Application of circular RNA circRftn1 in myocardial hypertrophy diagnosis

Abstract

The invention discloses application of circular RNA circRftn1 in diagnosis of cardiac hypertrophy. According to the application, research finds that the circular RNA circRftn1 is highly expressed in cardiac muscle tissues of a cardiac hypertrophy model mouse and is highly expressed in peripheral blood and cardiac muscle tissues of a cardiac hypertrophy patient; circRftn1 is a circular RNA (Ribonucleic Acid) which is found for the first time; the circular RNAcircRftn1 in the tail venous blood of the mouse can be used as a diagnostic marker of the cardiac hypertrophy of the mouse; the circular RNA circRftn1 in human peripheral blood can be used as a diagnostic marker of the human cardiac hypertrophy, and the circular RNA circRftn1 can be used for early large-scale screening of the human cardiac hypertrophy.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of circular RNA circRftn1 in diagnosis of cardiac hypertrophy. BACKGROUND

[0002] Pathological cardiac hypertrophy is an adverse reaction of myocardium to pathological pressure overload or neurohormonal stimulation, which reduces myocardial wall pressure by increasing myocardial wall thickness to maintain normal cardiac function. Pathological cardiac hypertrophy is often accompanied by adaptive cardiac remodeling, cardiac morphological changes and abnormal cardiac gene expression, and is a key inducible factor for the progression of diastolic heart failure (HFpEF). Long-term cardiac hypertrophy will develop into diastolic heart failure and even induce malignant arrhythmia to sudden death, which is a global public health problem with high morbidity and mortality.

[0003] At present, according to the diagnosis and treatment guidelines for cardiac hypertrophy published by the European Society of Cardiology (ESC) in 2014, the diagnostic criteria for cardiac hypertrophy are: any imaging (echocardiography, cardiac magnetic resonance imaging or computed tomography) detection results show that the left ventricular myocardial wall thickness of one or more segments is not completely caused by abnormal cardiac load and is greater than or equal to 14 mm; in children, the left ventricular wall thickness is greater than or equal to the predicted mean value + 2 x standard deviation; for the first-degree relatives of patients with cardiac hypertrophy, if the cardiac imaging detection results show that the left ventricular wall thickness of one or more segments is greater than or equal to 13 mm without other known causes, the patient can be diagnosed as having cardiac hypertrophy.

[0004] The precise diagnosis and / or differential diagnosis of cardiac hypertrophy mainly rely on imaging examination, which has the disadvantages of long time consumption, relatively high cost, inability to dynamically observe the development of the disease, and difficulty in being used for early large-scale screening. Therefore, it is of important clinical significance to seek new, reliable, non-invasive and rapid early large-scale screening methods for cardiac hypertrophy.

[0005] REFERENCES

[0006] 1. Mishra S, Kass DA. Cellular and molecular pathobiology of heart failure with preserved ejection fraction. Nat Rev Cardiol. Jun 2021; 18(6): 400-423. doi: 10.1038 / s41569-020-00480-6

[0007] 2. Heidenreich PA, Bozkurt B, Aguilar D, et al. 2022 AHA / ACC / HFSA Guideline for the Management of Heart Failure: A Report of the American College of Cardiology / American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation. May 3 2022; 145(18):e895-e1032. doi: 10.1161 / cir.0000000000001063

[0008] 3. McDonagh TA, Metra M, Adamo M, et al. 2021 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. Eur Heart J. Sep 21 2021; 42(36):3599-3726. doi: 10.1093 / eurheartj / ehab368

[0009] 4. Triposkiadis F, Xanthopoulos A, Butler J. Cardiovascular Aging and Heart Failure: JACC Review Topic of the Week. J Am Coll Cardiol. Aug 13 2019; 74(6):804-813. doi: 10.1016 / j.jacc.2019.06.053 SUMMARY

[0010] In view of the problems existing in the early large-scale screening of myocardial hypertrophy disease at the present stage, the expression of circular RNA in the myocardial tissue of a myocardial hypertrophy model mouse, the peripheral blood and myocardial tissue of a myocardial hypertrophy disease patient is researched, it is found that the circular RNA circRftn1 is highly expressed in the myocardial tissue of the myocardial hypertrophy model mouse, and is highly expressed in the peripheral blood and myocardial tissue of the myocardial hypertrophy disease patient, the circular RNA circRftn1 is a circular RNA found for the first time, it is researched and verified that the circular RNA circRftn1 in the tail vein blood of a mouse can be used as a diagnostic marker of a myocardial hypertrophy disease of the mouse, it is researched and verified that the circular RNA circRftn1 in the peripheral blood of a human can be used as a diagnostic marker of a myocardial hypertrophy disease of the human, and the circular RNA circRftn1 can be used for early large-scale screening of a myocardial hypertrophy disease of the human, in order to achieve the above purpose, the specific technical scheme of the present application is as follows:

[0011] The first aspect is to construct and verify a myocardial hypertrophy model of a mouse

[0012] A pressure overload-induced myocardial hypertrophy model is established by abdominal aortic constriction (AAC) operation, and a sham operation (sham) group is used as a control group.

[0013] Eight weeks after the AAC operation, the mice are subjected to echocardiography, the weight of the heart of the mice is examined, the histological analysis of the heart of the mice is performed, and the myocardial biomarkers are detected by Western blotting, all of which confirm that the myocardial hypertrophy mouse model is successfully constructed.

[0014] The second aspect is to identify the differentially expressed circRNA and target miRNA in the myocardial tissue of the myocardial hypertrophy model of a mouse

[0015] Among all the detected circRNAs, compared with the control group (sham operation group), the expression of 5 circular RNA molecules is increased in the myocardial hypertrophy model mouse, and the expression of 25 circular RNA molecules is reduced;

[0016] We particularly pay attention to 2 highest expression up-regulated circRNAs [circular RNA circRftn1 (i.e. novel_circRNA_0004641), novel_circ_000915] and 4 lowest expression down-regulated circRNAs (novel_circ_0001213, novel_circ_0002423, novel_circ_0003046 and novel_circ_0009685) for further research.

[0017] The third aspect is to investigate the expression of circRftn1 and other circular RNAs in the myocardial tissue of the myocardial hypertrophy model mouse

[0018] Real-time quantitative PCR (RT-qPCR) was used to verify the expression of circular RNAs in the myocardial tissue of mice with myocardial hypertrophy.

[0019] Results: The expression of circ_0004641 and circ_0009153 was increased, while the expression of circ_0003026 was decreased, and the expression of other circular RNAs had no significant difference.

[0020] Fourth aspect, the differentially expressed circular RNAs in the peripheral blood and myocardial tissue of patients with myocardial hypertrophy and healthy controls

[0021] Compared with the peripheral blood of healthy controls, the transcription level of circRNA_0004641 in the peripheral blood of patients with myocardial hypertrophy was significantly increased, and there was no significant difference in circ_0003046, circ_0002423, circ_0001213, circ_0009685 and circ_0009153.

[0022] Compared with the myocardial tissue samples of healthy controls, the transcription level of circRNA_0004641 in the myocardial samples of patients with myocardial hypertrophy was significantly increased, and there was no significant difference in circ_0003046, circ_0002423, circ_0001213, circ_0009685 and circ_0009153.

[0023] Fifth aspect, the diagnostic efficiency of circular RNA circRftn1 for myocardial hypertrophy in mice

[0024] Peripheral blood samples of 35 sham-operated C57BL / 6 mice and tail vein blood of 52 myocardial hypertrophy model mice were collected respectively, and the ROC-AUC of circular RNA circRftn1 for diagnosing myocardial hypertrophy model mice was 0.809 (95% CI: 0.718-0.899), the sensitivity was 0.790, and the specificity was 0.827; in addition, the tail vein blood of 15 sham-operated C57BL / 6 mice and 23 myocardial hypertrophy model mice were collected respectively, and the ROC-AUC of circular RNA circRftn1 for diagnosing myocardial hypertrophy model mice was 0.850 (95% CI: 0.735-0.965), the sensitivity was 0.800, and the specificity was 0.660; the results showed that the circular RNA circRftn1 in the tail vein blood of mice could be used as a diagnostic marker for myocardial hypertrophy in mice.

[0025] Sixth aspect, the diagnostic efficiency of circular RNA circRftn1 for human myocardial hypertrophy

[0026] The ROC-AUC of circRftnl in the peripheral blood of 30 healthy controls and 30 patients with myocardial hypertrophy is 0.916 (95% CI: 0.843-0.988), the sensitivity is 0.880, and the specificity is 0.926; in addition, the peripheral blood of 20 healthy controls and 20 patients with myocardial hypertrophy is collected respectively, and the ROC-AUC of circRftnl in the peripheral blood of patients with myocardial hypertrophy is 0.918 (95% CI: 0.835-1.000), the sensitivity is 0.780, and the specificity is 0.950; the results show that circRftnl in the peripheral blood of human can be used as a diagnostic marker for human myocardial hypertrophy.

[0027] Compared with the prior art, the beneficial effects of the present application are:

[0028] 1. The present application first discovers that circRftnl is highly expressed in the myocardial tissue of a myocardial hypertrophy model mouse, and is highly expressed in the peripheral blood and myocardial tissue of a patient with myocardial hypertrophy;

[0029] 2. circRftnl is a newly discovered circular RNA;

[0030] 3. It is first discovered that circRftnl in the tail vein blood of a mouse can be used as a diagnostic marker for a mouse myocardial hypertrophy disease, and circRftnl in the peripheral blood of a human can be used as a diagnostic marker for a human myocardial hypertrophy disease. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 , myocardial hypertrophy mouse modeling evaluation, wherein,

[0032] A is the HE, Congo red and WGA staining of the myocardial cross section of the left ventricle of the model group (Model group) and the sham operation group (Sham group) mice, and the enlarged view of the HE staining of the myocardial cross section,

[0033] B is a graph of the left ventricular posterior wall thickness (LVPW) of the Model group and the Sham group by ultrasonic examination,

[0034] C is a statistical graph of the left ventricular posterior wall thickness (LVPW) of the Model group and the Sham group by ultrasonic examination, the left is a statistical graph of the left ventricular end-systolic diameter (LVID), and the right is a statistical graph of the interventricular septal thickness (IVS),

[0035] D is a statistical graph of the ejection fraction (EF) of the Model group and the Sham group by ultrasonic examination,

[0036] E is a statistical graph of the cardiac output (CO) of the Model group and the Sham group by ultrasonic examination,

[0037] F is a graph of Western blotting detecting myocardial biomarkers β-actin heavy chain (β-MHC) and atrial natriuretic peptide (ANP) of Model group and Sham group,

[0038] G is a statistical graph of Western blotting detecting myocardial biomarkers β-MHC (left) and ANP (right) of Model group and Sham group,

[0039] Figure 2 , circRNA classification of Model group and Sham group, wherein,

[0040] A is a statistical graph of circRNA classification,

[0041] B is a volcano plot of circRNA classification,

[0042] C is a graph of circRNA distribution on chromosomes;

[0043] Figure 3 , circRNAs with differential expression in Model group compared with Sham group, wherein, there are 5 circRNAs with increased expression and 25 circRNAs with decreased expression;

[0044] Figure 4 , 5 Expression regulation and genomic information of 5 circRNAs with increased expression and 25 circRNAs with decreased expression;

[0045] Figure 5 , CeRNA interaction network and circ-miR interaction prediction group;

[0046] Figure 6 , Gene ontology (GO) analysis suggests that biological processes are enriched in transferase activity and ubiquitination;

[0047] Figure 7 , KEGG signaling pathway graph;

[0048] Figure 8 , The up-regulated genes and down-regulated genes in the sequencing results were verified by real-time fluorescence quantitative PCR (RT-qPCR) to verify the differences between Model group and Sham group, wherein,

[0049] A is the difference of up-regulated circRNAs circ_0004641 and circ_0009153 between Model group and Sham group,

[0050] B is the difference of down-regulated genes circ_0003046 and circ_0002423 between Model group and Sham group,

[0051] C represents the difference in expression of downregulated genes circ_0001213 and circ_0009685 between the Model group and the Sham group;

[0052] Figure 9 qPCR replication validation results of circular RNA in the same batch of mouse ventricular tissue that did not undergo circRNA sequencing;

[0053] Figure 10 Comparison of transcription levels of six circular RNAs in peripheral blood of patients with hypertrophic cardiomyopathy and healthy controls;

[0054] Figure 11 Comparison of transcription levels of six circular RNAs in the myocardium of patients with hypertrophic cardiomyopathy and normal individuals (healthy controls);

[0055] Figure 12 ROC curve of circular RNA circRftn1 in mouse tail vein blood for diagnosing hypertrophic cardiomyopathy in mice;

[0056] Figure 13 1. Verify the ROC of circular RNA circRftn1 in mouse tail vein blood for diagnosing hypertrophic cardiomyopathy in mice;

[0057] Figure 14 The ROC curve of circular RNA circRftn1 in human peripheral blood for diagnosing hypertrophic cardiomyopathy;

[0058] Figure 15 To verify the ROC of circular RNA circRftn1 in human peripheral blood for diagnosing human hypertrophic cardiomyopathy.

[0059] in Figures 1-15 middle,

[0060] *, **, ***, and **** represent P<0.05, P<0.01, P<0.001, and P<0.0001, respectively, indicating that the difference between the two groups is statistically significant. Detailed Implementation

[0061] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present application in any way.

[0062] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0063] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0064] Main reagents and materials

[0065]

[0066]

[0067]

[0068] Main instruments

[0069]

[0070] Example 1: Establishing a mouse model of myocardial hypertrophy through abdominal aortic coarctation surgery.

[0071] SPF C57BL / 6 mice (male, 6-8 weeks old, 20-22g). An average of 8 mice were used in the model group and the sham-operated group to establish a pressure overload-induced myocardial hypertrophy model through abdominal aortic coarctation (AAC) surgery.

[0072] AAC surgical plan:

[0073] Mice were anesthetized with 2.5% isoflurane gas followed by tracheal ventilation to maintain 1% isoflurane anesthesia. The mice were then fixed to a 37°C constant-temperature operating table, and the abdominal aorta was exposed by a midline abdominal incision. The vessel was bluntly dissected, narrowed using a 27mm needle and 5-0 suture, and then sutured. Control group mice underwent a similar "sham surgery"—a midline abdominal incision without vessel narrowing. Postoperatively, the incision site was closely monitored to assess for factors such as infection and bleeding that could lead to modeling failure.

[0074] Example 2: Validation of the constructed mouse myocardial hypertrophy model

[0075] I. Methods

[0076] (I) Echocardiography

[0077] Mice were fed a normal homeothermic diet and were evaluated by echocardiography every two weeks. Mice (2% isoflurane anesthesia followed by 0.5% maintenance) were examined by an echocardiogram in a supine position using a Vevo 2100 (Canada).

[0078] Eight weeks after AAC surgery, echocardiography was performed on mice using a Vevo 2100 imaging system. Mice were anesthetized, and left ventricular parameters were measured during diastole and systole. These indices in supplement 2 were statistically analyzed using Prisma 9 software.

[0079] Two-dimensional ultrasound parameters of the left ventricle during systole and diastole were calculated using the Simpson algorithm. The main parameters are as follows:

[0080] Left ventricular posterior wall thickness, end-diastolic (LVPW; d)

[0081] Left ventricular posterior wall thickness, end-systolic (LVPW; s)

[0082] End-diastolic interventricular septal thickness (IVS; d)

[0083] End-systolic interventricular septal thickness (IVS s)

[0084] Left ventricular internal diameter at end-diastolic (LVID; d)

[0085] Left ventricular internal diameter at the end of systole (LVID; s)

[0086] Left ventricular end-diastolic volume (LV Vol; d)

[0087] Left ventricular end-systolic volume (LV Vol; s)

[0088] Left ventricular mass (LV Mass)

[0089] Ejection Fraction (EF)

[0090] Fraction of shortening (FS)

[0091] Cardiac Output (CO)

[0092] Cardiac output = (Left ventricular end-diastolic volume - Left ventricular end-systolic volume) × Heart rate

[0093] (II) Heart Weight Examination

[0094] Mice were euthanized by cervical dislocation after echocardiography, and their hearts were removed and weighed.

[0095] (III) Histological Analysis

[0096] Hearts were removed, fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. The paraffin blocks were cut into 4–5 μm thick sections. Subsequently, the crossed heart sections were stained with hematoxylin-eosin (HE) and thionine to assess heart size and morphological changes. Cardiac cell boundaries were delineated using 1:500 FITC-linked wheat germ lectin (WGA), and nuclei were labeled with DAPI. Staining images were captured using a light microscope (Leica).

[0097] (iv) Western blot method for detecting myocardial biomarkers

[0098] Total protein was isolated and quantified using the ImageQuant LAS 4000 kit. A 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel was prepared, and 40 μg of protein was loaded for electrophoresis. The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in 5% skim milk at room temperature for 2 hours, and then incubated overnight with primary antibodies β-MHC (1:1,000), ANP (1:2,000), and GAPDH (1:10,000), followed by incubation with rabbit secondary antibody (1:5,000) for another 2 hours. Images were captured using a fluorescence imaging system, and the data were analyzed using ImageJ software.

[0099] II. Results

[0100] (a) such as Figure 1 As shown in Figure A, HE, Congo red, and WGA staining revealed that, compared with the sham-operated group, the AAC-operated group of mice had an increased myocardial cross-sectional area in the left ventricle and increased myocardial hypertrophy.

[0101] (ii) Ultrasound Figure 1 As shown in BE, compared with the sham group, the AAC group mice had significantly increased left ventricular posterior wall thickness (LVPW), left ventricular end-systolic diameter (LVID), interventricular septal thickness (IVS), and left ventricular volume during systole and diastole, and significantly decreased ejection fraction (EF) and cardiac output (CO).

[0102] (III) Results of Western blot analysis for detecting myocardial biomarkers, such as Figure 1As shown in FG: Compared with the sham-operated group, the expression of β-actin heavy chain (β-MHC) and atrial natriuretic peptide (ANP) was increased in the hearts of mice in the AAC-operated group.

[0103] III. Conclusion

[0104] Based on mouse cardiac histochemical staining, echocardiographic assessment, and combined quantitative expression analysis of myocardial protein peptides, the mouse model of myocardial hypertrophy after AAC surgery was successfully established.

[0105] Example 3: Identification of differentially expressed circRNAs and target miRNAs in myocardial tissue of sham-operated mice and myocardial hypertrophy model mice.

[0106] I. Methods

[0107] A total of 8 samples (4 mice in each group) from the sham-operated group and the hypertrophic cardiomyopathy model group were subjected to circRNA sequencing. The experimental methods for constructing the animal models in the sham-operated group and the hypertrophic cardiomyopathy model group are as described in Example 1.

[0108] (I) RNA extraction, library preparation and circular RNA sequencing

[0109] Total RNA was isolated from left ventricular tissue of mice in the sham-operated group and the hypertrophic cardiomyopathy model group using Trizol reagent. The specific method was as follows: After thoroughly grinding the tissue sample, 1 mL of Trizol reagent was added, and the mixture was vortexed vigorously for 15 seconds, then incubated at room temperature for 5 min; centrifuged at 12000 rpm for 15 min at 4°C; 500 μL of isopropanol was added, and the mixture was inverted and mixed, then incubated at room temperature for 10 min; centrifuged at 12000 rpm for 10 min at 4°C; the supernatant was discarded; 75% ethanol was prepared using RNAase-free ddH2O and pre-cooled at 4°C; 1 mL of 75% ethanol was added to an EP tube, and the mixture was inverted and mixed; centrifuged at 12000 rpm for 15 min at 4°C; the supernatant was discarded, and the mixture was incubated at room temperature for 5 min; 12 μL of RNAase-free water was added to dissolve the precipitate to obtain total RNA. RNA integrity was assessed using the RNA Nano 6000 analysis kit on a Bioanalyzer 2100 system before library preparation. Qualified RNA samples were desorbed for ribosomal RNA (rRNA) using the Ribo-Zero™ Gold kit. Follow the manufacturer's recommendations. Ultra™ RNA Library Preparation Kit Sequencing library.

[0110] The prepared library was sequenced on the Illumina HiSeq 4000 platform, producing 125bp / 150bp paired end reads.

[0111] (II) Circular RNA Identification and Target Prediction

[0112] 1. To ensure the quality and reliability of data analysis, the raw sequencing data was filtered.

[0113] (1) Remove readings with sequence adaptors;

[0114] (2) Remove readings with more than 10% content, where N (N indicates that the base information cannot be determined);

[0115] (3) Remove low-quality readings with Q_phred <= 5, which account for more than 50% of the total reading length.

[0116] 2. The Hisat2 algorithm is used to accurately align clean reads with the reference genome and obtain location information.

[0117] 3. The Find_Circle algorithm and CIRI algorithm were used to identify candidate circular RNAs, and the TMM algorithm was applied to standardize the reading data before differential analysis.

[0118] 4. Two levels of differentially expressed circular RNA were screened using volcano plot visualization: fold change and corrected significance level (p_adj / p_value), to infer the expression of differentially expressed circular RNA.

[0119] 5. Based on the correspondence between circular RNA and parental genes, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the parental genes with differential expression of circular RNA in each group.

[0120] 6. Using miRanda software, we analyzed the miRNA binding sites targeted by the identified circular RNAs to further investigate their functions. Based on the targeted miRNAs, we used miRDB software to predict downstream mRNAs.

[0121] II. Results

[0122] Ultimately, 9780 circRNAs were identified in both the sham surgery group and the myocardial hypertrophy model group.

[0123] Based on their location in the host genome, these circRNAs are divided into three categories:

[0124] like Figure 2As shown in Figure A, in the sham surgery group, 76.08% were exonic, 18.71% were intronic, and 5.23% were intergenic circular molecules; in the myocardial hypertrophy model group, 76.08% were exonic, 18.71% were intronic, and 5.23% were intergenic circular molecules.

[0125] like Figure 2 As shown in B, the length of most circRNAs in both groups ranged from 31 to 1970 bp;

[0126] like Figure 2 As shown in Figure C, 9780 circRNAs are distributed across the entire chromosome.

[0127] like Figure 3 As shown, among all the detected circRNAs, 5 molecules showed increased expression in the mouse myocardial hypertrophy model, and 25 molecules showed decreased expression (based on thresholds p_adj <= 0.05 and log2FoldChange >= 1.0). These included a known circRNA (circRNA_000121) and an unknown molecule such as circRftn1, a circular RNA with a circBase ID of hsa_circ_0004641, hereinafter referred to as circRftn1 or circRNA_0004641.

[0128] circRNA_0004641 is a newly discovered circular RNA, spliced ​​end to end, and its sequence is shown in SEQ ID NO:1.

[0129] like Figure 4 As shown, based on the expression regulation of the 5 upregulated molecules and 25 downregulated molecules detected, as well as genomic information, and according to the P-value and the new expression levels, we particularly focused on the 2 most upregulated circRNAs (novel_circRNA_0004641, novel_circRNA_0009153) and the 4 least downregulated circRNAs (novel_circRNA_0001213, novel_circRNA_0002423, novel_circRNA_0003046, and novel_circRNA_0009685) for further research.

[0130] like Figure 5 The diagram shown is a circRNA-miRNA interaction diagram.

[0131] like Figure 6As shown, gene ontology (GO) analysis suggests that biological processes are enriched in transferase activity and ubiquitination;

[0132] like Figure 7 As shown, pathway analysis by the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that functional circular RNAs were mainly enriched in transcription factors, the MAPK signaling pathway, the Apelin signaling pathway, and genomic systems.

[0133] Example 4: Real-time quantitative PCR (RT-qPCR) to verify the expression level of circular RNA molecules in the remaining myocardial tissue.

[0134] I. Methods

[0135] Myocardial tissue was ground, and total RNA was extracted using Trizol reagent (see Example 3 for specific method). RNA was quantified using a micro-UV-Vis spectrophotometer. 1 μL of RNA was added to the instrument probe, the instrument was turned off, and the measurement was initiated to obtain the RNA concentration. The total RNA volume was calculated by multiplying the RNA concentration by the RNA volume. 2000 ng of RNA was reverse transcribed into cDNA using the Tiangen reverse transcription kit (FastKing gDNA Dispelling RT SuperMix FastKing one-step method for removing genomic cDNA) according to its instructions. cDNA, qPCR primers, 2x SuperReal PreMixPlus reagent [Tiangen's SuperReal PreMix Plus (SYBR Green) SuperReal fluorescent quantitative premixed reagent], and RNase-free dd H2O were added to a 96-well plate. The 96-well plate was placed in the qPCR instrument, parameters were set, and the instrument was started. After the reaction, the melting and amplification curves were analyzed using the corresponding instrument software, and the results were calculated using the 2-ΔΔCt method.

[0136] The nucleotide sequences of the qPCR primers for the circular RNA circRftn1 (circ_0004641) are shown in the table below.

[0137]

[0138] II. Results

[0139] like Figure 8 As shown in A, among the genes upregulated in the sequencing results, the expression of circRNA_0004641 increased, while circRNA_0009153 showed no significant difference.

[0140] like Figure 8As shown in B, among the downregulated genes in the sequencing results, circRNA_0002423 and circRNA_0003046 showed higher expression.

[0141] like Figure 8 As shown in C, there was no significant difference in expression between circRNA_0001213 and circRNA_0009685.

[0142] III. Conclusion

[0143] We extracted nuclei from the remaining mouse myocardial tissue samples submitted for sequencing and validated the results using RT-qPCR. The results showed that the increased expression of circRNA_0004641 among the differentially expressed circRNA molecules was consistent with the sequencing results; no significant differences were observed in circRNA_0001213 and circRNA_0009658; while the expression of circRNA_0002423 and circRNA_0003046 was contrary to the sequencing results.

[0144] Therefore, we further performed a replication validation experiment on the same batch of mouse ventricular tissue that was not submitted for circRNA sequencing (see Example 5).

[0145] Example 5: Replication validation using the same batch of mouse ventricular tissue without circRNA sequencing

[0146] I. Methods

[0147] See Example 4

[0148] II. Results

[0149] like Figure 9 As shown, in mice from the same batch that were not sent for sequencing, qPCR results showed that the transcriptional expression of circRNA_0004641 and circRNA_0009153 was increased, while the expression of circRNA_0009685 was decreased, and there was no significant difference in the expression of other circular molecules.

[0150] Example 6: Differences in circular RNA expression in peripheral blood samples from healthy individuals (healthy controls) and patients with hypertrophic cardiomyopathy.

[0151] I. Methods

[0152] The samples consisted of peripheral blood samples from 30 healthy individuals (health controls) and 30 patients with hypertrophic cardiomyopathy. Real-time quantitative PCR (RT-qPCR) was used, as described in Example 4.

[0153] II. Results

[0154] like Figure 10As shown, compared with the peripheral blood of normal individuals (healthy controls), the transcription level of circRNA_0004641 in the peripheral blood of patients with hypertrophic cardiomyopathy was significantly increased, while there were no significant differences in circRNA_0003046, circRNA_0002423, circRNA_0001213, circRNA_0009685, and circRNA_0009153.

[0155] III. Conclusion

[0156] Compared with normal peripheral blood, the transcription level of circRNA_0004641 in the peripheral blood of patients with hypertrophic cardiomyopathy was significantly increased.

[0157] Example 7: Differences in circular RNA expression in myocardial tissue between normal individuals (healthy controls) and patients with hypertrophic cardiomyopathy

[0158] I. Methods

[0159] The samples consisted of myocardial tissue from 5 healthy individuals (healthy controls) and 5 patients with hypertrophic cardiomyopathy. Real-time quantitative PCR (RT-qPCR) was used, as described in Example 4.

[0160] II. Results

[0161] like Figure 11 As shown, compared with normal human (healthy control) myocardial samples, the transcription level of circRNA_0004641 was significantly increased in myocardial samples from patients with hypertrophic cardiomyopathy, while there were no significant differences in circRNA_0003046, circRNA_0002423, circRNA_0001213, circRNA_0009685, and circRNA_0009153.

[0162] III. Conclusion

[0163] Compared with normal human (healthy controls) myocardial tissue samples, the transcription level of circRNA_0004641 was significantly increased in myocardial tissue samples from patients with hypertrophic cardiomyopathy.

[0164] Example 8: Diagnostic efficacy of circular RNA circRftn1 in mouse hypertrophic cardiomyopathy I. Sample collection

[0165] Blood samples (300 μL each) from the tail veins of 35 sham-operated C57BL / 6 mice and 52 mice with myocardial hypertrophy were collected, anticoagulated with EDTA, and stored frozen at -80°C. The myocardial hypertrophy model mice were confirmed by cardiac histochemical staining, echocardiography, and quantitative analysis of myocardial protein peptide expression.

[0166] II. Detection of circular RNA circRftn1

[0167] (I) Extraction of total RNA

[0168] After thawing the blood sample on ice, take 100-250 μL of whole blood and add it to a new RNase-free centrifuge tube. Add 3 times the blood volume of Trizol reagent, vortex to mix evenly, and then extract total RNA. For other details, see Example 3.

[0169] (II) Quality analysis, reverse transcription, and qPCR amplification of RNA samples

[0170] The method is described in Example 4.

[0171] III. Diagnostic efficacy of circular RNA circRftn1 in mouse hypertrophic cardiomyopathy

[0172] (I) ROC Curve

[0173] 1. Plotting ROC curves: Using Graphpad Prism 8.0 software, input the relative expression level of circular RNA circRftn1 in each sample to plot the ROC curve of circular RNA circRftn1 recognizing myocardial hypertrophy;

[0174] 2. Calculate AUC: Based on AUC, obtain the optimal threshold and the corresponding sensitivity and specificity, so as to evaluate the recognition ability of circular RNA circRftn1 on hypertrophic cardiomyopathy model mice.

[0175] 3. We also obtained the optimal relative expression threshold of circular RNA circRftn1 for recognizing hypertrophic cardiomyopathy model mice, as well as the sensitivity and specificity at this threshold.

[0176] (II) Results

[0177] like Figure 12 As shown, the ROC-AUC of circular RNA circRftn1 in mouse tail vein blood for diagnosing hypertrophic cardiomyopathy in mice was 0.809 (95% CI: 0.718-0.899), with a sensitivity of 0.790 and a specificity of 0.827.

[0178] Example 9: Verification of the diagnostic efficacy of circular RNA circRftn1 for hypertrophic cardiomyopathy in mice.

[0179] I. Sample Collection

[0180] In addition, 300 μL of tail vein blood samples were collected from 15 sham-operated C57BL / 6 mice and 23 mice with myocardial hypertrophy models, respectively. The samples were anticoagulated with EDTA and stored frozen at -80°C. The myocardial hypertrophy models were confirmed by cardiac histochemical staining, echocardiography, and quantitative expression analysis of myocardial protein peptides.

[0181] II. Detection of circular RNA circRftn1

[0182] (I) Extraction of total RNA

[0183] Same as Example 8

[0184] (II) Quality analysis, reverse transcription, and qPCR amplification of RNA samples

[0185] Same as Example 8.

[0186] III. Verification of the diagnostic efficacy of circular RNA circRftn1 for hypertrophic cardiomyopathy in mice.

[0187] (I) ROC Curve

[0188] Same as Example 8.

[0189] (II) Results

[0190] like Figure 13 As shown, the ROC-AUC of circular RNA circRftn1 in mouse tail vein blood for diagnosing hypertrophic cardiomyopathy in mice was 0.850 (95% CI: 0.735-0.965), with a sensitivity of 0.800 and a specificity of 0.660.

[0191] The results of Examples 8 and 9 show that the circular RNA circRftn1 in mouse tail vein blood has high accuracy in diagnosing a mouse model of myocardial hypertrophy, and that the circular RNA circRftn1 in mouse tail vein blood can be used as a diagnostic marker for a mouse model of myocardial hypertrophy.

[0192] Example 10: Diagnostic efficacy of circular RNA circRftn1 in patients with myocardial hypertrophy

[0193] I. Sample Collection, Processing and Preservation

[0194] Peripheral blood samples (3 mL each) were collected from 30 healthy controls and 30 patients with hypertrophic cardiomyopathy. The samples were anticoagulated with EDTA and stored frozen at -80°C. Informed consent was obtained from all participants. All patients with hypertrophic cardiomyopathy underwent echocardiography and were clinically diagnosed. All samples were obtained with the approval of the organization's ethics committee.

[0195] The inclusion and exclusion criteria for the hypertrophic cardiomyopathy group are as follows:

[0196] Inclusion criteria: Interventricular septum or left ventricular wall thickness ≥14mm, or interventricular septum to left ventricular posterior wall ratio ≥1:3, as measured by echocardiography.

[0197] Exclusion criteria: patients with congenital heart disease, patients who have undergone surgery for congenital heart disease, patients with amyloid heart disease, patients with rheumatic heart disease, patients with pacemaker implantation, patients with atrioventricular block, and patients with secondary ventricular hypertrophy caused by a history of hypertensive pre-excitation syndrome, etc.

[0198] II. Detection of circular RNA circRftn1

[0199] (I) Extraction of total RNA

[0200] After thawing the blood sample on ice, take 100-250 μL of whole blood and add it to a new RNase-free centrifuge tube. Add 3 times the blood volume of Trizol reagent, vortex to mix evenly, and form uniform lysis. For other total NRA extraction methods, see Example 3.

[0201] (II) Quality analysis, reverse transcription, and qPCR amplification of RNA samples

[0202] The method is described in Example 4.

[0203] III. Diagnostic efficacy of circular RNA circRftn1 in human hypertrophic cardiomyopathy

[0204] (I) ROC

[0205] The method is described in Example 8.

[0206] (II) Results

[0207] like Figure 14 As shown, the ROC-AUC of circular RNA circRftn1 in human peripheral blood for diagnosing hypertrophic cardiomyopathy was 0.916 (95% CI: 0.843-0.988), with a sensitivity of 0.880 and a specificity of 0.926.

[0208] Example 11: Verifying the diagnostic efficacy of circRNA_0004641 in patients with myocardial hypertrophy I. Sample collection, processing and preservation

[0209] In addition, 3 mL of peripheral blood samples were collected from 20 healthy controls and 20 patients with myocardial hypertrophy, and the rest was the same as in Example 10.

[0210] II. Detection of circular RNA circRftn1

[0211] (I) Extraction of total RNA

[0212] Same as in Example 10.

[0213] (II) Quality analysis, reverse transcription, and qPCR amplification of RNA samples

[0214] Same as in Example 10.

[0215] III. Verifying the diagnostic efficacy of circular RNA circRftn1 in human hypertrophic cardiomyopathy

[0216] (I) ROC

[0217] Same as Example 8.

[0218] (II) Results

[0219] like Figure 15 As shown, the ROC-AUC of circular RNA circRftn1 in human peripheral blood for diagnosing hypertrophic cardiomyopathy was 0.918 (95% CI: 0.835-1.000), with a sensitivity of 0.780 and a specificity of 0.950.

[0220] The results of Examples 10 and 11 indicate that circRNA_0004641 in human peripheral blood has high diagnostic accuracy for hypertrophic cardiomyopathy, and circRNA_0004641 can serve as a diagnostic biomarker for hypertrophic cardiomyopathy in humans.

[0221] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

[0222] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The use of a circular RNA circRftn1 in the preparation of a human / mouse myocardial hypertrophy disease diagnosis product, characterized in that, The circBase ID of the circular RNA circRftn1 is hsa_circ_0004641, and the nucleotide sequence is shown as SEQ ID NO:

1.

2. Use according to claim 1, wherein The transcription level of the circular RNA circRftn1 is increased in the peripheral blood sample of a patient with cardiomegaly compared with a healthy control.

3. The use according to claim 1, wherein The transcription level of the circular RNA circRftn1 is increased in the myocardial tissue of a patient with cardiomegaly / mouse with cardiomegaly compared with a healthy control / cardiomegaly mouse.

4. The use according to claim 1, wherein The detection reagent of the circular RNA circRftn1 comprises total RNA extraction reagent, reverse transcription reagent, qPCR primer, and fluorescent quantitative premix reagent.

5. The use according to claim 4, wherein the compound is ###0002### The nucleotide sequence of the qPCR primer is shown as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO:

7.

6. Use according to any one of claims 1 or 4, wherein The product comprises a kit, a chip, and a test paper.

7. A product for the diagnosis of a human cardiac hypertrophy disease, characterized by, The product comprises a reagent for detecting the expression of the circular RNA circRftn1 in a sample, wherein the sample is peripheral blood of a human, the circBase ID of the circular RNA circRftn1 is hsa_circ_0004641, and the nucleotide sequence is shown as SEQ ID NO: 1.

Images