CN111411152A - Application of humanized circular RNA in preparation of heart disease related products - Google Patents

Abstract

The invention provides application of humanized circular RNA in preparation of heart disease related products, and relates to the technical field of biological medicines. The humanized circular RNA applied to preparing heart disease related products is nucleic acid containing a sequence shown as SEQ ID NO.1 or bioactive functional fragments, variants or derivatives of the nucleic acid of the sequence shown as SEQ ID NO. 1. The humanized circular RNA is applied to the preparation of heart disease related products and can have detection sensitivity and specificity when being used as a diagnosis marker.

Description

Application of humanized circular RNA in preparation of heart disease related products

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of humanized circular RNA in preparation of heart disease related products.

Background

At present, the diagnosis of coronary heart disease mainly comprises three modes of clinical manifestation, electrocardiogram and blood marker represented by troponin, however, the above means have defects of specificity, sensitivity, poor stability and the like, are not beneficial to doctors to make diagnosis in time, and intervene and treat before irreversible necrosis occurs in the myocardium. Therefore, the development of an improved coronary heart disease diagnosis product is imperative.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide an application of a human circular RNA in preparing heart disease related products.

The second objective of the invention is to provide a method for detecting the human circular RNA for non-diagnostic and therapeutic purposes.

The third purpose of the invention is to provide a kit containing a primer for detecting the human circular RNA and/or a nucleic acid probe targeting the human circular RNA.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one aspect of the invention, the invention provides the use of a circular RNA of human origin comprising a nucleic acid comprising the sequence shown as SEQ ID No.1, or a biologically active functional fragment, variant or derivative of a nucleic acid comprising the sequence shown as SEQ ID No.1, for the preparation of a product related to a heart disease.

Optionally, the heart disease-related product comprises a product for diagnosing, preventing or treating a heart disease;

optionally, the heart disease-related product comprises a medicament or a kit.

Optionally, the cardiac disorder comprises myocardial infarction, heart failure, or myocardial injury.

Optionally, the circular RNA of human origin is used as a diagnostic marker for heart disease;

optionally, the human circular RNA serves as a myocardial infarction blood diagnosis marker.

According to another aspect of the invention, the invention also provides a method for detecting the human circular RNA for non-diagnostic and therapeutic purposes, comprising detecting the human circular RNA using primers and/or nucleic acid probes targeting the human circular RNA;

optionally, the primer and the nucleic acid probe each independently comprise a modified derivative.

Optionally, the detection method comprises amplifying a target sequence using a primer to detect the human circular RNA;

optionally, the amplification comprises amplification by a PCR method, preferably a real-time quantitative PCR method;

optionally, the primer comprises a circHED specific primer, and the sequence of the circHED specific primer is shown as SEQ ID No.4 and SEQ ID No. 5;

optionally, the primers further comprise an internal reference primer;

optionally, the sequence of the internal reference primer is shown as SEQ ID NO.6 and SEQ ID NO. 7.

Optionally, the detection method comprises using a nucleic acid probe to bind to the target sequence to detect the circular RNA of human origin;

optionally, binding of nucleic acid probes to the target sequence is achieved using Northern blotting or E L ISA methods to detect the circular RNA of human origin.

Alternatively, the RNA in the sample is directly detected by using the nucleic acid probe, or the RNA in the sample to be detected is firstly reversely transcribed to obtain cDNA, and then the cDNA is detected by using the primer and/or the nucleic acid probe.

According to another aspect of the present invention, the present invention also provides a kit comprising a primer for detecting the above-mentioned human circular RNA and/or a nucleic acid probe targeting the human circular RNA.

Optionally, the kit comprises primers for detecting the human circular RNA;

optionally, the primer comprises a circHED specific primer, and the sequence of the circHED specific primer is shown as SEQ ID No.4 and SEQ ID No. 5;

optionally, the primers further comprise an internal reference primer;

optionally, the sequence of the internal reference primer is shown as SEQ ID NO.6 and SEQ ID NO. 7.

Compared with the prior art, the invention has the following beneficial effects:

in the application of the human-derived circular RNA in preparing heart disease related products, the human-derived circular RNA is one of the circular RNAs with the highest expression level in the heart, has higher conservation, has functions closely related to myocardial damage, and has great significance in the diagnosis and treatment of coronary heart disease. Meanwhile, due to the stable and specific properties of the circular RNA, compared with other types of markers, the circular RNA has larger abundance and more obvious change in body fluid (blood, urine, saliva and the like) of a patient, and therefore has the potential of becoming a novel diagnostic marker. The high stability and tissue cell specificity of circHED in body fluid enables the circHED to have detection sensitivity and specificity as a diagnostic marker of heart diseases.

The kit provided by the invention comprises a primer for detecting the human circular RNA and/or a nucleic acid probe targeting the human circular RNA, can be used for detecting the circular RNA as a heart disease diagnosis marker circHED, and has good detection sensitivity and specificity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the results of amplification of cDNA and gDNA using the forward and reverse primers, respectively, in example 1;

FIG. 2 shows the sequencing results of the reverse product in example 1;

FIG. 3 is the expression level of circHED in blood of a normal human and a myocardial infarction patient in example 2;

FIG. 4 is a ROC analysis of blood circHED levels in example 2.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

According to one aspect of the invention, the invention provides the use of a circular RNA of human origin comprising a nucleic acid comprising the sequence shown as SEQ ID No.1, or a biologically active functional fragment, variant or derivative of a nucleic acid comprising the sequence shown as SEQ ID No.1, for the preparation of a product related to a heart disease.

Circular RNA is a novel RNA with a covalent closed structure, is derived from reverse cleavage of precursor mRNA (back-cleaving), has a stable structure and a long half-life, and can still exist for a long time through cell death release or exosome transport to body fluid. The expression of the circular RNA has specificity on the tissue and cell level, and most of the circular RNA can regulate the development process of organs and maintain the normal physiological function of tissue cells through binding protein or miRNA. Research has shown that the development of various diseases is related to the expression disorder of tissue-specific circular RNA, such as various tumors, neurological diseases, etc.

RNA sequencing data show that a large amount of specifically expressed circular RNA exists in the heart, the expression disorder of the heart circular RNA is closely related to heart diseases, such as excessive circ-Foxo3 can limit ID-1, E2F1, FAK and HIF1 α in cytoplasm to promote senescence of myocardial cells by combining with the RNA, HRCR can inhibit the function of miR-223, myocardial hypertrophy can be caused when the HRCR is underexpressed, and miR-7 can be inhibited to promote apoptosis of the myocardial cells when the expression of CDR1as is increased in the myocardial infarction process.

In the application provided by the invention, the human circular RNA: RNA-Heart-erihed (abbreviated circHED), is one of the most highly expressed circular RNAs in the Heart, and the circHED comprises a nucleic acid having a sequence as shown in SEQ ID NO.1, or a biologically active functional fragment, variant or derivative of the nucleic acid having a sequence as shown in SEQ ID NO. 1. Biologically active functional fragments refer to fragments of circular rna of human origin (circHED), which have biological or physiological activity substantially similar to that of circHED and are also characterized by overexpression in samples of heart disease compared to normal samples. A variant of circHED refers to a circular RNA in the nucleic acid sequence of circHED that is different from circHED due to deletion or mutation, but that retains the biological and physiological activity of circHED, i.e. also has the characteristic of being overexpressed in samples of heart disease compared to normal samples. Derivatives of circHED include nucleic acids containing chemically or biochemically modified, non-natural or derivatized nucleotide bases including, but not limited to, single, double or multiple stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids.

The circHED applied to preparing heart disease related products is one of the cyclic RNAs with the highest expression quantity in the heart, has higher conservation, and the function of the circHED is closely related to myocardial damage. In addition, due to the stable and specific properties of the circular RNA, compared with other types of markers, the circular RNA has larger abundance and more remarkable change in body fluid (blood, urine, saliva and the like) of a patient, and therefore has the potential of becoming a novel diagnostic marker. The high stability and tissue cell specificity of circHED in body fluid enables the circHED to have detection sensitivity and specificity as a diagnostic marker of heart diseases.

In the product related to heart diseases, the heart diseases include but are not limited to diseases such as myocardial infarction, heart failure or other types of myocardial injuries. The function of the product includes, but is not limited to, use in the diagnosis, prevention or treatment of heart disease; types of products include, but are not limited to, pharmaceuticals or kits.

Some alternative examples of applications are as follows: circHED can be used as a diagnostic marker of heart diseases, preferably as a blood diagnostic marker of myocardial infarction. Therefore, the circHED can be applied to preparing a kit for diagnosing heart diseases, wherein the circHED is used as a diagnostic marker of the diagnostic kit, and the diagnostic kit judges whether a patient has the heart diseases or not by judging the expression level of the circHED in a sample to be detected; meanwhile, the circHED can also be used as a control sample and/or a quality control sample in a diagnostic kit; the circHED is applied to preparing a medicine for preventing or treating heart diseases, and can be used as a marker for evaluating the effect of the medicine.

According to another aspect of the invention, the invention also provides a detection method for detecting the human circular rna (circHED) for non-diagnostic and therapeutic purposes, the detection method comprising detecting circHED using a primer and/or a nucleic acid probe targeting circHED. A nucleic acid probe refers to a polymer of nucleotides of any length, including ribonucleotides and/or deoxyribonucleotides, capable of hybridizing or partially hybridizing to circHED. The primer and the nucleic acid probe respectively and independently contain one or more of natural purine base and/or pyrimidine base, chemically or biochemically modified purine base and/or pyrimidine base; the primer and the nucleic acid probe may also independently comprise a label, including but not limited to a luminescent group and/or one of a quenching group, an isotope, a receptor and a ligand thereof of the luminescent group, one of an antigen and an antibody thereof, or one of an enzyme and a substrate thereof, respectively.

Some examples of detection methods for detecting the human circular rna (circHED) for non-diagnostic and therapeutic purposes include, but are not limited to, methods for assessing or determining the effect of the presence or amount of circHED on the presence or amount of other biologically active components in a test sample; or circHED is taken as a marker for researching physiological and biochemical processes, so as to research the mechanism of some signal paths or other physiological processes, and the like.

In some alternative embodiments, examples of detecting circHED using primers include, but are not limited to, using PCR to amplify nucleic acids in a test sample using primers to detect whether circHED is contained in the test sample. In some alternative embodiments, the primer comprises a circHED-specific primer having a sequence as shown in SEQ ID No.4 and SEQ ID No. 5. The PCR preferably adopts a real-time quantitative PCR method which has high detection efficiency and can carry out quantitative analysis, when the real-time quantitative PCR method is adopted, the primers preferably also comprise internal reference primers, and optionally, the sequences of the internal reference primers are shown as SEQ ID NO.6 and SEQ ID NO. 7. It is understood that the reference primer is not limited to real-time fluorescent quantitative PCR, and may be used when other types of detection methods are used and the reference primer is used, or may have other sequences.

In some alternative embodiments, nucleic acid probes are used to bind to the target sequence to detect circHED, examples of which include, but are not limited to, Northern blotting or E L ISA methods.

In some alternative embodiments, the RNA in the sample is directly detected by using the nucleic acid probe, or the RNA in the sample to be detected is reverse transcribed to obtain cDNA, and then the cDNA is detected by using the primer and/or the nucleic acid probe.

According to another aspect of the invention, the invention further provides a kit, which can detect circHED as a cardiac disease diagnosis marker by using the primer of the human circular RNA and/or the nucleic acid probe targeting the human circular RNA, and has good detection sensitivity and specificity. The primer and the nucleic acid probe respectively and independently comprise one or more of natural purine base and/or pyrimidine base, chemically or biochemically modified purine base and/or pyrimidine base; the primer and the nucleic acid probe may also independently comprise a label, including but not limited to a luminescent group and/or one of a quenching group of a luminescent group, an isotope, a receptor and a ligand thereof, one of an antigen and an antibody thereof, or one of an enzyme and a substrate thereof.

Examples of conventional reagents include, but are not limited to dNTPs, salts, enzymes, fluorochromes, buffers, etc. for PCR amplification, enzymes, primers, etc. for reverse transcription, reagents for Northern blotting or E L ISA experiments, receptors, ligands, enzymes, enzyme substrates, antibodies or antigens, etc. for detecting labels on the probes.

In some alternative embodiments, the kit comprises primers for amplifying the human circular RNA, the primers comprising a circHED-specific primer having a sequence as shown in SEQ ID No.4 and SEQ ID No. 5. The kit can also comprise an internal reference primer, and the sequence of the internal reference primer can be optionally shown as SEQ ID NO.6 and SEQ ID NO. 7.

The technical solution and the advantages of the present invention will be further explained with reference to the preferred embodiments.

Example 1

Identification of circHED in human blood:

and identifying circHED in blood by adopting an inverse PCR method. Collecting 500 μ l of human blood, adding 500 μ l Trizol (Takara RNAioso Plus 9108), inverting the centrifuge tube for several times, standing at room temperature for 15min, extracting total RNA according to the instruction, and further performing reverse transcription on RNA by using Takara RR0037A reverse transcription kit and Random primer Random 6-mer to synthesize cDNA; another 300. mu.l of blood was passed through the TIANGEN blood genomic DNA extraction system (DP349) to extract genomic DNA (gDNA). The sequence of circHED end junction amplified by reverse primer PCR using blood cDNA or gDNA as template and forward primer as control was used. And carrying out agarose gel electrophoresis detection on the amplified product, and carrying out sanger sequencing on the amplified product with cDNA as a template.

The primer sequences are as follows:

circHED forward primer:

F:5’-TGACAGCAGCCTGGAGCATCTT-3’(SEQ ID NO.2)；

R:5’-TGCTTGCCAGCTCGATACCTCT-3’(SEQ ID NO.3)。

circHED reverse primer (circHED specific primer):

F:5’-TGTGAGTGAGAGCATTGGCA-3’(SEQ ID NO.4)；

R:5’-ATCCCATTGAAAAGGTGGGTGA-3’(SEQ ID NO.5)。

the experimental results are as follows:

identification of circHED expression in blood: since the ends of the circular RNA sequences are covalently linked, reverse primers need to be designed to amplify the end-linked portions. The PCR results showed that the forward primer was able to amplify a fragment of the expected size from the cDNA and gDNA; fragments of the expected size can be amplified from cDNA using the reverse primer, but since reverse cleavage to form circular RNA is performed after transcription, the product cannot be amplified from gDNA (FIG. 1). The sequencing results of the reverse products showed that the ends of the circHED sequence were linked (fig. 2), and these identification results demonstrated that circHED was indeed present in blood and had a high expression level.

Example 2

Detection of circHED levels in myocardial infarction patient blood:

the experiment respectively collects 50 blood samples of normal persons without cardiovascular disease history and acute myocardial infarction patients, and the myocardial infarction patients need to accord with the following clinical manifestations: (1) angina pectoris lasts more than 30 min; (2) the electrocardiogram shows typical myocardial infarction characteristics; (3) the blood content of molecular markers such as creatine kinase, lactate dehydrogenase and troponin T or I is increased. Blood RNA was extracted and cDNA was synthesized as described in example 1, using 18SrRNA as an internal reference, using qPCR for detection, 2^-ΔΔCTThe method calculates the relative expression level of circHED in blood. The qPCR kit used the naughty zmq Universal SYBR qPCR Master Mix, and the experimental procedures were performed as described. The primer sequences are as follows:

circHED qPCR primers (circHED specific primers):

F:5’-TGTGAGTGAGAGCATTGGCA-3’(SEQ ID NO.4)；

R:5’-ATCCCATTGAAAAGGTGGGTGA-3’(SEQ ID NO.5)。

18SrRNA qPCR primers (internal reference primers):

F：5’-TTCGATGGTAGTCGCCGTGCCTA-3’(SEQ ID NO.6)；

R：5’-CCTGCTGCCTTCCTTGGATGTGGTA-3’(SEQ ID NO.7)。

and detecting the effect of blood circHED levels on differentiating normal persons from myocardial infarction patients using a Receiver Operating Characteristic (ROC) curve, and assessing the accuracy of the circHED diagnosis of myocardial infarction by the area under the ROC curve (AUC).

The experimental results are as follows:

detecting the expression level of circHED in the blood of a normal human and a myocardial infarction patient: the detection result shows that the average value of the blood circHED level of the myocardial infarction patient is obviously higher than that of a normal person and is more than 2 times of that of the normal person (figure 3). The results were further subjected to ROC analysis, which showed that the AUC value obtained by differentiating normal humans from myocardial infarction patients with the blood circHED level was 0.8004 (fig. 4), indicating that circHED is an ideal diagnostic marker of myocardial infarction.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and such modifications and substitutions do not depart from the spirit and scope of the present invention as defined by the appended claims.

SEQUENCE LISTING

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Claims (10)

1. Use of a circular RNA of human origin comprising a nucleic acid comprising the sequence shown as SEQ ID No.1 or a biologically active functional fragment, variant or derivative of a nucleic acid comprising the sequence shown as SEQ ID No.1 for the preparation of a product related to a heart disease.

2. The use according to claim 1, wherein the heart disease-related product comprises a product for diagnosing, preventing or treating a heart disease;

optionally, the heart disease-related product comprises a medicament or a kit.

3. The use according to claim 1, wherein the heart disease comprises myocardial infarction, heart failure or myocardial injury.

4. The use according to any one of claims 1 to 3, wherein the circular RNA of human origin is used as a diagnostic marker for heart disease;

optionally, the human circular RNA serves as a myocardial infarction blood diagnosis marker.

5. A method of detecting the human circular RNA of any one of claims 1 to 4 for non-diagnostic and therapeutic purposes, comprising detecting the human circular RNA using primers and/or nucleic acid probes targeting the human circular RNA;

optionally, the primer and the nucleic acid probe each independently comprise a modified derivative.

6. The method for detecting the human circular RNA according to claim 5, wherein the method for detecting the human circular RNA comprises amplifying a target sequence using a primer to detect the human circular RNA;

optionally, the amplification comprises amplification by a PCR method, preferably a real-time quantitative PCR method;

optionally, the primer comprises a circHED specific primer, and the sequence of the circHED specific primer is shown as SEQ ID NO.4 and SEQ ID NO. 5;

optionally, the primers further comprise an internal reference primer;

optionally, the sequence of the internal reference primer is shown as SEQ ID NO.6 and SEQ ID NO. 7.

7. The detection method according to claim 5, wherein the detection method comprises binding a nucleic acid probe to a target sequence to detect the human circular RNA;

optionally, binding of nucleic acid probes to the target sequence is achieved using Northern blotting or E L ISA methods to detect the circular RNA of human origin.

8. The detection method according to any one of claims 5 to 7, wherein the RNA in the sample is directly detected using the nucleic acid probe, or the RNA in the sample to be detected is reverse-transcribed to obtain cDNA, which is then detected using the primer and/or the nucleic acid probe.

9. Kit comprising primers for the detection of the circular RNA of human origin according to any one of claims 1 to 4 and/or nucleic acid probes targeting said circular RNA of human origin.

10. The kit of claim 9, wherein the kit comprises primers for detecting the human circular RNA;

optionally, the primer comprises a circHED specific primer, and the sequence of the circHED specific primer is shown as SEQ ID NO.4 and SEQ ID NO. 5;

optionally, the primers further comprise an internal reference primer;

optionally, the sequence of the internal reference primer is shown as SEQ ID NO.6 and SEQ ID NO. 7.

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