CircRNA marker and application thereof in preparation of diagnostic kit and therapeutic drug for myocardial fibrosis after myocardial infarction

Abstract

The invention relates to a circRNA marker and application of the circRNA marker in preparation of a diagnostic kit and a therapeutic drug for myocardial fibrosis after myocardial infarction, and belongs to the technical field of biological medicine. In order to solve the problem that a sensitive circRNA marker for diagnosing myocardial infarction related diseases is lacked, the invention provides a circRNA biomarker circ-CELF1, the nucleotide sequence of the circRNA biomarker circ-CELF1 is as shown in SEQ ID NO.1, and the expression of the circRNA biomarker circ-CELF1 in myocardial infarction mouse heart tissues and human cardiac fibroblasts treated by transforming growth factor-beta1 is remarkably up-regulated. The circ-CELF1 can be used as a novel biomarker for clinical diagnosis of myocardial fibrosis. By knocking down the circ-CELF1, expression of MI and TGF-beta1 induced myocardial fibrosis markers can be remarkably reduced, myocardial fibrosis is improved, and it is prompted that the medicine taking circ-CELF1 as the target spot can inhibit myocardial fibrosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a circRNA marker and application thereof in preparation of a myocardial fibrosis diagnosis kit after myocardial infarction and a therapeutic drug. BACKGROUND

[0002] Myocardial infarction (MI) refers to a process that ischemic necrosis occurs in a corresponding myocardial region due to a sharp decrease or interruption of coronary artery blood flow. Myocardial fibrosis plays an important role in the occurrence and development of myocardial infarction. When myocardial fibroblasts are damaged or subjected to inflammatory stress, collagen expression significantly increases, which can initially enhance the integrity of the tissue and help stabilize the heart function, but sustained stimulation leads to a large number of proliferation of myocardial fibroblasts and transformation into myofibroblasts, accompanied by excessive deposition of extracellular matrix (ECM) in the heart, which reduces the compliance of the heart tissue and promotes heart dysfunction, and finally, causes heart failure. The pathological mechanism of myocardial fibrosis is complex and involves the participation of multiple regulatory factors. At present, the research field still lacks in-depth understanding of effective intervention molecules and key targets for myocardial fibrosis.

[0003] CircRNAs are a newly discovered class of non-coding RNAs, which are formed by exon or intron circularization, have a covalent closed loop structure, do not have 5' cap and 3' poly(A) tail structure, have high conservation, and have expression specificity of species, tissue, disease and developmental stage. CircRNAs have a variety of important biological functions, such as acting as a “sponge” for miRNA, interacting with proteins, regulating gene splicing or transcription, and epigenetic regulation, etc. At present, circRNAs have been found to have potential value as biomarkers in a variety of diseases. Studies have shown that circRNAs have certain correlation with the occurrence and development of myocardial fibrosis, however, the specific regulatory mechanism of a large number of circRNAs in cardiac fibrosis after myocardial infarction is still in the initial research stage and needs to be further clarified, and there is a lack of sensitive circRNA markers for diagnosing myocardial infarction related diseases. SUMMARY

[0004] In order to solve the problem of lacking sensitive circRNA markers for diagnosing myocardial infarction related diseases, the application provides a circRNA marker and application thereof in preparation of a myocardial fibrosis diagnosis kit after myocardial infarction and a therapeutic drug.

[0005] The technical scheme of the application is as follows:

[0006] A circRNA biomarker circ-CELF1, the nucleotide sequence of the circ-CELF1 is shown as SEQ ID NO. 1.

[0007] A use of a circRNA biomarker circ-CELF1 in preparing a diagnostic kit for myocardial fibrosis after myocardial infarction, wherein the diagnostic kit contains reverse transcription quantitative PCR primers that specifically amplify the circ-CELF1 backsplicing junction as claimed in claim 1.

[0008] Furthermore, the nucleotide sequence of the upstream primer of the PCR primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.3.

[0009] A diagnostic kit for myocardial fibrosis after myocardial infarction, comprising reverse transcription quantitative PCR primers for specifically amplifying the circ-CELF1 reverse splicing junction as described in claim 1, wherein the upstream primer nucleotide sequence of the PCR primer is shown in SEQ ID NO. 2, and the downstream primer nucleotide sequence is shown in SEQ ID NO. 3.

[0010] A use of a circRNA biomarker circ-CELF1 in the preparation of a drug for treating myocardial fibrosis after myocardial infarction, wherein the drug contains a nucleic acid fragment or a recombinant vector that inhibits the expression of circ-CELF1 as claimed in claim 1 or reduces its activity.

[0011] Furthermore, the nucleic acid fragment is a circ-CELF1 interferor, and the circ-CELF1 interferor is an siRNA that inhibits the back-splicing and cyclization of circ-CELF1 according to claim 1, and the nucleotide sequences of the sense chain and antisense chain of the siRNA are shown as SEQ ID NO.4 and SEQ ID NO.5, respectively, or the nucleotide sequences of the sense chain and antisense chain of the siRNA are shown as SEQ ID NO.6 and SEQ ID NO.7, respectively, or the nucleotide sequences of the sense chain and antisense chain of the siRNA are shown as SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0012] Furthermore, the recombinant vector is a viral packaging plasmid containing shRNA that inhibits the reverse splicing and circularization of circ-CELF1 as claimed in claim 1.

[0013] Furthermore, the virus is an adeno-associated virus AAV9 containing a cardiac fibroblast-specific periostin core promoter.

[0014] A drug for treating myocardial fibrosis after myocardial infarction, comprising a nucleic acid fragment or a recombinant vector that inhibits the expression of circ-CELF1 or reduces its activity as claimed in claim 1.

[0015] Furthermore, the nucleic acid fragment is a circ-CELF1 interferor, and the circ-CELF1 interferor is an siRNA that inhibits the back-splicing and cyclization of circ-CELF1 according to claim 1, and the nucleotide sequences of the sense chain and antisense chain of the siRNA are shown as SEQ ID NO.4 and SEQ ID NO.5, respectively, or the nucleotide sequences of the sense chain and antisense chain of the siRNA are shown as SEQ ID NO.6 and SEQ ID NO.7, respectively, or the nucleotide sequences of the sense chain and antisense chain of the siRNA are shown as SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0016] Beneficial effects of the present invention:

[0017] This study successfully identified a unique circular circRNA, named circ-CELF1, which was significantly upregulated in the heart tissue of mice with myocardial infarction and in cardiac fibroblasts treated with transforming growth factor-β1, suggesting that circ-CELF1 can serve as a new biomarker for the clinical diagnosis of myocardial fibrosis.

[0018] Knockdown of circ-CELF1 significantly reduced the expression of myocardial fibrosis markers induced by MI and TGF-β1, improving myocardial fibrosis. This suggests that drugs targeting circ-CELF1 could inhibit the development of myocardial fibrosis, providing an effective molecular target for drug development. Furthermore, circ-CELF1 is highly conserved at 91.6%, and its human conserved circular RNA (hsa-circ-CELF1) is significantly upregulated in TGF-β1-induced human cardiac fibroblasts (HCFs). Knockdown of hsa-circ-CELF1 significantly inhibited TGF-β1-induced expression of myocardial fibrosis markers and proliferation of human cardiac fibroblasts, suggesting that this circular RNA could serve as a new biomarker for the clinical diagnosis of myocardial fibrosis. Drugs targeting this circular RNA could be used as anti-myocardial fibrosis drugs, with potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the screening process of circ-CELF1 in the circAtlas database in Example 1;

[0020] Figure 2 This is a comparison chart of the level changes of circRNAs detected by qPCR at 1 day, 1 week, and 4 weeks after myocardial infarction (MI) in Example 1;

[0021] Figure 3Figure 2A. Comparison of the expression levels of 5 selected circRNAs in TGF-β1 -treated cardiac fibroblasts (CFs) detected in Example 1;

[0022] Figure 4 Figure 2B. Schematic diagram of circ-CELF1 genomic information in Example 2;

[0023] Figure 5 Figure 2C. Comparison of the expression changes of circ-CELF1 and linear Celf1 mRNA levels in CFs after RNase R treatment in Example 2;

[0024] Figure 6 Figure 2D. Comparison of the relative expression levels of circ-CELF1 in different tissues of mice in Example 2;

[0025] Figure 7 Figure 2E. Comparison of the expression levels of circ-CELF1 in cardiac fibroblasts (CFs) and cardiomyocytes (CMs) detected by qPCR in Example 2;

[0026] Figure 8 Figure 2F. Comparison of the expression levels of circ-CELF1 in mouse plasma in Example 2;

[0027] Figure 9 Figure 2G. Comparison of the subcellular localization of circ-CELF1 in CFs after cytoplasm / nucleus separation verified by qPCR in Example 2;

[0028] Figure 10 Figure 3A. Echocardiogram of each group of mice in Example 3;

[0029] Figure 11 Figure 3B. Comparison of ejection fraction (EF) of each group of mice in Example 3;

[0030] Figure 12 Figure 3C. Comparison of fractional shortening (FS) of each group of mice in Example 3;

[0031] Figure 13 Figure 3D. Comparison of left ventricular internal dimension at end-systole (LVID; s) of each group of mice in Example 3;

[0032] Figure 14 Figure 3E. Comparison of left ventricular internal dimension at end-diastole (LVID; d) of each group of mice in Example 3;

[0033] Figure 15 Figure 3F. Comparison of mRNA levels of fibrosis-related biomarkers Collagen 1 and FN1 in myocardial tissue of each group of mice in Example 3;

[0034] Figure 16Representative images of heart sections stained by MASSON staining method (top) and images of α-SMA expression detected by immunohistochemistry (bottom) of mice in each group in Example 3;

[0035] Figure 17 This is a comparison of the knockdown efficiency of different circ-CELF1 siRNAs in Example 4;

[0036] Figure 18 This is a comparison of the mRNA levels of Collagen1 and FN1 in CFs after 24 hours of TGF-β1 treatment in Example 4, after knockdown of circ-CELF1;

[0037] Figure 19 This is a comparison of the WB analysis results of the Collagen1 expression level in CFs after 24 hours of TGF-β1 treatment in Example 4, which knocked down circ-CELF1;

[0038] Figure 20 This is a comparison of the WB analysis results of FN1 expression levels in CFs after 24 hours of TGF-β1 treatment in Example 4, after knockdown of circ-CELF1;

[0039] Figure 21 This is a comparison of the WB analysis results of the MMP2 expression level in circ-CELF1 knockdown CFs after TGF-β1 treatment for 24 hours in Example 4;

[0040] Figure 22 These are images and fluorescence intensity comparisons of immunofluorescence staining of α-SMA in fibroblasts after knockdown of circ-CELF1 in Example 4;

[0041] Figure 23 This is the EdU staining image of fibroblasts after knockdown of circ-CELF1 in Example 4;

[0042] Figure 24 Schematic diagram of the hsa-circ-CELF1 genome information in Example 5;

[0043] Figure 25 This is a comparison of the expression changes of hsa-circ-CELF and linear Celf1 mRNA levels in HCFs after RNaseR treatment in Example 5;

[0044] Figure 26 This is a comparison chart of the knockdown efficiency of different hsa-circ-CELF1 siRNAs in Example 6;

[0045] Figure 27This is a comparison of the mRNA levels of Collagen1 and FN1 in hsa-circ-CELF1 knockdown HCFs after 24 hours of TGF-β1 treatment in Example 6;

[0046] Figure 28 This is a comparison of the WB analysis results of the Collagen1 expression level in hsa-circ-CELF1 knockdown cells after TGF-β1 treatment of HCFs for 24 hours in Example 6;

[0047] Figure 29 This is a comparison of the WB analysis results of FN1 expression levels in hsa-circ-CELF1 knockdown cells after TGF-β1 treatment of HCFs for 24 hours in Example 6;

[0048] Figure 30 This is the EdU staining image of fibroblasts after knockdown of hsa-circ-CELF1 in Example 6. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described below with reference to the examples, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. The process equipment or devices not specifically noted in the following examples are all conventional equipment or devices in the art. Unless otherwise specified, the raw materials used in the examples of the present invention can be obtained commercially. The experimental methods used in the examples are operated according to conventional conditions or the instructions provided by the manufacturer unless otherwise specified.

[0050] Example 1

[0051] This example provides a validation experiment showing that circ-CELF1 expression is upregulated in a myocardial fibrosis model.

[0052] To demonstrate the role of circular RNA (circRNAs) in the progression of cardiac fibrosis, this example analyzed the circAtlas database. CircRNA expression data were obtained from the circAtlas dataset and analyzed and validated according to the following steps. The screening process is shown in the figure below. Figure 1 shown.

[0053] First, a total of 932 circRNAs were screened for high conserved expression between mice and humans. Subsequently, 274 circRNAs were selected from exonic regions. A further 68 circRNAs were identified that were both highly conserved and derived from exonic regions. Finally, the five circRNAs with the highest abundance in mouse heart and high expression in their host genes were identified: circ-ADAM10, circ-MED13, circ-MAP2K4, circ-GLYR1, and circ-CELF1.

[0054] This example conducts an in-depth analysis of the five selected circRNAs to examine their expression changes at different time points after myocardial infarction.

[0055] 1. Research Methods

[0056] (1) Establishment of mouse MI model:

[0057] Experimental animals: Male C57BL / 6 mice, weighing 25 ± 5 g, were provided by the Experimental Animal Center of Harbin Medical University.

[0058] Male C57BL / 6 mice were randomly divided into two groups: a sham-operated (Sham) group and a myocardial infarction (MI) model group. After anesthesia with intraperitoneal injection of aflototin (10 mg / kg), mice were immobilized in the supine position and connected to a ventilator. A thoracotomy was performed at the 4th-5th intercostal space. The pericardium was incised, and the left anterior descending coronary artery was ligated 2 mm below the left atrial appendage. The thorax was then closed, and electrocardiographic changes were observed. Significant ST-segment elevation was considered a successful model. No ligation was performed in the sham-operated group. The sham and MI groups were then housed under the same conditions for 1 day, 1 week, and 4 weeks before heart samples were obtained.

[0059] (2) Real-time PCR:

[0060] First, tissue RNA was extracted: heart tissue was cut to an appropriate size and placed in an RNase-free imported EP tube. Work on ice, add 1 ml of Trizol, and grind thoroughly with an electric grinder. Add 250 µl of chloroform to the EP tube, shake vigorously for 30 seconds, and then place on ice for 15 minutes. Centrifuge at 13,500 rpm at 4°C for 15 minutes. Carefully aspirate the supernatant and transfer it to a new EP tube. Add an equal volume of isopropanol to the supernatant, mix thoroughly, and incubate at 4°C for 1 hour. Centrifuge at 13,500 rpm at 4°C for 10 minutes. After centrifugation, discard the supernatant, add 1 ml of 75% ethanol (ethanol:DEPC water = 3:1) to the EP tube, and centrifuge at 10,600 rpm at 4°C for 5 minutes. Discard the supernatant and invert onto filter paper to dry. Add 10 µl of DEPC water to each tube to fully dissolve the RNA precipitate and store at -80°C. Reverse transcription was then performed.

[0061] The real-time PCR reverse transcription reaction system is shown in Table 1:

[0062] Table 1

[0063]

[0064] After detecting the RNA concentration, the RNA reverse transcription system was prepared according to the above system, and reverse transcription was performed at 37°C for 10 minutes, 85°C for 5 seconds, and 4°C for ∞; the obtained cDNA was stored at -20°C.

[0065] The following experiments were performed according to the operating procedures of the Applied Biosystems SYBR Green PCR Master Mix kit.

[0066] The SYBR Green dye-based real-time PCR reaction system is as follows: SYBR Green PCR Master Mix (2×), 10µl; 10µM Forward Primer, 1µl; 10µM Reverse Primer, 1µl; RT product, 1µl; Nuclease-free water, 7µl; total volume 20µl.

[0067] Specific back-to-back primers were designed for the reverse cleavage site of circular RNA circ-CELF1. Their base sequences are shown in SEQ ID NO. 11 and SEQ ID NO. 12 and were synthesized by RiboBio Co., Ltd. The experiment was performed using an AppliedBiosystems 7500 FAST Real-time PCR instrument.

[0068] The reaction conditions for SYBR Green dye-based real-time PCR are as follows:

[0069] Stage 1: 95°C for 10 minutes;

[0070] Stage 2: 95°C for 15 seconds; 60°C for 30 seconds; 72°C for 30 seconds (40 cycles in total).

[0071] The experimental results were calculated using the Ct value method to calculate the amount of the target gene. -∆∆CT Analyze relative changes in gene expression.

[0072] 2. Observation results:

[0073] A mouse myocardial infarction (MI) model at multiple time points was constructed, and mouse heart tissues were taken at different time points to detect the expression changes of the above five circRNAs at different time points after myocardial infarction. The results are shown in Figure 2 As shown in Table 1, compared with the heart of the sham operation group, circ-CELF1 was significantly up-regulated in the mouse heart at 1 day, 1 week and 4 weeks after myocardial infarction. In addition, as shown in Figure 3 circ-CELF1 also showed a significant up-regulation trend in CFs treated with transforming growth factor-β1 (TGF-β1). Therefore, this embodiment demonstrates that circ-CELF1 is up-regulated in the myocardial fibrosis model.

[0074] Example 2

[0075] This embodiment investigates the basic properties of the mouse circ-CELF1 screened in Example 1.

[0076] 1. Research method:

[0077] (1) Sequencing of the circularization site

[0078] In order to verify the circularization properties of circ-CELF1, primers were designed for the sequence at the junction site of circ-CELF1 and PCR amplification was performed. The nucleotide sequence of the upstream primer of the PCR primer is shown in SEQ ID NO. 11, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 12.

[0079] The product amplified by trans-PCR was subjected to sanger sequencing to accurately confirm the reverse splicing site and determine the correct linker sequence of circ-CELF1.

[0080] (2) RNase experiment:

[0081] RNase R enzyme can digest linear RNA substrates, but cannot digest circular RNA. After total RNA of myocardial fibroblasts was extracted and digested by RNase R enzyme, the expression levels of circ-CELF1 and Celf1 mRNA were detected by qPCR.

[0082] (3) Nucleus and cytoplasm separation experiment

[0083] According to the PARIS™ kit instructions. The myocardial fibroblasts were treated with 500 μl of pre-cooled cell separation buffer, resuspended, and the resuspended cells were incubated on ice for 5-10 minutes. Then centrifuged and washed the nuclear pellet with pre-cooled cell separation buffer, and the cytoplasmic component was separated from the nuclear pellet, and the sample was divided into aliquots, then reverse transcribed to obtain cDNA according to the above method, and qPCR was performed to determine that circ-CELF1 is mainly located in the cytoplasm.

[0084] 2. Observation results:

[0085] like Figure 4 As shown, Circ-CELF1 is generated by back-splicing of exons 2 and 8 of the Celf1 gene (Chr2:90998580-91004862), with a length of 562 nucleotides and a nucleotide sequence as shown in SEQ ID NO.10.

[0086] like Figure 5 As shown, ribonuclease R (RNase R) treatment experiments showed that compared with Celf1 linear mRNA, circ-CELF1 was more resistant to RNase R digestion. RNase R can digest linear RNA but has no effect on circular RNA, proving that circ-CELF1 is a circular RNA.

[0087] Subsequently, the expression of circ-CELF1 in different mouse tissues was evaluated, e.g. Figure 6 The results showed that circ-CELF1 was expressed at the highest abundance in the heart.

[0088] In addition, cardiomyocytes and cardiac fibroblasts were isolated from the hearts of normal adult mice and myocardial infarction model mice to detect the expression level of circ-CELF1. Figure 7 As shown in the Figure 3, circ-CELF1 was mainly expressed in CFs, and the expression level of circ-CELF1 was significantly upregulated under MI conditions.

[0089] At the same time, if Figure 8 As shown in the results, circ-CELF1 expression was significantly upregulated in the plasma of mice 4 weeks after MI compared with that in the sham-operated group, suggesting that circ-CELF1 could be used as a clinical diagnostic marker.

[0090] Then, if Figure 9 As shown in the figure, subcellular fractionation experiments combined with qPCR experiments revealed that circ-CELF1 was mainly localized in the cytoplasm of CFs.

[0091] Example 3

[0092] This example investigates the effects of circ-CELF1 on mice with myocardial infarction.

[0093] 1. Research Methods

[0094] (1) Adeno-associated virus vector construction

[0095] Constructing recombinant vector containing circ-CELF1 siRNA; extracting and purifying high-quality endotoxin-free recombinant plasmid for sequencing-corrected recombinant plasmid; co-transfecting HEK293T cells with high-efficiency recombinant vector and virus packaging plasmid (purchased) to perform virus packaging and collect virus liquid; after concentration and purification, infecting cells (293T cells) with high-quality virus liquid; accurately determining virus titer and analyzing experimental results by quantitative PCR, and entrusting Shanghai Jikai Biotechnology Co., Ltd. for synthesis.

[0096] First, according to the Circ-CELF1-shRNA adeno-associated virus target sequence as shown in SEQ ID NO. 13, the shRNA sequence is designed by the conventional method in the art and prepared by Jikai Biotechnology Co., Ltd.

[0097] GV390 adeno-associated virus is selected as the vector skeleton. The designed shRNA sequence is synthesized into a double-stranded DNA fragment, which is directly connected to the enzyme-digested RNA interference adeno-associated virus vector through the enzyme digestion sites at both ends. Then, the ligation product is transformed into E. coli competent cells, and plasmid extraction and positive clone sequencing verification are performed. Then, AAV virus packaging is performed, virus liquid is collected, and after concentration and purification, 293T cells are infected with high-quality virus liquid. The virus titer is accurately determined by quantitative PCR, and the experimental results are analyzed, and the AAV9-sh-circ-CELF1 adeno-associated virus construction is entrusted to Shanghai Jikai Biotechnology Co., Ltd. for synthesis.

[0098] (2) Heart function detection by echocardiography

[0099] The mice were anesthetized by intraperitoneal injection of aflurit (10 mg / kg), and the mice were fixed on the experimental table. The mouse heart in the middle of the chamber was detected by ultrasound in the B mode measurement obtained in the parasternal short-axis view of the sternal muscle level with a 30 MHz probe Vevo2100 high-resolution imaging system (VisualSonics, Toronto, Canada), and the heart rate (HR), ejection fraction (EF), fractional shortening (FS), left ventricular end-systolic diameter (LVIDs), and left ventricular end-diastolic diameter (LVIDd) were measured.

[0100] (3) Masson staining

[0101] The procedure was performed according to the Solarbio (G1340) Masson trichrome staining kit instructions. Paraffin sections were dewaxed and washed sequentially with tap water and then distilled water. Nuclei were stained with hematoxylin for 1-2 minutes, then blued with warm water for 5 minutes. Ponceau acid fuchsin solution was then used for 10 minutes. Staining was then performed with phosphomolybdic acid solution for 2 minutes, followed by aniline blue staining for 2 minutes, and then washed with 1% glacial acetic acid working solution for 1 minute. Dehydration was performed with 95% ethanol and then anhydrous ethanol three times. Clearing was performed with xylene three times. The sections were mounted with neutral gum, sealed, and dried at 65°C before being photographed under a microscope.

[0102] (4) Immunohistochemistry

[0103] Heart tissue was paraffin-dehydrated, embedded, and sliced. The slices were preheated for 30 minutes and then dewaxed. After eliminating endogenous peroxidase activity with 3% H2O2, antigen retrieval and blocking were performed. The primary antibody (α-SMA) was incubated overnight and then incubated again with a horseradish peroxidase-labeled secondary antibody. After DAB color development and hematoxylin anti-blueing, the slides were quickly dehydrated with 95% ethanol, dehydrated three times with anhydrous ethanol, and transparentized with xylene. The slides were sealed with neutral gum, dried, and photographed.

[0104] 2. Observation results:

[0105] To evaluate the function of circ-CELF1 in myocardial infarction, we constructed an adeno-associated virus type 9 (AAV9) system driven by the periostin core promoter to regulate the expression of circ-CELF1. This system was used to silence circ-CELF1 in mouse CFs (AAV9-sh-circ-CELF1). The specific operation was as follows: 5×10 10 vg of virus per mouse was diluted to 20ul with normal saline, and AAV9-sh-circ-CELF1 or AAV9-sh-NC was injected into the mice through the tail vein for 3 weeks. Subsequently, the mouse myocardial infarction model was established by ligating the left anterior descending artery and maintained for 4 weeks.

[0106] Echocardiographic analysis results Figure 10-12 As shown, the ejection fraction (EF) and fractional shortening (FS) of MI mice treated with AAV9-sh-circ-CELF1 were significantly increased compared with those of MI mice treated with AAV9-sh-NC.

[0107] like Figure 13-14 As shown, the left ventricular end-systolic diameter (LVIDs) and left ventricular end-diastolic diameter (LVIDd) of the AAV9-sh-circ-CELF1 group mice were reduced compared with those of the AAV9-sh-NC-treated MI mice.

[0108] like Figure 15As shown, compared with the AAV9-sh-NC control group, the expression of fibrosis-related biomarkers in the heart tissue of the AAV9-sh-circ-CELF1-treated MI mice was down-regulated.

[0109] By Figure 16 Histological examination by Masson staining showed that knockdown of circ-CELF1 improved collagen deposition in the heart of the MI mice compared with the AAV9-sh-NC group of mice. Immunohistochemical analysis showed that knockdown of circ-CELF1 significantly reduced the transformation of fibroblasts to myofibroblasts in the heart tissue after MI surgery.

[0110] The above results show that knockdown of circ-CELF1 at the in vivo level can inhibit myocardial fibrosis and cardiac function impairment caused by MI.

[0111] Example 4

[0112] This example investigates the therapeutic effect of knockdown of circ-CELF1 in a TGF β1-induced myocardial fibrosis model.

[0113] 1. Research method:

[0114] (1) Construction of circRNA small interfering RNA

[0115] To confirm the biological function of circ-CELF1 in neonatal rat primary cardiac fibroblasts (CFs), siRNAs were designed targeting the circularization site of circ-CELF1, and were synthesized by Guangzhou Ribo Biological Technology Co., Ltd.

[0116] The target sequences combined in this example are shown in SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16, respectively. Three small interfering RNAs (siRNAs) targeting the reverse splicing site of circ-CELF1 were designed according to the three binding target points, and were named si-circ-CELF1-1, si-circ-CELF1-2 and si-circ-CELF1-3, respectively.

[0117] (2) Western blot experiment

[0118] Add 150 μl of pre-prepared cell lysis buffer (the amount of lysis buffer can be adjusted based on cell density) to the cells to be extracted. Lyse the cells in an ice bath for 5 minutes. Use a cell scraper to remove adherent cells and pipette the protein lysate into a 1.5 ml EP tube. Centrifuge at 13,500 rpm at 4°C for 15-20 minutes. After centrifugation, pipette the supernatant into a new 1.5 ml EP tube. This represents the total protein extracted. First, place the prepared gel into the electrophoresis tank and fill it to the appropriate mark with electrophoresis buffer. Load the protein samples in the order specified for the experiment, then add the protein marker. Connect the electrophoresis apparatus and adjust the stacking gel voltage to 70 V. Run the gel for approximately 30 minutes. Once the protein samples have passed through the stacking gel and formed a straight line, adjust the voltage to 110 V. Remove the gel using a gel remover and arrange it in the order of white sponge, filter paper, NC membrane, gel, filter paper, and black sponge, gently removing any bubbles. Keep the transfer chamber on ice to keep it cold. Transfer the membrane at a constant current of 300mA for 120 minutes. Place the NC membrane in a pre-prepared blocking buffer containing 10% skim milk and block for 1.5 hours at room temperature on a shaker. Cut the target band from the blocked NC membrane according to the molecular weight of the target protein. Place the membrane in a pre-diluted primary antibody solution and incubate overnight at 4°C in a ziplock bag. Remove the NC membrane and wash it four times with PBST (7 minutes each time). Then, incubate with a diluted secondary antibody solution at room temperature for 50 minutes before developing and analyzing.

[0119] (3) Immunofluorescence

[0120] Mouse cardiac fibroblasts were seeded into 24-well plates covered with glass slides at a density of 50% and cultured in a cell culture incubator for 24 hours. The cell culture medium was discarded with a pipette and the cells were washed three times with PBS for 5 minutes each on a shaker. The cells were fixed with pre-chilled 4% paraformaldehyde for 20 minutes. A permeabilization buffer was prepared: 30 μl of Triton dissolved in 10 ml of PBS was added to each well to cover the cell fragments and allowed to stand at room temperature for 1 hour. The cells were blocked with goat serum and incubated at 37°C for 1 hour. After blocking, the blocking buffer was discarded and the cells were washed three times with PBST for 5 minutes each. α-SMA was mixed with a 1:50 ratio of blocking buffer. 50 μl of the prepared primary antibody was added to each well and the cells were refrigerated at 4°C overnight. The primary antibody was washed three times with PBST for 5 minutes each. Prepare fluorescent secondary antibody according to the appropriate ratio and add 50 μl of the prepared fluorescent secondary antibody to each well in the dark. Incubate at 37°C in the dark for 1.5 hours. Wash the secondary antibody three times with PBST for 5 minutes each. Prepare DAPI according to the appropriate ratio and add 50 μl of the prepared DAPI to each well in the dark. Incubate at room temperature for 15 minutes. Discard the DAPI stain and wash the cells three times with PBST for 5 minutes each. Imaging the treated cells was performed using a live cell imaging station in the dark.

[0121] (4) EdU staining

[0122] According to the operation of the EdU Cell Proliferation Detection Kit of Ribobio (C10310) EdU Cell Proliferation Detection Kit. The cells were cultured in a 24-well plate, and circ-CELF1 small interfering RNA was transfected. Prepare EdU working solution with a final concentration of 10 μM, prepare Click Additive Solution, and gently shake the culture plate to ensure that the reaction mixture can evenly cover the sample. Incubate at room temperature for 30 minutes in the dark. Wash 3 times, then stain the nucleus, and observe under a fluorescence microscope.

[0123] 2. Observation results:

[0124] The knockdown efficiency of different circ-CELF1 siRNAs is shown in Figure 17 After treatment with three circ-CELF1 siRNAs, the level of circ-CELF1 in CFs decreased, and the level of circ-CELF1 in CFs after treatment with si-circ-CELF1-1 significantly decreased, so si-circ-CELF1-1 was selected for subsequent knockdown experiments.

[0125] The functional index is shown in Figures 18-20 After silencing circ-CELF1, the fibrosis-related markers induced by TGF-β1 were significantly down-regulated.

[0126] The extracellular matrix remodeling in the process of cardiac fibrosis was detected. The results are shown in Figure 21 After knocking down circ-CELF1, the level of matrix metalloproteinase 2 (MMP2) in cells treated with TGF-β1 was significantly reduced.

[0127] The results of α-smooth muscle actin (α-SMA) immunostaining are shown in Figure 22 After knocking down circ-CELF1, the fluorescence activity of TGF-β1-induced α-SMA was significantly reduced.

[0128] Through 5-ethynyl-2'-deoxyuridine (EdU) staining experiment, as shown in Figure 23 Silencing circ-CELF1 can inhibit the proliferation of CFs induced by TGF-β1.

[0129] The above results show that knocking down circ-CELF1 can inhibit the collagen expression and cell proliferation of cardiac fibroblasts.

[0130] Example 5

[0131] This example investigates the gene information of human circular RNA hsa-circ-CELF1.

[0132] In this example, human-derived circular CELF1 (hsa-circ-CELF1) was screened through the circAtlas database.

[0133] 1. Research Methods

[0134] The structure of hsa-circ-CELF1 was clarified by Sanger sequencing, and the circular RNA characteristics were verified by RNase R assay. The PCR primers designed in this example for human circ-CELF1, i.e., the sequence at the hsa-circ-CELF1 junction site, have an upstream primer nucleotide sequence as shown in SEQ ID NO. 2, and a downstream primer nucleotide sequence as shown in SEQ ID NO. 3. The remaining specific experimental steps were the same as in Example 1.

[0135] 2. Observation results:

[0136] Sanger sequencing results Figure 24 As shown, the structure of hsa-circ-CELF1 was clarified, and the nucleotide sequence of hsa-circ-CELF1 was shown in SEQ ID NO.1.

[0137] The circular structure of hsa-circ-CELF1 was identified by exoribonuclease RNase R. The results of RNase R experiment are shown in Figure 2. Figure 25 As shown in the results, compared with linear RNA, RNase R had no degradation effect on hsa-circ-CELF1, proving that hsa-circ-CELF1 is indeed a circular structure.

[0138] Example 6

[0139] In this example, hsa-circ-CELF1 knockdown siRNA was constructed to investigate the therapeutic effect of hsa-circ-CELF1 knockdown in a TGF-β1-induced human cardiac fibroblast model.

[0140] 1. Research Methods

[0141] The effect of hsa-circ-CELF1 in human fibroblasts was verified by qPCR, WB, and EDU experiments. The specific steps are as described in Example 4.

[0142] 2. Observation results:

[0143] To investigate whether hsa-circ-CELF1 has the same pro-fibrotic properties as mouse circ-CELF1 in human fibroblasts (HCFs), three siRNAs targeting the backsplicing sites of hsa-circ-CELF1 were designed and named si-hsa-circ-CELF1-1, si-hsa-circ-CELF1-2, and si-hsa-circ-CELF1-3.

[0144] The nucleotide sequences of the sense chain and antisense chain of si-hsa-circ-CELF1-1 are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, the nucleotide sequences of the sense chain and antisense chain of si-hsa-circ-CELF1-2 are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively, and the nucleotide sequences of the sense chain and antisense chain of si-hsa-circ-CELF1-3 are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0145] To comply with the requirements of WIPO ST.26, the six nucleotide sequences shown in SEQ ID NO.4 to SEQ ID NO.9 all use T instead of U to represent uracil in RNA. The specific replacements are as follows:

[0146] In the nucleotide sequence shown in SEQ ID NO.4, T is used to replace U at positions 11 and 17 of the 5' end, which represents uracil in RNA. In the nucleotide sequence shown in SEQ ID NO.5, T is used to replace U at positions 1, 6, 10, 13, 15 and 17 of the 5' end, which represents uracil in RNA.

[0147] The specific sense strand of si-hsa-circ-CELF1-1 is: GCACAGACCAUGGAGCUCATT;

[0148] The antisense strand of si-hsa-circ-CELF1-1 is: UGAGCUCCAUGGUCUGUGCTT.

[0149] In the nucleotide sequence shown in SEQ ID NO. 6, T is used to replace the U at positions 8 and 14 of the 5' end, which represents uracil in RNA. In the nucleotide sequence shown in SEQ ID NO. 7, T is used to replace the U at positions 2, 3, 4, 9, 13, 16 and 18 of the 5' end, which represents uracil in RNA.

[0150] The specific sense strand of si-hsa-circ-CELF1-2 is: CAGACCAUGGAGCUCAAAGTT;

[0151] The antisense strand of si-hsa-circ-CELF1-2 is specifically: CUUUGAGCUCCAUGGUCUGTT.

[0152] In the nucleotide sequence shown in SEQ ID NO. 8, the U at the 6th and 12th positions of the 5' end is replaced by T, which represents uracil in RNA. In the nucleotide sequence shown in SEQ ID NO. 9, the U at the 1st, 2nd, 4th, 5th, 6th, 11th, 15th and 18th positions of the 5' end is replaced by T, which represents uracil in RNA.

[0153] The sense strand of si-hsa-circ-CELF1-2 is specifically: GACCAUGGAGCUCAAAGAATT.

[0154] The antisense strand of si-hsa-circ-CELF1-2 is specifically: UUCUUUGAGCUCCAUGGUCTT.

[0155] The knockdown efficiency of different hsa-circ-CELF1 siRNAs was detected, and the results are shown in Table 1. Figure 26 As shown in Table 1, siRNA-1 significantly reduced the expression level of hsa-circ-CELF1 in HCFs, and was therefore used in subsequent experiments. Figure 27-Figure 29 As shown in Table 2, silencing of hsa-circ-CELF1 in HCFs effectively inhibited the up-regulation of fibrosis-related proteins and mRNA levels induced by TGF-β1. Figure 30 As shown in Table 3, under the condition of TGF-β1 stimulation, silencing of hsa-circ-CELF1 could inhibit the proliferation of HCFs.

[0156] In summary, the above results show that circ-CELF1 is expected to become a potential new therapeutic target for cardiac fibrosis, and has certain clinical significance.

Claims

1. A circRNA biomarker circ-CELF1, characterized by: The nucleotide sequence of circ-CELF1 is shown in SEQ ID NO.

1.

2. A use of the circRNA biomarker circ-CELF1 according to claim 1 in preparing a diagnostic kit for myocardial fibrosis after myocardial infarction, characterized in that: The diagnostic kit contains reverse transcription quantitative PCR primers for specifically amplifying the circ-CELF1 reverse splicing junction as claimed in claim 1.

3. The use of the circRNA biomarker circ-CELF1 in preparing a diagnostic kit for myocardial fibrosis after myocardial infarction according to claim 2, characterized in that: The nucleotide sequence of the upstream primer of the PCR primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

3.

4. A diagnostic kit for myocardial fibrosis after myocardial infarction, characterized in that: Contains reverse transcription quantitative PCR primers for specifically amplifying the circ-CELF1 reverse splicing junction as claimed in claim 1, wherein the upstream primer nucleotide sequence of the PCR primer is shown in SEQ ID NO.2, and the downstream primer nucleotide sequence is shown in SEQ ID NO.

3.

5. A use of the circRNA biomarker circ-CELF1 according to claim 1 in preparing a drug for treating myocardial fibrosis after myocardial infarction, characterized in that: The drug contains a nucleic acid fragment or a recombinant vector that inhibits the expression of circ-CELF1 as claimed in claim 1 or reduces its activity.

6. The use of the circRNA biomarker circ-CELF1 in the preparation of a drug for treating myocardial fibrosis after myocardial infarction according to claim 5, characterized in that: The nucleic acid fragment is a circ-CELF1 interferor, and the circ-CELF1 interferor is an siRNA that inhibits the back-splicing and cyclization of circ-CELF1 according to claim 1, and the nucleotide sequences of the sense chain and antisense chain of the siRNA are shown as SEQ ID NO.4 and SEQ ID NO.5, respectively, or the nucleotide sequences of the sense chain and antisense chain of the siRNA are shown as SEQ ID NO.6 and SEQ ID NO.7, respectively, or the nucleotide sequences of the sense chain and antisense chain of the siRNA are shown as SEQ ID NO.8 and SEQ ID NO.9, respectively.

7. The use of the circRNA biomarker circ-CELF1 in the preparation of a drug for treating myocardial fibrosis after myocardial infarction according to claim 5, characterized in that: The recombinant vector is a viral packaging plasmid containing shRNA that inhibits the reverse splicing and circularization of circ-CELF1 as claimed in claim 1.

8. The use of the circRNA biomarker circ-CELF1 in the preparation of a drug for treating myocardial fibrosis after myocardial infarction according to claim 7, characterized in that: The virus is an adeno-associated virus AAV9 containing a cardiac fibroblast-specific periostin core promoter.

9. A drug for treating myocardial fibrosis after myocardial infarction, characterized in that: The drug contains a nucleic acid fragment or a recombinant vector that inhibits the expression of circ-CELF1 as claimed in claim 1 or reduces its activity.

10. The drug for treating myocardial fibrosis after myocardial infarction according to claim 9, characterized in that: The nucleic acid fragment is a circ-CELF1 interferor, and the circ-CELF1 interferor is an siRNA that inhibits the back-splicing and cyclization of circ-CELF1 as claimed in claim 1, and the nucleotide sequences of the sense chain and antisense chain of the siRNA are shown as SEQ ID NO.4 and SEQ ID NO.5, respectively, or the nucleotide sequences of the sense chain and antisense chain of the siRNA are shown as SEQ ID NO.6 and SEQ ID NO.7, respectively, or the nucleotide sequences of the sense chain and antisense chain of the siRNA are shown as SEQ ID NO.8 and SEQ ID NO.9, respectively.

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