Application of human-derived circular RNA (Ribonucleic Acid) in preparation of detection kit or medicine for diagnosing pathological myocardial hypertrophy, and derivative peptide generated by translation of circular RNA and application of derivative peptide

Abstract

The invention provides an application of human circular RNA (Ribonucleic Acid) in preparation of a detection kit or a medicine for diagnosing pathological myocardial hypertrophy, and the human circular RNA comprises nucleic acid containing a sequence as shown in SEQ ID NO.1, or a bioactive functional fragment, a variant or a derivative of the nucleic acid containing the sequence as shown in SEQ ID NO.1. The invention also provides a derivative peptide generated by circular RNA translation. The invention also provides a kit for diagnosis or prognosis evaluation of pathological myocardial hypertrophy and a pharmaceutical composition for treatment or / and prevention of pathological myocardial hypertrophy. Compared with other types of markers, the human source circular RNA provided by the invention has stable and specific properties, has larger abundance in body fluid of a patient and more obvious change, and has the potential of becoming a novel diagnostic marker. The specific RNA interference adenovirus is prepared, and the expression efficiency is high; the virus genome cannot be integrated into the host cell genome, the controllability is good, and the safety is high.

Description

Technical Field

[0001] The present invention relates to use of human circular RNA in preparing a detection kit or medicine for diagnosing, preventing or treating heart diseases, as well as a derivative peptide produced by translation of the circular RNA and use thereof. Background Art

[0002] As cardiovascular diseases such as myocardial hypertrophy, myocardial infarction, heart failure, etc. have the characteristics of complex pathogenesis and hidden disease process, there is a lack of effective diagnosis and treatment methods.

[0003] Circular RNA is a type of RNA with a covalently closed structure that can stably exist in cells and even body fluids, regulating gene expression by acting as miRNA / protein sponges, protein scaffolds, and protein translation templates. In addition, dysregulation of circular RNA has been shown to be closely related to the onset of heart disease. For example, Circular RNAs in Cardiovascular Diseases: Regulation and Therapeutic Applications, DOI: 10.34133 / research.0038, discloses the regulatory and therapeutic applications of circular RNA in cardiovascular diseases. Yuan Jinsong, et al., Research Progress on the Relationship between Circular RNA and Cardiovascular Diseases, Shandong Medicine, Vol. 58, No. 6, 2018, found that circRNAs are closely related to the pathophysiological mechanisms of cardiovascular diseases, and play an important role in the occurrence and development of cardiovascular diseases such as heart failure, myocardial infarction, cardiomyopathy, hypertension, atherosclerosis, and cardiac aging. Liu Yehong, et al., Research progress of circRNA in cardiovascular diseases, Chin Heart J, 2020, 32(3). Studies have confirmed that circRNA is related to the occurrence and development of cardiovascular diseases such as human atherosclerosis, myocardial infarction, and heart failure, and may become a more ideal biomarker for disease diagnosis.

[0004] At present, there are examples of circular RNA being used in the diagnosis of myocardial damage in China. For example, application number 202211216356.X, invention name: circular RNA markers, kits and applications for diagnosing myocardial damage caused by breast cancer chemotherapy, discloses an application of a substance for detecting circular RNA markers, including one or more of the following applications: A1) application in the preparation of products for diagnosing myocardial damage caused by breast cancer chemotherapy; A2) application in the preparation of products for screening myocardial damage caused by breast cancer chemotherapy; A3) application in the preparation of products for assessing the risk of myocardial damage caused by breast cancer chemotherapy; A4) application in the preparation of products for prognosis assessment of myocardial damage caused by breast cancer chemotherapy; A5) application in the preparation of products for distinguishing myocardial damage caused by breast cancer chemotherapy from other diseases. The circular RNA marker disclosed in this patent document is suitable for myocardial damage caused by breast cancer chemotherapy. However, this study only uses the relative expression level difference (fold change of expression amount) of circular RNA in normal samples and damaged samples as the screening criterion, while ignoring the absolute expression level of circular RNA. If the basic expression level of the screened circular RNA is low, the detection results are easily affected by individual differences, sample quality, operational errors and other factors, resulting in false positives or false negatives. Therefore, there are still some imperfections.

[0005] The inventor of this application previously applied for an invention for the application of circular RNA in the diagnosis of myocardial infarction (application number 202010400681.6, invention name: Application of human circular RNA in the preparation of heart disease related products). The invention provides a human circular RNA that is highly expressed in the heart and can be used as a stable blood diagnostic marker for myocardial infarction. The application of this human circular RNA in the preparation of heart disease related products as a diagnostic marker can have both detection sensitivity and specificity.

[0006] Pathological myocardial hypertrophy and myocardial infarction are two different heart diseases, which differ in pathophysiological mechanisms, clinical manifestations and treatments. Pathological myocardial hypertrophy refers to the increase in the volume of myocardial cells, which may be due to the excessive load on the heart for a long time, such as hypertension, valvular disease, etc., which leads to the proliferation and enlargement of myocardial cells. Pathological myocardial hypertrophy can be an adaptive response of the heart to long-term load increase, but long-term myocardial hypertrophy may lead to decreased heart function or even heart failure. Myocardial infarction is caused by a sharp decrease or interruption of coronary blood flow, resulting in severe and persistent myocardial ischemia, and ultimately myocardial necrosis. Myocardial infarction usually presents with symptoms such as severe chest pain, dyspnea, sweating, etc., and requires emergency medical intervention. The causes of myocardial infarction may include coronary atherosclerosis, thrombosis, vasospasm, etc.

[0007] There are currently no reports on the specific detection of human circular RNA for pathological cardiac hypertrophy. Summary of the invention

[0008] The present invention provides use of human circular RNA in preparing a detection kit or medicine for diagnosing, preventing or treating pathological myocardial hypertrophy, as well as a derivative peptide produced by translation of the circular RNA and use thereof.

[0009] The present invention provides a use of a human circular RNA in preparing a detection kit or a drug for diagnosing pathological myocardial hypertrophy, wherein the human circular RNA comprises a nucleic acid containing a sequence as shown in SEQ ID NO.1, or a biologically active functional fragment, variant or derivative of a nucleic acid containing a sequence as shown in SEQ ID NO.1.

[0010] The present invention provides a derivative peptide produced by translation of a circular RNA, comprising a polypeptide having a sequence as shown in SEQ ID NO.2, or a fragment, variant or derivative of the polypeptide having a sequence as shown in SEQ ID NO.2.

[0011] The present invention provides the use of the derivative peptide in preparing a detection kit or medicine for diagnosing pathological myocardial hypertrophy.

[0012] The present invention provides a kit for diagnosing or prognosticating pathological myocardial hypertrophy, the kit comprising a probe or primer for specifically detecting circular RNA, or an antibody or small molecule compound for specifically detecting a polypeptide; wherein the circular RNA is a nucleic acid comprising a sequence shown in SEQ ID NO.1, and the polypeptide is a polypeptide comprising a sequence shown in SEQ ID NO.2.

[0013] The present invention also provides a use of a specific RNA interference adenovirus in the preparation of a drug for treating or / and preventing pathological myocardial hypertrophy, wherein the specific RNA interference adenovirus is obtained by integrating a shRNA expression sequence specifically targeting circular RNA into an adenovirus expression vector and packaging the resultant vector into a recombinant adenovirus vector, i.e., using adenovirus as a vector to express the interference shRNA specifically targeting circular RNA;

[0014] The nucleic acid sequence of the circular RNA is shown in SEQ ID NO.1.

[0015] The present invention also provides a pharmaceutical composition for treating and / or preventing pathological myocardial hypertrophy, which contains an effective amount of a specific RNA interference adenovirus, wherein the specific RNA interference adenovirus is obtained by integrating a shRNA expression sequence specifically targeting circular RNA into an adenovirus expression vector and packaging it into a recombinant adenovirus vector, that is, using adenovirus as a vector to express the interference shRNA specifically targeting circular RNA;

[0016] The nucleic acid sequence of the circular RNA is shown in SEQ ID NO.1.

[0017] The pharmaceutical composition of the present invention contains an effective amount of specific RNA interference adenovirus, and is added with pharmaceutically acceptable carriers or excipients to prepare a commonly used pharmaceutical preparation; the pharmaceutically acceptable carriers or excipients are selected from plasmid expression vectors, chitosan, cholesterol, liposomes, and nanoparticles.

[0018] Wherein, the preparation is an oral preparation or an injection preparation.

[0019] Most proteins are produced by the translation of messenger RNA (mRNA) in cells. The circHDR described in the present invention is different from most other non-coding circular RNAs in that it has a protein translation ability similar to that of mRNA. The derivative peptide is a natural translation product of circHDR in cardiomyocytes, rather than artificially translated. Its function and expression are identical to circHDR, and its translation can be inhibited by circHDR adenovirus. In addition, although circular RNA is more stable than other linear RNAs, the detection of circHDR can further reduce the error caused by detecting a single indicator.

[0020] The circHDR used in the present invention is one of the circular RNAs with the highest expression in the heart and can reflect the condition of cardiac pathology in a relatively stable and accurate manner.

[0021] The human circular RNA provided by the present invention is used in the preparation of heart disease related products, especially for pathological myocardial hypertrophy. The heart disease related circular RNA (circ-Heart Disease-Related, circHDR) involved is one of the most highly expressed circular RNAs in the heart, with high conservatism, and its function is also closely related to heart disease, which is of great significance in the diagnosis and treatment of heart disease. At the same time, due to the stable and specific nature of circular RNA, compared with other types of markers, it is more abundant in the patient's body fluids and changes more significantly, and has the potential to become a new diagnostic marker. Prepared into a specific RNA interference adenovirus, the expression efficiency is high; the viral genome will not be integrated into the host cell genome, and it has good controllability and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The expression level changes of circHDR during Ang II (angiotensin type II)-induced cardiomyocyte hypertrophy;

[0023] Figure 2Figure 1 shows the changes in the level of cardiomyocyte hypertrophy after interfering with circHDR. Figure A shows the relative average surface area of ​​cardiomyocytes; Figure B shows the expression level of ANP mRNA in cardiomyocytes; Figure C shows the expression level of BNP mRNA in cardiomyocytes. “-” means that this reagent was not added or used, and “+” means that this reagent was added or used.

[0024] Figure 3 The expression level of HDRP in cardiomyocytes after overexpression of circHDR;

[0025] Figure 4 The expression level of HDRP changes after Ang II induced cardiomyocyte hypertrophy;

[0026] Figure 5 The changes in the level of cardiomyocyte hypertrophy after expressing HDRP (Figure A is the relative average surface area of ​​cardiomyocytes; Figure B is the expression level of ANP mRNA in cardiomyocytes; Figure C is the expression level of BNP mRNA in cardiomyocytes);

[0027] Figure 6 Figure 3 shows the changes in cardiac hypertrophy levels after knocking down circHDR in the mouse myocardium (Figure A shows the relative expression level of circHDR in the mouse heart. Figure B shows the heart weight / body weight ratio of the mouse. Figure C shows the cardiac shortening fraction. "-" indicates that the reagent was not added or used, and "+" indicates that the reagent was added or used). DETAILED DESCRIPTION

[0028] Experimental reagents and materials:

[0029] The animals used in this experiment were purchased from Vitallife, the cell culture medium was purchased from SciGen, and the cell transfection reagent lipo3000 was purchased from Thermofisher; the expression vector was constructed by Liuhe BGI, the interfering adenovirus was constructed and synthesized by Heyuan Bio, RNA extraction, reverse transcription, and qPCR reagents were purchased from Novozymes, Ang II was purchased from MCE, Actin antibody was purchased from ABclonal, and HDRP antibody was prepared by ABclonal.

[0030] Data source:

[0031] circHDR was screened from high-throughput sequencing data of cardiac circular RNA published by Werfel et al. Werfel S, Nothjunge S, Schwarzmayr T, Strom TM, Meitinger T, Engelhardt S. Characterization of circular RNAs in human, mouse and rat hearts. J Mol Cell Cardiol. 2016 Sep; 98: 103-7.

[0032] The beneficial effects of the present invention are demonstrated by efficacy tests as follows.

[0033] Experimental Example 1 Changes in the expression level of circHDR during Ang II-induced cardiomyocyte hypertrophy

[0034] Rat primary cardiomyocytes were treated with 150 nM Ang II, and the cells were collected after 0 h, 3 h, 6 h, 12 h, 24 h and 48 h, respectively. Total RNA was extracted with TRIzol, and the expression level of circHDR in cardiomyocytes was detected by PT-qPCR.

[0035] The results showed that as the Ang II treatment time increased, the expression level of circHDR in cardiomyocytes showed a significant upward trend. Figure 1 .

[0036] Experimental Example 2: Changes in cardiomyocyte hypertrophy levels after interfering with circHDR

[0037] After 12 hours of infection of cardiomyocytes with circHDR interfering adenovirus (and yuan biosynthesis), the cells were treated with 150nM Ang II. After 48 hours, the cardiomyocytes were stained by immunofluorescence and their surface areas were measured by ImageJ software. Cell RNA was extracted and the expression levels of ANP and BNP mRNA were detected by RT-qPCR to evaluate the degree of cardiomyocyte hypertrophy.

[0038] The results showed that Ang II treatment could significantly increase the surface area of ​​cardiomyocytes and increase the expression of ANP and BNP. After knocking down circHDR, the effects of Ang II on the surface area of ​​cardiomyocytes and the expression of ANP and BNP were significantly inhibited. Figure 2 .

[0039] The role of circHDR interference adenovirus is to produce shRNA that specifically targets the circHDR sequence and is complementary to it in cardiomyocytes, thereby causing the degradation of circHDR. The experimental results showed that after treatment with angiotensin II, the expression of circHDR was significantly upregulated; and after interference with circHDR, the degree of cardiomyocyte hypertrophy was significantly reduced, indicating that abnormally high levels of circHDR are one of the causes of cardiomyocyte hypertrophy, indicating that circHDR is a pathogenic molecule of pathological myocardial hypertrophy and can be used as a target for the treatment of pathological myocardial hypertrophy.

[0040] Experimental Example 3 Derivative peptides produced by circular RNA translation

[0041] The peptide derivatives of the present invention are produced by natural translation of circular RNA in cells, rather than artificially stimulating cell production through an extracellular reaction system or by adding additional substances. The mechanism of circular RNA translation protein can be referred to in the following literature:

[0042] 1.Pamudurti NR, Bartok O, Jens M, Ashwal-Fluss R, Stottmeister C, Ruhe L, Hanan M, Wyler E, Perez-Hernandez D, Ramberger E, Shenzis S, Samson M, Dittmar G, Landthaler M, Chekulaeva M, Rajewsky N, Kadener S. Translation of CircRNAs.MolCell.2017Apr 6;66(1):9-21.e7.

[0043] 2. Yang Y, Fan X, Mao M, Song X, Wu P, Zhang Y, Jin Y, Yang Y, Chen LL, Wang Y, Wong CC, Xiao

[0044] 3.Legnini I,Di Timoteo G,Rossi F,Morlando M,Briganti F,Sthandier O,Fatica A,Santini T,Andronache A,Wade M,Laneve P,Rajewsky N,Bozzoni I.Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions inMyogenesis.Mol Cell.2017Apr 6;66(1):22-37.e9.

[0045] circHDR has translation function. Therefore, a large amount of HDRP can be produced in cells by transfecting cells with circHDR expression vectors through liposomes or infecting cells with circHDR overexpression adenovirus. For example, the circHDR expression vector was transfected into cardiomyocytes, and the empty vector was used as a control. Cells were collected 48 hours after transfection to extract proteins, and the expression level of HDRP in cells was detected by Western-blot using HDRP antibodies. The results showed that compared with the expression level of endogenous HDRP in the control group cells, the level of HDRP in the cardiomyocytes of the circHDR overexpression group was significantly increased (see Figure 3 ).

[0046] Experimental Example 4 Changes in the expression level of HDRP after Ang II induced cardiomyocyte hypertrophy

[0047] Rat primary cardiomyocytes were treated with 150 nM Ang II for 48 h, and then the cells were collected. The total cell protein was extracted with RIPA lysis buffer, and the expression level of HDRP in cardiomyocytes was detected by Western-blot.

[0048] The results showed that as the Ang II treatment time increased, the expression level of HDRP in cardiomyocytes increased significantly. Figure 4 .

[0049] Experimental Example 5 Effect of HDRP on the level of cardiomyocyte hypertrophy

[0050] HDRP expression vector (the HDRP open reading frame in the circHDR sequence was inserted into the expression vector pcDNA3.1), circHDR expression vector and circHDR expression vector with a start codon mutation (the start codon of the HDRP open reading frame in the circHDR sequence was mutated to make it lose the ability to translate protein) were transfected into cardiomyocytes. After 48 hours, the cardiomyocytes were stained with immunofluorescence and their surface areas were measured using ImageJ software. Cell RNA was extracted and the expression levels of ANP and BNP mRNA were detected by RT-qPCR to evaluate the degree of cardiomyocyte hypertrophy.

[0051] The results showed that overexpression of HDRP produced similar effects to overexpression of circHDR, which could significantly increase the surface area of ​​cardiomyocytes and increase the expression of ANP and BNP, while mutant circHDR showed almost no function in promoting cardiomyocyte hypertrophy, indicating that the derivative peptide HDRP performed the function of circHDR, and both it and circHDR were pathogenic and promoted myocardial hypertrophy; in other words, degradation of circHDR by interfering adenovirus or small interfering RNA could inhibit the production of derivative peptides, thereby inhibiting the development of myocardial hypertrophy (see Figure 5 ).

[0052] The above results show that the expression of circHDR and its derivative peptide HDRP increases significantly with the development of cardiomyocyte hypertrophy, and plays a role in promoting myocardial hypertrophy, suggesting that the development of myocardial hypertrophy can be evaluated based on their expression. Simultaneous detection of HDRP and circHDR can avoid the error caused by detecting a single indicator to a certain extent. In addition, the results also suggest that specific inhibitors can be developed with circHDR or HDRP as targets as preventive and therapeutic drugs for pathological myocardial hypertrophy.

[0053] Experimental Example 6: circHDR interfering adenovirus inhibits pathological myocardial hypertrophy by interfering with the expression of circHDR at the animal level

[0054] 2×10 12 The circHDR interference adenovirus (and yuan biosynthesis) was injected into 8-week-old male C57 / BL / 6 mice through the jugular vein. Then a slow-release pump was implanted subcutaneously on the back of the mouse and AngII was given at a dose of 0.6 mg / kg body weight / day. After 2 weeks of continuous administration, the mouse was anesthetized and the slow-release pump was surgically removed. The mouse was weighed, heart function was tested, and then the cervical dislocation was performed. The heart was weighed, RNA was extracted, and circHDR levels were detected by qPCR.

[0055] The results showed that Ang II significantly induced the expression of circHDR in the heart, increased heart weight, and significantly reduced the shortening fraction of the mouse heart. The circHDR interference adenovirus significantly inhibited the expression of circHDR and the increase in heart weight, effectively protecting the heart function. See the attached Figure 6 .

[0056] Knockdown of circHDR can significantly reduce the enlarged cardiomyocyte surface area and elevated ANP and BNP expression levels caused by Ang II treatment, proving that silencing circHDR can inhibit cardiomyocyte hypertrophy and suggesting that using appropriate vectors to package circHDR interfering RNA into drugs and deliver it to cardiac tissue will play a role in treating and preventing pathological myocardial remodeling. Silencing circHDR in the mouse heart using circHDR interfering adenovirus can inhibit Ang II-induced heart enlargement and decreased cardiac function, further proving the therapeutic effect of circHDR interfering adenovirus on pathological myocardial hypertrophy at the animal level. In addition, circHDR can encode heart disease-related peptides (HDRP) and function through HDRP: HDRP expression increases after Ang II treatment of cardiomyocytes; transfection of HDRP expression vectors in cardiomyocytes can also cause cardiomyocyte hypertrophy, while transfection of circHDR expression vectors with start codon mutations that lose their ability to encode HDRP cannot induce myocardial hypertrophy, suggesting that neutralizing HDRP with specific antibodies or small molecule compounds can also have a preventive and therapeutic effect.

[0057] In summary, the inventors have demonstrated through experiments that detecting the expression levels of circHDR and its derivative peptide HDRP and inhibiting circHDR and its derivative peptide HDRP in the myocardium have important application values ​​for the diagnosis and prevention of heart diseases.

[0058] Therefore, the present invention provides a kit for diagnosing heart disease by detecting circHDR and HDRP levels, comprising circHDR-specific primers / probes and HDRP antibodies / small molecule compounds, as well as a drug formed by packaging circHDR small interfering RNA with appropriate carriers or excipients (such as mammalian expression plasmids, adenoviruses, adeno-associated viruses, chitosan, cholesterol, liposomes, nanoparticles, etc.) and an antibody or small molecule compound that specifically neutralizes HDRP, which are used for preventing and / or treating heart disease by intravenous injection.

[0059] SEQ ID No.1

[0060] Sequence

[0061]

[0062] SEQ ID No.2

[0063]

Claims

1. Use of a human circular RNA in the preparation of a detection kit or a drug for diagnosing pathological myocardial hypertrophy, wherein: The human circular RNA includes a nucleic acid containing a sequence as shown in SEQ ID NO.1, or a biologically active functional fragment, variant or derivative of a nucleic acid containing a sequence as shown in SEQ ID NO.

1.

2. A derivative peptide produced by translation of circular RNA, characterized in that: A polypeptide comprising the sequence shown in SEQ ID NO.2, or a fragment, variant or derivative of the polypeptide comprising the sequence shown in SEQ ID NO.

2.

3. Use of the derivative peptide according to claim 2 in the preparation of a detection kit or a drug for diagnosing pathological myocardial hypertrophy.

4. A kit for diagnosing or prognostic evaluation of pathological myocardial hypertrophy, the kit comprising a probe or primer for specifically detecting circular RNA, or an antibody or small molecule compound for specifically detecting a polypeptide; wherein the circular RNA is a nucleic acid comprising the sequence shown in SEQ ID NO.1, and the polypeptide is a polypeptide comprising the sequence shown in SEQ ID NO.

2.

5. Use of a specific RNA interference adenovirus in the preparation of a drug for treating and / or preventing pathological myocardial hypertrophy, characterized in that: The specific RNA interference adenovirus is obtained by integrating the shRNA expression sequence specifically targeting circular RNA into an adenovirus expression vector and packaging it into a recombinant adenovirus vector, that is, using adenovirus as a vector to express the interference shRNA specifically targeting circular RNA; The nucleic acid sequence of the circular RNA is shown in SEQ ID NO.

1.

6. A pharmaceutical composition for treating and / or preventing pathological myocardial hypertrophy, characterized in that: It contains an effective amount of specific RNA interference adenovirus, which is obtained by integrating the shRNA expression sequence specifically targeting circular RNA into an adenovirus expression vector and packaging it into a recombinant adenovirus vector; the nucleic acid sequence of the specific RNA is shown in SEQ ID NO.

1.

7. The pharmaceutical composition for treating and / or preventing pathological myocardial hypertrophy according to claim 6, characterized in that: The invention contains an effective amount of specific RNA interference adenovirus, and adds pharmaceutically acceptable carriers or auxiliary materials to prepare a commonly used pharmaceutical preparation; the pharmaceutically acceptable carriers or auxiliary materials are selected from plasmid expression vectors, chitosan, cholesterol, liposomes, and nanoparticles.

8. The pharmaceutical composition for treating and / or preventing pathological myocardial hypertrophy according to claim 7, characterized in that: The preparation is an oral preparation or an injection preparation.

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