Ctts, mimetics and uses thereof

Abstract

The application belongs to the field of gene drugs, and particularly relates to CTRTS, an analog thereof and application thereof. The CTRTS has a nucleotide sequence as shown in SEQ ID NO. 1. The CTRTS analog is CTRTS Agomir, and has a nucleotide sequence as shown in SEQ ID NO. 2. The present application finds that the expression level of CTRTS is reduced when doxorubicin induces cardiotoxicity; overexpression of CTRTS can significantly inhibit doxorubicin-induced cardiotoxicity, and plays an important role in the regulation of myocardial cell death. The existing drug has limited therapeutic effect, therefore, CTRTS plays an important role in the regulation of myocardial cell death, can regulate the doxorubicin-induced cardiotoxicity treatment by participating in the regulation of the myocardial cell death process, so as to improve the clinical prognosis effect.

Description

Technical Field

[0001] This invention belongs to the field of gene therapy, specifically involving CTRTS, mimics and their applications. Background Technology

[0002] Cardiovascular diseases, including coronary atherosclerotic heart disease, heart failure, hypertension, and arrhythmias, are currently the leading cause of death and disability worldwide. Despite significant progress in diagnosis and treatment, the incidence and mortality rates of cardiovascular diseases remain high. Therefore, the search for novel biomarkers with high sensitivity and specificity to achieve early warning, precise classification, and efficacy monitoring of cardiovascular diseases has become a key scientific problem and clinical need that urgently needs to be addressed in this field. In recent years, with the deepening of research on non-coding RNA, a new class of small RNAs—tsRNA (tRNA-derived small RNA)—has attracted widespread attention. tsRNA is a functional small RNA fragment derived from the cleavage of precursor tRNA or mature tRNA under specific conditions. Recent studies have shown that tsRNA plays a crucial role in the post-transcriptional regulation of gene expression, participating in various life processes such as cell proliferation, apoptosis, differentiation, and stress response. Increasing evidence suggests that tsRNA is specifically expressed in the pathophysiological processes of the cardiovascular system, and changes in its expression profile are closely related to the occurrence and development of diseases such as atherosclerosis, myocardial ischemia / reperfusion injury, and heart failure. Compared with other non-coding RNAs, tsRNAs possess unique advantages such as high expression abundance, structural stability, strong tissue specificity, and ease of detection in bodily fluids like blood, making them highly promising biomarkers. However, systematic and in-depth research is currently lacking on the specific functions, regulatory mechanisms, and clinical translational value of particular tsRNA molecules in specific cardiovascular diseases. This invention is proposed based on this technical background, aiming to address the shortcomings of existing technologies.

[0003] Doxorubicin is a broad-spectrum, highly effective anthracycline antibiotic widely used to treat various solid and hematologic malignancies, including leukemia, lymphoma, breast cancer, and sarcoma. However, its clinical application is severely limited by its dose-dependent cardiotoxicity, a side effect that can lead to irreversible myocardial damage, ultimately resulting in dilated cardiomyopathy and refractory heart failure, significantly impacting the long-term quality of life and overall survival of cancer patients. Currently, clinical monitoring of doxorubicin cardiotoxicity relies primarily on regular electrocardiograms and echocardiograms, as well as monitoring changes in left ventricular ejection fraction. However, these indicators typically only show significant abnormalities after irreversible structural and functional damage to the myocardium, failing to provide early warning. Dexrazoxane is the only FDA-approved drug for the prevention of doxorubicin cardiotoxicity, but its use may affect antitumor efficacy and cannot completely eliminate the risks. Therefore, there is an urgent need in this field to find novel biomarkers that can sensitively reflect myocardial cell damage in the early stages of cardiac injury, or even before functional abnormalities occur, in order to achieve risk stratification, early diagnosis and intervention for high-risk patients with doxorubicin cardiotoxicity, thereby maximizing the protection of patients' cardiac function while ensuring the effectiveness of tumor treatment.

[0004] Agomir is a specially labeled and chemically modified double-stranded small RNA that regulates the biological function of target genes by mimicking endogenous tsRNAs, and holds promise as a fundamental approach for treating various infectious diseases, hematological malignancies, cardiovascular diseases, and neurodegenerative diseases. Agomir is a double-stranded small RNA molecule designed based on the mature tsRNA sequence to mimic the endogenous mature tsRNA sequence. Agomir includes one sequence identical to the target mature tsRNA sequence and one complementary sequence. Specific Agomir can be introduced into cells expressing the corresponding tsRNA to mimic its function. As a tsRNA mimic, Agomir has become an important tool in molecular biology research and potential therapeutics due to its high efficiency, stability, and strong targeting capabilities. Summary of the Invention

[0005] Based on the aforementioned background, this invention provides a tsRNA named CTRTS (Cardiotoxicity Related tsRNA) according to its characteristics. This invention has found that CTRTS expression levels decrease during doxorubicin-induced cardiotoxicity; overexpression of CTRTS can significantly inhibit doxorubicin-induced cardiotoxicity, playing a crucial role in the regulation of cardiomyocyte death. Existing drug treatments have limited efficacy; therefore, CTRTS plays a vital role in the regulation of cardiomyocyte death and can regulate the treatment of doxorubicin-induced cardiotoxicity by participating in the regulation of cardiomyocyte death processes, thereby improving its clinical prognosis.

[0006] In a first aspect, the present invention provides a tsRNA, named CTRTS, the nucleotide sequence of which is shown in SEQ ID NO.1, and the sequence is as follows:

[0007] Furthermore, the CTRTS sequence is derived from mice, humans, or other mammals.

[0008] A second aspect of the invention provides the application of CTRTS in a product for detecting doxorubicin-induced cardiotoxicity.

[0009] In a third aspect, the present invention provides the application of CTRTS as a biomarker in the preparation of products for detecting cardiomyocyte death.

[0010] In a fourth aspect, the present invention provides a product for detecting cardiomyocyte death, comprising a reagent for detecting a biomarker, said biomarker being a CTRTS sequence, and said reagent for detecting the biomarker comprising an RNA primer pair for amplifying the CTRTS transcription of claim 1.

[0011] The present invention also provides a CRTTS mimic, namely CRTTS Agomir, having a nucleotide sequence as shown in SEQ ID NO.2, the sequence of which is:

[0012] In a fifth aspect, the present invention provides the application of CTRTS mimics as drugs for cardiomyocyte death and doxorubicin-induced cardiotoxicity. Overexpression of CTRTS can significantly reduce cardiomyocyte death and reverse the degree of doxorubicin-induced cardiotoxicity. Overexpression of CTRTS effectively alleviates the problems of poor prognosis and treatment of heart disease caused by doxorubicin-induced cardiotoxicity.

[0013] The present invention also provides the use of the CTRTS mimicry in the preparation of medicaments for the prevention and / or treatment of heart disease, including processes of cardiotoxicity following doxorubicin treatment or myocardial cell death.

[0014] A sixth aspect of the invention also provides a medicament for the prevention and / or treatment of heart disease, the medicament comprising a CTRTS mimic, the CTRTS mimic being CTRTS Agomir having a nucleotide sequence as shown in SEQ ID NO.2.

[0015] The heart disease mentioned includes doxorubicin-induced cardiotoxicity and heart failure.

[0016] The drug is based on the mature Agomir; relying on Agomir, a CTRTS analogue was constructed and delivered to the heart lesion site, which can be used as a drug to treat doxorubicin-induced cardiotoxicity and achieve effective treatment of the lesion.

[0017] Compared with the prior art, the beneficial effects of the present invention include: 1. This invention is the first to discover that CTRTS expression is reduced during doxorubicin-induced cardiotoxicity, is highly correlated with cardiomyocyte death, and that overexpression of CTRTS can significantly inhibit the cardiomyocyte death process; 2. Based on the mechanism of CTRTS in regulating cardiomyocyte death, this invention provides the application of CTRTS as a biomarker and provides a product for detecting cardiomyocyte death or cardiotoxicity; 3. This invention utilizes a CTRTS agomir to construct a CTRTS agomir, which is then delivered to the location of the heart lesion. It can be used as a drug to treat cardiotoxicity, achieving effective treatment of the lesion and providing a drug for the prevention and / or treatment of heart disease. Attached Figure Description

[0018] Figure 1 The expression of CTRTS in an doxorubicin-induced mouse model of cardiotoxicity; Figure 2 The expression of CTRTS in a mouse model of doxorubicin-induced primary cardiomyocyte cytotoxicity; Figure 3 The expression of CTRTS in patients with dilated cardiomyopathy; Figure 4 To improve the survival rate of cardiomyocytes after doxorubicin induction by overexpressing intracellular CTRTS; Figure 5 To overexpress intracellular CTRTS, the expression level of lactate dehydrogenase (LDH) was induced by doxorubicin. Detailed Implementation

[0019] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the invention are merely illustrative examples of specific implementations of the invention and are intended to explain the invention, and do not constitute a limitation thereof. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified by the manufacturer, are all commercially available conventional products.

[0020] The endpoints of the ranges and any values ​​disclosed herein are not limited to the exact ranges or values, which should be understood to include those close to them.

[0021] Example 1: Expression of CTRTS in an doxorubicin-induced mouse model of cardiotoxicity Doxorubicin-induced mouse model of cardiotoxicity Eight-week-old male mice (C57BL / 6) were selected and purchased from Jinan Pengyue Experimental Animal Breeding Center, Shandong Province. All animal experiments were conducted under the guidance of the Ethics Committee of the Experimental Animal Center of Qingdao University. Animals were allowed free access to food during the experiments. All animal studies complied with European Parliament Directive 2010 / 63 / EU and were approved by the Biomedical Research Ethics Committee of Qingdao University (License No.: 20200610C576620221208124). Mice were weighed and intraperitoneally injected with doxorubicin at a concentration of 20 mg / kg for 4 weeks to establish a mouse model of cardiotoxicity.

[0022] Real-time quantitative PCR (RT-qPCR) Total RNA was extracted from mouse cardiomyocytes or tissues using TRIZOL reagent, and its concentration and purity were determined using nanodrop One. The AG Evo M-MLV RT Kit was used to convert the total RNA into cDNA. Quantitative real-time PCR was performed using the SYBR Green Kit. RT-qPCR was performed using a CFX96 real-time quantitative PCR detection system. Reaction conditions: 95℃ for 10 min; 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles.

[0023] result: like Figure 1 As shown, compared with the control group, the expression level of CTRTS mRNA was significantly downregulated in the doxorubicin-induced mouse cardiotoxicity model. p<0.001 The study showed that CTRTS expression was significantly reduced in the hearts of mice after treatment with doxorubicin.

[0024] Example 2: Expression of CTRTS in an doxorubicin-induced mouse primary cardiomyocyte cytotoxicity model Extraction and culture of primary mouse cardiomyocytes Cardiac cells were isolated from 1-2 day old newborn mice (C57BL / 6). The newborn mice were washed with 75% alcohol. Under sterile conditions, the heart was removed with surgical scissors and placed in a culture dish containing pre-chilled PBS. The heart was washed three times with PBS. The heart tissue was minced with surgical scissors and transferred to 10 mL of digestion solution (1.2 mg / mL trypsin and 0.14 mg / mL collagenase II). The mixture was gently shaken in a 37°C water bath for 6 min. After each digestion, the supernatant was transferred to a centrifuge tube containing serum. The centrifuge tube was stored on ice. Fresh digestion solution was added again until the heart tissue disappeared. Centrifuge at 1000 rpm for 10 min, collect the supernatant, resuspend in F12 / DMEM containing 10% serum, centrifuge again at 1000 rpm for 10 min, collect the supernatant, filter through a 70-mesh cell filter, place the filtered cardiomyocytes in a 10 cm culture dish, and incubate at 37°C in a 5% CO2 humidified incubator for 1.5 h. Fibroblasts adhere to the culture medium. Collect the culture medium containing cardiomyocytes into a new centrifuge tube, centrifuge at 1000 rpm for 10 min, discard the supernatant, resuspend in fresh serum-containing medium, and add 0.1 mM 5-bromodeoxyuridine (BrdU). Plate according to the experimental design and incubate at 37°C in a 5% CO2 humidified incubator. On the second day, change the culture medium in the culture dish and continue incubation for one more day.

[0025] Doxorubicin-induced mouse primary cardiomyocyte model Primary mouse cardiomyocytes were extracted, and 1 μM doxorubicin was added to the cell culture dish at a volume ratio of 1:1000 to the culture medium. The cells were treated for 24 h, and total cell protein was extracted.

[0026] result: like Figure 2 As shown, compared with the control group, the expression level of CTRTS mRNA was significantly downregulated in the doxorubicin-induced mouse primary cardiomyocyte cytotoxicity model. p<0.001 The results showed that after treatment with doxorubicin, the expression of the CTRTS gene in primary mouse cardiomyocytes was significantly reduced, consistent with the results in heart tissue.

[0027] Example 3: Expression of CTRTS in patients with dilated cardiomyopathy Detection of CTRTS gene in patients with dilated cardiomyopathy (DCM) In clinical practice, heart tissue samples were collected from normal individuals and patients with dilated cardiomyopathy. Total RNA was extracted from the heart, and the expression levels of CTRTS in normal human heart tissue and patients with dilated cardiomyopathy were detected by reverse transcription and real-time quantitative PCR.

[0028] result: like Figure 3 As shown, compared with normal human heart tissue, the expression level of CTRTS gene mRNA is significantly downregulated in patients with dilated cardiomyopathy. p<0.001 The results showed that the expression of the CTRTS gene was significantly lower in patients with dilated cardiomyopathy than in normal individuals.

[0029] Example 4: Survival rate of cardiomyocytes after overexpression of intracellular CTRTS via doxorubicin induction. Cell viability detection Cell viability was assessed using a commercially available CCK-8 assay kit purchased from Shanghai Taoshu Biotechnology. Primary cardiomyocytes were isolated from each suckling mouse, enough to seed 10 wells of a 96-well plate. After transfection and doxorubicin treatment for the required time, the culture medium in the 96-well plates was discarded, and each well was refilled with a mixture of 90 μL DMEM / F12 and 10 μL CCK-8. The plates were then placed back into the cell culture incubator and cultured for 2–4 h. The absorbance at 450 nm was then measured using a microplate reader. Cell viability was calculated as: (Experimental wells / Control wells) * 100%.

[0030] result: like Figure 4 As shown, after treatment with doxorubicin, the cell viability of primary mouse cardiomyocytes was significantly increased compared to the control group. However, after transfection with CTRTS-Agomir and treatment with doxorubicin, the cell viability was significantly decreased compared to the doxorubicin-only treatment group. p<0.001 The results suggest that CTRTS can effectively reverse the doxorubicin-induced death of primary cardiomyocytes in mice.

[0031] Example 5: Overexpression of intracellular CTRTS and the expression level of lactate dehydrogenase (LDH) after doxorubicin induction. lactate dehydrogenase detection Lactate dehydrogenase was detected using a commercial lactate dehydrogenase assay kit from Solarbio. The processed cell culture medium was collected into centrifuge tubes. The test sample and reagents were added step-by-step according to the instructions of the lactate dehydrogenase assay kit, as shown in the table below.

[0032]

[0033] Mix the sample and reagent thoroughly and let stand at room temperature for 5 min. Use an ELISA reader to measure the absorbance at 450 nm and assess the lactate dehydrogenase level.

[0034] result: like Figure 5 As shown, after treatment of mouse primary cardiomyocytes with doxorubicin, the content of lactate dehydrogenase in the cell supernatant was significantly increased compared with the control group. However, after transfection with CTRTS-Agomir and subsequent treatment with doxorubicin, the content of lactate dehydrogenase in the cell supernatant was significantly decreased compared with the doxorubicin-only treatment group. p<0.001 The results suggest that CTRTS can effectively reduce doxorubicin-induced death of primary cardiomyocytes in mice and alleviate the damage caused by cardiotoxicity.

Claims

1. A tsRNA, characterized in that: It is named CTRTS, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The application of CTRTS as described in claim 1 in a product for detecting doxorubicin-induced cardiotoxicity.

3. The application of CTRTS as a biomarker as described in claim 1 in the preparation of products for detecting myocardial cell death.

4. A product for detecting cardiomyocyte death, comprising reagents for detecting biomarkers, characterized in that: The reagent for detecting the biomarker contains an RNA primer pair for amplifying the CTRTS transcription as described in claim 1.

5. A CTRTS simulator, characterized in that: The CTRTS mimic is CTRTS Agomir, which has the nucleotide sequence shown in SEQ ID NO.

2.

6. The use of the CTRTS mimicry as described in claim 5 as a drug for myocardial cell death and doxorubicin-induced cardiotoxicity.

7. The use of the CTRTS mimicry as described in claim 5 in the preparation of a medicament for the prevention and / or treatment of heart disease.

8. The application as described in claim 7, characterized in that: The heart disease mentioned includes the process of cardiotoxicity following doxorubicin treatment or the death of myocardial cells.

9. A medicament for the prevention and / or treatment of heart disease, characterized in that: The drug includes the CTRTS analogue as described in claim 5.

10. The medicament as described in claim 9, characterized in that: The heart disease mentioned includes doxorubicin-induced cardiotoxicity and heart failure.

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