agomir in inhibiting ferroptosis in myocardial ischemia-reperfusion injury; Figure 4 It shows the role of FASR agomir in protecting cardiac function in myocardial ischemia-reperfusion injury. Detailed Embodiments

[0016] The following further elaborates the present invention in combination with specific embodiments. The examples are only for illustrating the present invention and not for limiting it. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0017] Example 1 Detect the expression difference of FASR in the hearts of mice in the Sham group and the I / R group The technical methods involved in this example are as follows: (1) The surgical subjects were C57BL / 6J mice with normal physiological indices. Heart tissues were dissected from the mice in the Sham group, I / R group, I / R + negative control (NC) group, and I / R + FASR agomir group respectively. An appropriate amount of each tissue was placed into a 2 ml clean centrifuge tube, and 4℃ pre-cooled Trizol Reagent and 4 mm zirconia grinding beads were added to each tube. A high-throughput tissue grinder was used to homogenize and break each tissue; (2) The tissue homogenate was aspirated into a 1.5 mL centrifuge tube, and the total RNA of each sample was extracted by the Trizol method. The RNA precipitate was dissolved in deionized water to make the RNA concentration about 500 µg / mL; (3) According to one aspect of the present invention, the present invention provides a ferroptosis-related small RNA, named FASR. FASR contains the nucleotide sequence shown in SEQ ID NO: 1. Using EvoM-MLV Reverse Transcription Kit II (AccurateBiology) to reverse the RNA into cDNA, and the amplification conditions were based on the instructions. The reverse transcription primer sequence of FASR is: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGACAAA-3', that is, SEQ ID NO: 2; (4) Mouse U6 was selected as the internal reference gene, and Reverse Transcription Kit II (AccurateBiology) was used to reverse the internal reference gene, and the amplification conditions were based on the instructions. Using a real-time fluorescence quantitative PCR instrument (ThermoFisher Scientific QuantStudio 6), according to the 2 EvoM-MLV method, the cDNA obtained after reverse transcription was quantitatively detected, and the amplification conditions were based on the two-step method of the SYBR Green qPCR Mix (Monad) instructions. The real-time fluorescence quantitative PCR primer sequences of FASR are: -ΔΔCt Forward primer sequence: 5'-GCTGCATTCAATAAGGCAGAAT-3', that is, SEQ ID NO: 3; Reverse primer sequence: 5'-AGTGCAGGGTCCGAGGTATT-3', that is, SEQ ID NO: 4. The results are as

[0018] shown in Figure 1As shown, the relative expression levels of FASR in the hearts of the sham surgery (Sham) group and the I / R group in mice. The expression level of FASR in the hearts of the I / R group was significantly lower than that in the hearts of the Sham group (P<0.05 indicates that the difference is statistically significant). The experimental results show that FASR is significantly down-regulated in the myocardial tissue of ischemia-reperfusion injury, showing a connection with the regulation of myocardial ischemia-reperfusion injury.

[0019] Example 2 Detect ferroptosis marker genes in cardiomyocytes and heart tissues Ptgs2 mRNA expression The technical methods involved in this example are as follows: (1) Heart tissues were taken from the hearts of mice in the Sham group, I / R group, I / R+NC group, and I / R+FASR agomir group, placed in 2 ml clean centrifuge tubes, and 4°C pre-cooled Trizol Reagent and 4 mm zirconia grinding beads were added simultaneously. A high-throughput tissue grinder was used to homogenize and disrupt each tissue. Cell samples were taken from cardiomyocytes in the control group, hypoxia / reoxygenation (H / R) group, H / R+NC group, and H / R+FASR mimics group; (2) Absorb the heart tissue homogenate into a 1.5 mL centrifuge tube, collect the cardiomyocytes into a 1.5 mL centrifuge tube, and extract the total RNA of each sample by the Trizol method. Dissolve the RNA precipitate with deionized water to make the RNA concentration about 500 µg / mL; (3) Select mouse U6 as the internal reference gene and use EvoM-MLV Reverse Transcription Kit II (AccurateBiology) to reverse the internal reference gene, and the amplification conditions are based on the instructions. Use a real-time fluorescence quantitative PCR instrument (ThermoFisher Scientific QuantStudio 6), and according to the 2 -ΔΔCt method to quantitatively detect the cDNA obtained after reverse transcription, and the amplification conditions are based on the two-step method of the SYBR Green qPCR Mix (Monad) instructions.

[0020] Ptgs2 The primer sequences for real-time fluorescence quantitative PCR are as follows: Forward primer sequence: 5'-TGGGGGAAGAAATGTGCCAA-3', that is, SEQ ID NO: 9; Reverse primer sequence: 5'-CAGCCATTTCCTTCTCTCCTGT-3, that is, SEQ ID NO: 10.

[0021] The results are as follows Figure 2 shown in (A). The mRNA expressions of cardiomyocytes in the control group, H / R group, H / R+NC group, and H / R+FASR mimics group Ptgs2 The mRNA expression level of cardiomyocytes in the H / R+FASR mimics group Ptgs2 was significantly lower than that in the NC group, indicating the alleviation of ferroptosis and reflecting the inhibitory effect of FASR mimics on ferroptosis in cardiomyocytes.

[0022] The results are as follows Figure 3 shown in (A). The mRNA expressions of the hearts in the Sham group, I / R group, I / R+NC group, and I / R+FASR agomir group Ptgs2 The mRNA expression level of the heart tissue in the I / R+FASR agomir group Ptgs2 was significantly lower than that in the NC group, indicating the alleviation of ferroptosis and reflecting the inhibitory effect of overexpressed FASR agomir on ferroptosis in the heart.

[0023] Example 3 Detect the Fe 2+ levels in cardiomyocytes and heart tissues. Use the ferrous ion colorimetric assay kit (Elabscience), which is applicable to detect the content of ferrous ions in serum, animal and plant tissue samples, and cell samples. The ferrous ions in the sample bind to the probe, and the generated substance has a strong absorption peak at a wavelength of 593 nm. Within a certain range, its optical density value is linearly correlated with the ferrous ion concentration. For tissue samples, according to the Fe 2+ detection kit, take 0.1 g of fresh tissue blocks, add 0.9 mL of reagent I, and then use a high-throughput tissue grinder to break and homogenize. After centrifuging the homogenate at 10,000 rpm for 10 min, take the supernatant for standby. For cell samples, mix the collected cells with reagent I, place them on ice for lysis, and after centrifuging the lysate at 10,000 rpm for 10 min, take the supernatant for standby. Dilute the Fe 2+ standards to make the standard concentrations (μmol / L) be 0, 5, 10, 15, 20, 30, 40, 50 for subsequent preparation of the standard curve. Continue the subsequent operations according to the instructions. The ferrous ions in the sample bind to the probe, and the generated product has a strong absorption peak at a wavelength of 593 nm. Its optical density value is linearly correlated with the ferrous ion concentration.

[0024] The results are as follows Figure 2 shown in (B). The Fe 2+ levels of cardiomyocytes in the control group, H / R group, H / R+NC group, and H / R+FASR mimics group. The Fe levels of the cells in the H / R+FASR mimics group2+ The level was significantly lower than that of the NC group, indicating the alleviation of ferroptosis, reflecting the inhibitory effect of FASR mimics on ferroptosis in cardiomyocytes.

[0025] The results were as Figure 3 shown in (B), which were the Fe 2+ levels in the hearts of the Sham group, I / R group, I / R+NC group, and I / R+FASR agomir group. The Fe 2+ level in the heart tissue of the I / R+FASR agomir group was significantly lower than that of the NC group, indicating the alleviation of ferroptosis, reflecting the inhibitory effect of FASR agomir on ferroptosis in the heart.

[0026] Example 4 Echocardiography was used to detect the left ventricular fractional shortening (FS) and ejection fraction (EF). EF is the percentage of the volume of blood ejected from the left ventricle during each cardiac contraction to the volume of the left ventricle at the end of diastole. FS is the ratio of the internal diameter of the left ventricle during systole to that during diastole. Both EF and FS of the left ventricle are important parameters for evaluating cardiac health, and they reflect the functional state of the heart from different angles. EF mainly measures the pumping efficiency of the heart, while FS mainly reflects the contractile ability of the ventricular muscle.

[0027] The specific implementation process was as follows: 24 hours after the operation of the mouse heart I / R injury model, the mice were anesthetized and the hair on their chest and abdomen was removed, and the mice were fixed in the supine position on a warming plate. A 6 LAB portable small animal ultrasonic imaging system (VINNO), equipped with a probe and coupling agent, was used for transthoracic echocardiography. Two-dimensional-guided M-mode images were recorded in the parasternal long-axis and short-axis views at the level of the mouse papillary muscle. The FS and EF of the left ventricular echocardiogram of the mice were calculated using established standard formulas, and the FS and EF values of each group were analyzed to evaluate the cardiac function of the mice.

[0028] The results were as Figure 4 shown in (A), which were the FS of the hearts of the Sham group, I / R group, I / R+NC group, and I / R+FASR agomir group. The FS of the I / R+FASR agomir group was significantly higher than that of the I / R+NC group, indicating that FASR agomir played a role in protecting cardiac function and increasing the left ventricular FS.

[0029] The results were as Figure 4As shown in (B), the EF of the hearts in the Sham group, I / R group, I / R+NC group, and I / R+FASR agomir group. The EF in the I / R+FASR agomir group was significantly higher than that in the I / R+NC group, indicating that FASR agomir played a role in protecting cardiac function and increasing the left ventricular EF.

[0030] Example 5 Treatment and culture of cardiomyocytes This example involves the culture of primary cardiomyocytes from mice in the control group, H / R group, H / R+NC group, and H / R+FASR mimics group. In this experiment, primary cardiomyocytes were used as the model, and a random sequence was transfected as the NC group. FASR mimics were transfected using liposomes to increase the expression of FASR in cells, serving as the FASR mimics group.

[0031] The FASR mimics sequence includes SEQ ID NO: 5 and SEQ ID NO: 6: 5'-UAGCUGCAUUCAAUAAGGCAGAAUUUUGUC-3', namely SEQ ID NO: 5; 5'-CAAAAUUCUGCCUUAUUGAAUGCAGCUAUU-3', namely SEQ ID NO: 6.

[0032] The control group was cultured under normoxic conditions for 24 h, and the average oxygen concentration was approximately 21%; the H / R group, H / R+NC group, and H / R+FASR mimics group were first cultured in an anaerobic incubator under hypoxic conditions for 18 h, with the average oxygen concentration being approximately below 1%. After 18 h, they were transferred to a normoxic incubator for 6 h of reoxygenation. Subsequent experiments were carried out after the construction of the in vitro cardiomyocyte ischemia-reperfusion injury model.

[0033] Example 6 Overexpression of FASR in animals The overexpression of FASR in mice was achieved by FASR agomir. The FASR agomir sequence includes SEQ ID NO: 7 and SEQ ID NO: 8: 5'-UAGCUGCAUUCAAUAAGGCAGAAUUUUGUC-3', namely SEQ ID NO: 7; 5'-CAAAAUUCUGCCUUAUUGAAUGCAGCUAUU-3', namely SEQ ID NO: 8.

[0034] Inject FASR agomir into the caudal vein of mice using a clean insulin needle, with each injection ranging from 1 OD to 5 OD according to the overexpression effect. Complete 3 injections continuously within one week, and conduct relevant experiments within 7 days after the last injection.

[0035] Example 7 Construction of myocardial ischemia-reperfusion model Establish an adult mouse myocardial I / R injury model by ligating the left anterior descending coronary artery (LAD) of mice. The specific operation is as follows: After anesthetizing the mice, perform endotracheal intubation and use an animal ventilator to assist the normal breathing of the mice. Open the thoracic cavity of the mice at an appropriate position and expose most of the heart using a rib spreader. Ligate the LAD with a 6-0 nylon suture (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. During the LAD surgery, ensure the integrity of the pericardial membrane structure, and properly close the thoracic cavity incision and skin wound. After the surgery, the mice are placed on a warming plate for recovery. The Sham group does not undergo the LAD ligation step, and the remaining surgical steps are the same.

[0036] In summary, FASR is expressed in cardiomyocytes, participates in the regulation of myocardial ischemia-reperfusion injury, and is involved in the ferroptosis pathway. Therefore, the FASR, FASR mimics, and FASR agomir provided by the present invention can be used as markers for predicting, diagnosing, and differentiating myocardial ischemia-reperfusion injury, and can be used as active ingredients in products for treating myocardial ischemia-reperfusion injury. By using FASR mimics and / or FASR agomir, it is possible to increase the expression of FASR in the heart, significantly inhibit ferroptosis, thereby reducing myocardial ischemia-reperfusion injury, and achieving the effect of protecting cardiac function, so as to achieve the purpose of preparing drugs for preventing and / or treating myocardial ischemia-reperfusion injury as a new gene therapy technology.

[0037] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and 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. A marker for myocardial ischemia-reperfusion injury, characterized in that, The biomarker is FASR, and the nucleic acid sequence of FASR is shown as SEQ ID NO:

1.

2. A kit for detecting myocardial ischemia-reperfusion injury, the kit includes primer sequences for FASR detection, and the primer sequences are shown as SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

4. The kit is used to detect the biomarker as described in claim 1.

3. The application of the biomarker FASR as described in claim 1 in the preparation of drugs for preventing and / or treating myocardial ischemia-reperfusion injury.

4. The component of the drug for preventing and / or treating myocardial ischemia-reperfusion injury in the application as described in claim 3 is at least one of FASR, FASR mimics and FASR agomir.

5. The FASR mimics according to claim 4, characterized in that, The FASR mimics is a FASR mimic, and is transfected into cardiomyocytes using LipofectamineTM 8000 to achieve overexpression of FASR in cardiomyocytes; the FASR mimics sequence includes SEQ ID NO: 5 and SEQ ID NO:

6.

6. The FASR agomir according to claim 4, characterized in that, The FASR agomir is a FASR activator, and after being diluted with enzyme-free water, it is directly injected into the mouse body via the tail vein, which can achieve overexpression of FASR in the mouse body; the FASR agomir sequence includes SEQ ID NO: 7 and SEQ ID NO: 8.

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