Circular RNA gene circRNA and application thereof

Abstract

The invention discloses a circular RNA (Ribonucleic Acid) gene circTMCC1 and application of the circular RNA gene circTMCC1 in preparation of a kit for diagnosing and screening coronary atherosclerosis and evaluating the severity of the coronary atherosclerosis. The circular RNA provided by the research can be used as a potential biomarker for coronary atherosclerosis, diagnosis is carried out by detecting the expression level of the gene in peripheral blood mononuclear cells, the circular RNA has good stability, and the circular RNA can be used for predicting coronary heart disease in cooperation with traditional coronary atherosclerosis risk factors, so that coronary heart disease patients can be found in the early stage, and the risk of coronary heart disease is reduced. The coronary heart disease death rate is reduced and the patient prognosis is improved. At present, the main diagnosis method for coronary atherosclerotic heart disease has the risks of high danger coefficient, large radiation and the like, has the advantages of convenience, small trauma, good repeatability, no radiation and the like by collecting peripheral blood of a patient for detection, and is easier to accept.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical diagnostic markers, and particularly relates to a circular circTMCC1 molecule and application thereof that can be used for screening and diagnosing coronary atherosclerosis. Background Art

[0002] Circular RNA (circRNA) is an endogenous non-coding RNA that is reverse spliced ​​and forms a closed loop in structure. The expression of circRNAs is tissue-specific and relatively stable because their closed loop structure prevents them from being degraded by RNA exonucleases. circRNAs have multiple cellular functions, and the functional mechanisms of circRNAs are as follows: 1. CircRNAs can act as sponges for miRNA-Ago2 complexes, weakening the function of miRNAs. 2. Exon-intron circular RNAs (EIciRNAs) interact with U1 snRNP and polymerase II in the promoter region of EIciRNAs host genes to induce their transcription (bottom); 3. CircRNAs can act as decoys for proteins and regulate their functions. 4. CircRNAs can act as translatable RNAs encoding polypeptides. 5. Reverse splicing and linear splicing compete with each other to produce circRNAs or mRNAs. Changes in the expression of circular RNAs are associated with the occurrence and development of a variety of pathological physiologies.

[0003] The regulatory mechanisms and networks of circRNAs are complex and three-dimensional. It can bind to DNA, RNA, and proteins at multiple levels and regulate gene expression at different stages such as gene transcription and translation. Given the complexity of circRNAs functions, it can regulate the functions and protein expression of multiple miRNAs. Therefore, changing the expression of a circRNA may simultaneously activate multiple vascular growth factor-mediated signaling pathways, thereby promoting angiogenesis and maturation. Due to its tissue specificity and functional complexity, circRNA has broad prospects in the treatment of angiogenesis and may become a new therapeutic target and potential biomarker for coronary heart disease.

[0004] In recent years, researchers have found that circular RNA can be involved in the occurrence and development of a variety of cardiovascular diseases. However, its specific mechanism of action is still unclear. Therefore, we tried to find a circRNA biomarker to diagnose coronary atherosclerosis, and at the same time, synergize with traditional coronary atherosclerosis risk factors to improve the diagnostic efficiency. Summary of the invention

[0005] The purpose of the present invention is to provide a circRNA diagnostic marker for coronary atherosclerosis. The circRNA is useful in the screening, diagnosis, and prognosis evaluation of coronary atherosclerosis.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a circular RNA gene circTMCC1, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The preparation for detecting the circular RNA gene circTMCC1 is used in the preparation of any of the following products: (1) Application in the preparation of diagnostic reagents for screening coronary atherosclerosis; (2) Application in the preparation of diagnostic reagents for diagnosing coronary atherosclerosis; (3) Application in the preparation of diagnostic reagents for preliminary determination of the severity of coronary atherosclerosis; (4) Application in the preparation of diagnostic reagents for the prognosis evaluation of coronary atherosclerosis.

[0008] Furthermore, the preparation is used to detect the expression level of circTMCC1 in PBMC cells of the subject, or to specifically identify circTMCC1.

[0009] A marker for diagnosing coronary atherosclerosis, wherein the marker is a circular RNA gene circTMCC1, and the nucleotide sequence is shown in SEQ ID NO.1.

[0010] A diagnostic reagent for coronary atherosclerosis, comprising: a preparation for detecting the expression amount of the circular RNA gene circTMCC1, wherein the nucleotide sequence of the circTMCC1 is shown as SEQ ID NO.1.

[0011] A diagnostic kit for coronary atherosclerosis, comprising the diagnostic reagent according to claim 5.

[0012] Further, the diagnostic kit uses the following steps for detection: (1) Isolate PBMCs from the blood sample to be tested, extract RNA and place at -80°C; (2) The RNA obtained in step 1 was reverse transcribed to obtain cDNA, and the designed circTMCC1 amplification primers were added to perform qPCR experiments; (3) The amplified product obtained in step 2 was subjected to Sanger sequencing, RNaseR resistance, actinomycin D treatment, and agarose gel electrophoresis to verify the circularization of circTMCC1; (4) Based on the expression level of circTMCC1 in step 4 and the normal control group, analyze whether the gene is differentially expressed in the test sample.

[0013] Furthermore, the amplification primers are specific primers, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3.

[0014] Furthermore, the kit also includes a fluorescent dye and an amplification primer for specifically detecting the expression level of the internal reference GAPDH.

[0015] Furthermore, the fluorescent dye is ChamQ Universal SYBR qPCR Master Mix, and the specific primers for GAPDH, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO.4, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.5.

[0016] The present invention provides a biomarker circRNA suitable for coronary atherosclerosis. By detecting the expression level of the gene in peripheral PBMCs, normal people and patients with coronary atherosclerosis can be effectively distinguished. The application of the gene expression level for diagnosis can screen patients with coronary heart disease at an early stage and evaluate the severity of coronary atherosclerosis, timely intervene, reduce the mortality rate of coronary heart disease, and improve the prognosis of patients. Compared with the current coronary artery enhanced CT, coronary angiography and other coronary artery atherosclerosis detection methods, the scheme of collecting peripheral blood from patients for detection has the characteristics of low radiation, low risk, good stability, and can reduce trauma. The marker designed by the present invention has good value in the diagnosis of coronary atherosclerosis (AUC=0.7557). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 and 2 It is the circRNA differentially expressed in the fatty streak stage of coronary atherosclerosis grade 1 VS the fibrous plaque stage of coronary atherosclerosis grade 4 in Example 1.

[0018] Figure 3 This is the expression characteristic of circTMCC1 in clinical specimens in Example 2.

[0019] Figure 4 The receiver operating characteristic curve analysis results of circTMCC1 in clinical specimens in Example 2 Figure 5 The receiver operating characteristic curve analysis results of circTMCC1 and traditional coronary atherosclerosis risk factors in clinical specimens in Example 2 Figure 6 The qPCR in Example 3 verified that circTMCC1 was successfully silenced in mouse heart tissue.

[0020] Figure 7This is the relationship between circTMCC1 knockdown in Example 3 and myocardial infarction, the most serious complication of coronary atherosclerosis. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application.

[0022] It should be noted that the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or apparatuses.

[0023] The research team collaborated with the Department of Human Anatomy of Nanjing Medical University to conduct transcriptomic sequencing analysis on human coronary artery specimens. A total of 1,640 circRNAs were detected, including 1,259 known circRNAs and 381 newly discovered circRNAs (Figure 1). The detected circRNAs are widely distributed on all human chromosomes (including autosomes and sex chromosomes). There are more circRNAs found on chromosomes 1 and 2 (162 and 170, respectively) than on other chromosomes. In addition, the Y chromosome has the least circRNAs among all chromosomes, which may be because the Y chromosome is the shortest in the human genome and contains the least number of genes. Through GO functional enrichment and KEGG pathway prediction analysis, we found that these differentially expressed circRNAs are involved in a series of pathways closely related to atherosclerosis. This study reveals that the relationship between circRNA and cardiovascular disease has laid a theoretical foundation and is an important technical work accumulation for this subject. We screened out differentially expressed circRNAs from the sequencing data of coronary atherosclerosis stage 1 fatty streak stage VS coronary atherosclerosis stage 4, and a total of 24 circRNAs were screened, among which circTMCC1 had the largest absolute value of log2FoldChange

[0024] Study subjects: 130 consecutive patients with coronary heart disease confirmed by coronary angiography and 43 normal control patients were enrolled. According to the angiography results, subjects with at least one coronary artery stenosis >50% were selected as the patient group, and subjects with all coronary artery stenosis <50% were selected as the control group. Peripheral blood was collected from the above enrolled patients, and the baseline data are shown in Table 1.

[0025] Table 1 Clinical baseline data of AS patients and control group patients

[0026] The specific steps are as follows: Separation of samples: PBMCs were separated by density gradient centrifugation with a sample to lymphocyte separation solution volume of 1:1, 2000rpm*20min. After stratification, a pipette was used to insert into the cloud layer to aspirate the mononuclear cells, which were then stored in TRIzol reagent. Total RNA was extracted by centrifugation in chloroform at 12000g for 15min, precipitated by centrifugation with an equal volume of isopropanol at 12000g×10min, purified by centrifugation with 75% ethanol at 7500g×5min, and finally dissolved in RNase-free water.

[0027] Primer design: PCR primers were designed based on the sequence shown in SEQ ID NO. 1. The primer sequences are as follows: Forward primer: 5'-AGGGAACTGCGGAGCGGCTT-3' Reverse primer: 5'-TGCCTATCTTCATCACTCTTCCT -3' Reverse transcription: Reverse transcription was performed using HiScript III All-in-one RT SuperMix for qPCR reagent and the reaction was performed on ice.

[0028] Amplification: The cDNA amplified in step (3) was used as a template and the reaction system was prepared with ChamQ Universal SYBR qPCR Master Mix reagent. Real-time quantitative PCR was performed in two steps. The reaction procedure was as follows: 95°C / 5 min pre-denaturation, 95°C / 10 s denaturation, 60°C / 30 s annealing / extension, 40 cycles were repeated, three replicate wells were set for each sample, and the relative expression level was calculated by the ΔΔCt method.

[0029] Verification results: Statistical analysis results showed that circTMCC1 (nucleotide sequence as shown in SEQID NO.1) had significant expression differences between the two groups (p<0.001), and the gene was highly expressed in the coronary heart disease group. The receiver operating characteristic curve analysis results showed that the area under the curve was greater than 0.5, indicating that the gene has a certain diagnostic ability for coronary atherosclerosis.

[0030] Acute myocardial infarction animal model: 10-week-old male C57BL / 6 mice were adaptively fed for 1 week, weighed, and anesthetized by inhalation of 1-2% isoflurane, with the anesthetic dose adjusted according to body weight. The mice were then intubated and ventilated using a small animal ventilator. A left thoracotomy was made between the third and fourth intercostal spaces, and the muscles and pericardium were cut to expose the left anterior descending branch. The anterior wall of the left ventricle was ligated with 8-0 silk suture 2 mm below the lower edge of the left atrial appendage. The ligated mice were randomly divided into two groups, and the lentiviral vectors (LVs) carrying LV-vector and LV-sh-circTMCC1 were injected into the mouse myocardium by orthotopic injection. The chest was then sutured layer by layer. Sham-operated mice underwent the same surgery without LAD ligation. On the 7th day after myocardial infarction, blood was collected from the retroorbital plexus for ELISA analysis, and heart tissue was obtained under deep anesthesia before the mice were euthanized.

[0031] qPCR ensures that circTMCC1 is successfully silenced in mouse heart tissue. After removing the mouse myocardial tissue, we used scissors to cut a small amount of myocardial tissue from the mouse apex. Through a series of experiments such as RNA extraction, primer design, reverse transcription, amplification, and the relative expression level was calculated by the ΔΔCt method. The silencing rate is as follows Figure 6 shown.

[0032] HE and Masson staining: Mouse heart tissue was collected and fixed with 4% paraformaldehyde. Heart tissue from 1 mm below the ligature to the apex was embedded in paraffin and sliced ​​at a thickness of 5 μm. The sections were dewaxed, hydrated, and stained using Masson's Trichrome staining reagent and hematoxylin and eosin staining kit according to the manufacturer's instructions. The stained sections were examined and imaged using an Olympus CKX53 microscope.

[0033] Verification results: Statistical analysis HE and Masson staining confirmed that the myocardial infarction area in LV-sh-circTMCC1 mice was smaller than that in LV-sh-NC mice (p<0.01)( Figure 7 These findings suggest that circTMCC1 is highly associated with cardiac damage associated with myocardial infarction, a fatal complication of atherosclerosis, and affects the prognosis of atherosclerosis.

[0034] It should be understood that the present invention is described by way of example only and can be modified within the scope and spirit of the present invention. The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary skills in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A circular RNA gene circTMCC1, characterized in that: The nucleotide sequence of circTMCC1 is shown in SEQ ID NO.

1.

2. The preparation for detecting the circular RNA gene circTMCC1 is used in the preparation of any of the following products, characterized in that: (1) Application in the preparation of diagnostic reagents for screening coronary atherosclerosis; (2) Application in the preparation of diagnostic reagents for diagnosing coronary atherosclerosis; (3) Application in the preparation of diagnostic reagents for preliminary determination of the severity of coronary atherosclerosis; (4) Application in the preparation of diagnostic reagents for the prognosis evaluation of coronary atherosclerosis.

3. The use according to claim 2, characterized in that: The preparation is used to detect the expression level of circTMCC1 in PBMC cells of a subject, or a reagent for specifically identifying circTMCC1.

4. A marker for diagnosing coronary atherosclerosis, characterized in that: The marker is the circular RNA gene circTMCC1, and the nucleotide sequence is shown in SEQ ID NO.

1.

5. A diagnostic reagent for coronary atherosclerosis, characterized in that include: A preparation for detecting the expression amount of the circular RNA gene circTMCC1, wherein the nucleotide sequence of the circTMCC1 is shown in SEQ ID NO.

1.

6. A diagnostic kit for coronary atherosclerosis, characterized in that: The diagnostic kit comprises the diagnostic reagent according to claim 5.

7. The diagnostic kit according to claim 6, characterized in that: The diagnostic kit uses the following steps to perform the test: (1) Isolate PBMCs from the blood sample to be tested, extract RNA and place at -80°C; (2) The RNA obtained in step 1 was reverse transcribed to obtain cDNA, and the designed circTMCC1 amplification primers were added to perform qPCR experiments; (3) The amplified product obtained in step 2 was subjected to Sanger sequencing, RNaseR resistance, actinomycin D treatment, and agarose gel electrophoresis to verify the circularization of circTMCC1; (4) Based on the expression level of circTMCC1 in step 4 and that of the normal control group, analyze whether there is differential expression of the gene in the test sample.

8. The diagnostic kit according to claim 7, characterized in that: The amplification primers are specific primers, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.

3.

9. The diagnostic kit according to claim 7, characterized in that: The kit also includes a fluorescent dye and an amplification primer for specifically detecting the expression level of internal reference GAPDH.

10. The diagnostic kit according to claim 9, characterized in that: The fluorescent dye is ChamQ UniversalSYBR qPCR Master Mix, and the specific primers for GAPDH, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO.4, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.5.

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