Application of miRNA in the prevention or treatment of postoperative atrial fibrillation

Abstract

This invention relates to the technical field of pharmaceutical compositions, specifically disclosing the application of miRNAs in the prevention or treatment of postoperative atrial fibrillation, wherein the miRNA is at least one of hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p. Compositions for diagnosing, preventing, or treating postoperative atrial fibrillation using miRNA inhibitors are also disclosed. A kit for diagnosing postoperative atrial fibrillation is also disclosed, comprising reagents capable of detecting miRNAs. A method for providing information for diagnosing postoperative atrial fibrillation is also disclosed, comprising detecting the expression level of miRNAs in a biological sample and comparing it with a control group; when the expression level of miRNAs in the biological sample is higher, a risk of postoperative atrial fibrillation is determined. The application of miRNAs in the prevention or treatment of postoperative atrial fibrillation of this invention can be effectively used for the prevention and treatment of postoperative atrial fibrillation.

Description

Technical Field

[0001] This invention relates to the technical field of pharmaceutical compositions, and more particularly to the use of miRNA in the prevention or treatment of postoperative atrial fibrillation. Background Technology

[0002] Postoperative atrial fibrillation (POAF) has consistently occurred in approximately 30% of patients following coronary artery bypass grafting (CABG). This arrhythmia is not transient and significantly impacts both short-term and long-term postoperative prognosis, but its complex pathogenesis remains poorly understood. POAF typically peaks between postoperative days 2 and 4 and is strongly associated with CABG. The prevailing hypothesis suggests that the brief surgical manipulation of the heart during surgery, coupled with the pre-existing fragility of the atrial matrix, interacts to induce postoperative cardiac remodeling, characterized by early atrial collagen deposition, which in turn leads to POAF.

[0003] Pericardial drainage fluid (PCF) provides a unique, non-invasive, and direct window into the heart, rich in bioactive factors from the surgical site that reflect underlying cardiac pathological conditions. Extracellular vesicles (EVs) play a crucial role in intercellular and tissue communication, particularly in the development of arrhythmias and fibrosis. Among EVs, microRNAs (miRNAs) stand out due to their high stability and potential role in upstream gene transcription. EVs from PCFs of patients with chronic atrial fibrillation carry specific miRNA biomarkers associated with cardiac fibrosis. Plasma-derived exosomal miRNAs such as hsa-miR-184 have been shown to be diagnostic biomarkers for cardiac surgery-related acute kidney injury; therapeutic EVs derived from human cardiac cells have effectively helped resist POAF in a rat model of aseptic pericarditis through anti-inflammatory and anti-fibrotic effects. Although these studies have confirmed the role of EV-miRNAs in chronic atrial fibrillation and their therapeutic potential for POAF, the role of PCF-derived EV-miRNAs in the pathogenesis of POAF has never been investigated. Summary of the Invention

[0004] The purpose of this invention is to provide a composition for diagnosing, preventing or treating postoperative atrial fibrillation using a miRNA inhibitor, which can be effectively used for the prevention and treatment of postoperative atrial fibrillation.

[0005] This invention is achieved through the following technical solution: the application of the miRNA of this invention in the prevention or treatment of postoperative atrial fibrillation, wherein the miRNA is at least one of hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p. The miRNA is preferably hsa-miR-4324.

[0006] The present invention also provides a pharmaceutical composition for the prevention or treatment of postoperative atrial fibrillation, the pharmaceutical composition comprising a miRNA inhibitor as an active ingredient; wherein the miRNA is at least one selected from hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p. Specifically, the database code for hsa-miR-4324 is MIMAT0016876, with a sequence of 5'-CCCUGAGACCCUAACCUUAA-3'; the database code for hsa-miR-1304-3p is MIMAT0022720, with a sequence of 5'-UCUCACUGUAGCCUCGAACCCC-3'; and the database code for hsa-miR-329-3p is MIMAT0001629, with a sequence of 5'-AACACACCUGGUUAACCUCUUU-3'.

[0007] Furthermore, the miRNA inhibitor regulates postoperative atrial fibrillation by inhibiting the upregulated expression of miRNAs in the pericardial sac fluid.

[0008] Furthermore, the miRNA inhibitor is selected from any one of miRNA-specific siRNA, aptamers, antisense oligonucleotides, ribozymes, and compounds.

[0009] The present invention also provides a kit for diagnosing postoperative atrial fibrillation, the kit containing reagents capable of detecting miRNAs.

[0010] Furthermore, the miRNA is at least one of hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p.

[0011] Furthermore, the kit is selected from microarrays, aptamer chip kits, enzyme-linked immunosorbent assay kits, gene expression serial analysis kits, quantitative real-time PCR kits, and combinations thereof.

[0012] The present invention also provides a method for providing information for diagnosing postoperative atrial fibrillation, including detecting the expression level of miRNA in a biological sample and comparing it with a control group, and determining the risk of postoperative atrial fibrillation when the expression level of miRNA in the biological sample is higher.

[0013] Furthermore, the miRNA is at least one of hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p.

[0014] Furthermore, the biological sample is extracellular vesicles (EVs) in pericardial drainage fluid (PCF).

[0015] The technical solution of the present invention has at least the following advantages and beneficial effects: The application of the miRNA of the present invention in the prevention or treatment of postoperative atrial fibrillation disease shows that the expression of hsa-miR-1304-3p, hsa-miR-4324, hsa-miR-329-3p, especially hsa-miR-4324, is clearly associated with postoperative atrial fibrillation disease (POAF). High expression of miRNA means a high probability of POAF. Therefore, these miRNAs can be used in clinics for POAF disease and can be further used for the prevention or treatment of POAF. Attached Figure Description

[0016] Figure 1 The expression of multiple miRNAs was shown at two postoperative time points;

[0017] Figure 2 The roles of various miRNAs in the TGF-β signaling pathway were shown;

[0018] Figure 3 The incidence of POAF after injection of three miRNAs was shown;

[0019] Figure 4 The induction rate of POAF by different concentrations of hsa-miR-4324 was shown;

[0020] Figure 5 The time of POAF onset after injection of hsa-miR-4324 was shown;

[0021] Figure 6 The expression of hsa-miR-4324 in PCF-EVs from patients with and without POAF was shown.

[0022] Figure 7 The predictive ability of three miRNAs for POAF was demonstrated;

[0023] Figure 8 This demonstrates the effect of hsa-miR-4324 on SKP1 gene levels;

[0024] Figure 9 The effect of hsa-miR-4324 on SKP1 protein levels was shown;

[0025] Figure 10 This shows the effect of hsa-miR-4324 on SKP1 protein levels after normalization with the internal reference protein GAPDH;

[0026] Figure 11 This study demonstrated the targeted inhibition of the SKP1 gene by hsa-miR-4324 in a dual-luciferase reporter gene assay. Detailed Implementation

[0027] Example 1

[0028] hsa-miR-4324 was significantly upregulated in the POAF group and was closely related to the profibrotic pathway.

[0029] Detection method: The quality of miRNAs in PCF exosomes from individuals with POAF and non-POAF was sequenced at 0.5 hours and 6 hours post-surgery.

[0030] Isolation of PCF-EVs: The isolation method for PCF-EVs followed the guidelines of the International Society for Extracellular Vesicle Separation (MISE2018). PCF-EVs were isolated using differential ultracentrifugation. Specifically, after thawing on ice, PCF samples were centrifuged sequentially at 4°C: 3000 g for 30 min, followed by 10000 g for 1 h. The supernatant was filtered through a 0.45 μm filter (CORNING). Subsequently, the supernatant was transferred to an ultracentrifuge tube (catalog number: 331372, BECKMAN) and centrifuged at 100000 g for 70 min at 4°C. The precipitate was washed with phosphate-buffered saline (PBS), followed by the same ultracentrifugation steps. The supernatant was discarded, the extracellular vesicle precipitate was resuspended in PBS, and stored at −80°C for further analysis.

[0031] miRNA Extraction and Library Preparation: Total RNA (including miRNAs) was purified from exosomes using the miRNeasy Micro Kit (217084, QIAGEN) according to its manual. miRNA-seq library preparation was performed using the gel-free workflow provided by the QIAseq miRNALibrary Kit (331505, QIAGEN). Briefly, the adapter was sequentially ligated to the 3' and 5' ends of the miRNA in an unbiased reaction. This was followed by universal cDNA synthesis with a unique molecular identifier (UMI), cDNA purification, library amplification, and library purification steps.

[0032] miRNA-seq data analysis: Adapter sequences were removed using Cutadapt (version 2.7). Low-quality reads, reads shorter than 16 bp, or UMI reads shorter than 12 bp were removed. Clean reads were mapped to mature human miRNA sequences downloaded from miRBase (version 22) using bowtie (version 1.2.2) with parameters -v 0 (disallow mismatches), -m 5 (allow up to 5 multi-map events), and --best --strata (return only the alignment with the best alignment score). SAM files were converted to sorted BAM files using SAMtools (version 1.15.1), and read counts were calculated using HTseq (version 2.0.2) in non-empty intersection mode. Dimensionality reduction was performed using the UMAP method, and visualization was performed using ggplot2 (version 2.2.1).

[0033] Differential expression analysis: The trimmed mean (TMM) of the M values ​​between each sample pair was normalized to library size using the R package edgeR (version 3.36.0)10, and the degree of difference in miRNA expression levels between samples was calculated. mRNAs with an absolute fold change greater than 1.5 and a p-value less than 0.05 were considered differentially expressed miRNAs and visualized using ggplot2 (version 2.2.1).

[0034] miRNA target gene prediction: The experimentally validated miRNA-target gene interaction database miRTarBase (version 9.0) and the predicted miRNA target gene database TargetScan (human, version 8.0) were used to identify potential miRNA-target gene regulatory relationships. To obtain highly reliable target genes regulated by differentially expressed miRNAs, only miRNA-target gene regulatory relationships supported by both experimental and predictive evidence were retained.

[0035] Functional enrichment analysis: Potential target genes of miRNAs were input into the R package clusterProfiler (version 4.6.2) to calculate the enrichment level (P-value) of the corresponding biological processes and KEGG pathways.

[0036] Results: The top ten miRNAs with persistently elevated expression levels at two time points (0.5 hours and 6 hours post-surgery) were examined, as shown in the attached table. Figure 1As shown, hsa-miR-4324 showed significantly higher expression levels than other miRNAs at 0.5h and 6h post-surgery. After removing duplicate miRNAs, a total of 24 miRNAs were found to have increased expression levels in the POAF group, of which 20 had more than 20 highly plausible potential target genes. Further functional enrichment analysis was performed on these miRNAs. The results are shown in the attached figure. Figure 2 As shown, these miRNAs play a key role in the transforming growth factor-β (TGF-β) signaling pathway, and can accelerate the fibrosis process by promoting fibroblast proliferation, collagen secretion and extracellular matrix deposition, with hsa-miR-4324 being the most significant.

[0037] Example 2

[0038] The incidence of POAF was significantly increased in the hsa-miR-4324 injection group of mice and showed a dose-dependent effect.

[0039] Detection method:

[0040] Intracardiac miRNA injection: Mice were anesthetized, intubated, and their hearts were exposed. To evaluate the proarrhythmic potential of three selected candidate microRNAs (hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p mimics (all from Sangon Biotech)), mice received two injections, each consisting of a pre-prepared mixture of miRNA mimic (20 μL, 10 μM) and lipids (Lipofectamine RNAiMAX, 1:1 volume ratio), injected into the anterior wall of the left ventricle using a 1 mL syringe with a 34-gauge needle, following the manufacturer's protocol. Control mice received injections of the non-targeted sequence (NC) mimic. Following injection, the intercostal spaces, muscles, and skin were sutured. Mice were then implanted with a wireless telemetry device for continuous monitoring; as previously described, sPOAF was observed within 5 days. Postoperative infection was prevented by intramuscular penicillin (20,000 units daily) for 3 days. Mice were extubated to restore normal breathing and placed on a heated pad until awake, after which they were transferred to their cages.

[0041] Three miRNAs, including hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p, were continuously upregulated at two time points (0.5 h and 6 h post-surgery), exhibiting the largest fold change and abundance at both times. Notably, hsa-miR-4324 was the only miRNA to show the largest continuous upregulation at both time points. Meanwhile, at 6 h post-surgery, hsa-miR-1304-3p showed the largest change in abundance. Therefore, further functional validation of these three miRNAs was performed. The three miRNAs were injected into the anterior wall of the left ventricle (LV) of 6-month-old male mice, with null control mimics used as a control. (See attached image) Figure 3 As shown, continuous heart rate monitoring over 5 days revealed that the incidence of POAF in the hsa-miR-4324 injection group was as high as 83%, while the incidence of POAF in the hsa-miR-329-3p and hsa-miR-1304-3p injection groups was 33% and 50%, respectively. No POAF was observed in the control group.

[0042] hsa-miR-4324 showed a significant dose-dependent effect in inducing POAF. The proportions of mice developing POAF after injection of 5, 10, and 20 μM hsa-miR-4324 were 50%, 83.3%, and 100%, respectively (see attached figure). Figure 4 (As shown).

[0043] In mice injected with 10 μM miR-4324, the timing of POAF attacks peaked 2–3 days post-injection (see attached figure). Figure 5 As shown in the figure, this phenomenon is closely related to the clinical manifestations of POAF after cardiac surgery.

[0044] Example 3

[0045] hsa-miR-4324 can effectively predict POAF.

[0046] RNA Extraction and Real-Time Quantitative PCR: Total RNA (including miRNA) was extracted using Trizol reagent (Invitrogen). Extracted miRNA was reverse transcribed into cDNA using the Mir-X miRNA First-Strand Synthesis Kit (Takara), followed by real-time quantitative PCR (qPCR) using hsa-miR-4324 specific primers and TB Green Advantage qPCR Premix (Takara). Total RNA was reverse transcribed into cDNA using One-Step gDNA Removal and cDNA Synthesis Super Mixture (TransGen Biotech), followed by real-time qPCR using PerfectStart Green qPCR Super Mixture (TransGen Biotech). Data were analyzed using the 2-ΔΔCt method. Primer information is as follows:

[0047]

[0048] ROC curve analysis: In SPSS software (version 20), select "Analyze" > "Binary Logistic Regression" > "ROC Curve," and then select the categorical and predictor variables for ROC analysis. The predictive performance of the identified biomarker miR-4324 was assessed using the area under the receiver operating characteristic (ROC) curve (AUC; discriminative power). The optimal cutoff value for miR-4324 was determined using the ROC curve to optimize sensitivity and specificity.

[0049] Quantitative PCR (qPCR) analysis showed that, as shown in the attached... Figure 6 As shown, compared with non-POAF patients, the level of hsa-miR-4324 in PCF-EVs of POAF patients was significantly increased. Furthermore, as shown in the attached... Figure 7 As shown, hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p can effectively predict POAF. The areas under the curve (AUC) for each miRNA predicting POAF individually are: hsa-miR-1304-3p AUC = 0.784, hsa-miR-4324 AUC = 0.853, and hsa-miR-329-3p AUC = 0.774. The AUC for the combined prediction of the three miRNAs is 0.903. All three miRNAs showed good predictive ability for POAF individually, with hsa-miR-4324 showing the best prediction performance. However, the combined prediction of the three miRNAs was even better than that of hsa-miR-4324.

[0050] Example 4

[0051] hsa-miR-4324 exerts its pro-fibrotic effect by targeting the SKP1 gene.

[0052] Detection method:

[0053] Isolation and Culture of Primary Cardiac Fibroblasts: Neonatal mouse cardiac fibroblasts (NMCFs) were isolated from 1-2 day old C57BL / 6J mice. The ventricles were washed with cold phosphate-buffered saline (PBS), cut into 1 mm³ fragments, and digested with 0.0625% trypsin (Sigma-Aldrich) for 5 minutes at room temperature, followed by digestion with type II collagenase (1 mg / mL, Solarbio) at 37°C with continuous shaking for 5 minutes. After precipitation, the supernatant was collected and added to Dulbecco's modified Eagle medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin / streptomycin (Hyclone). This process was repeated until the tissue was completely digested. The collected supernatant was filtered through a 70 μm cell filter to remove residual debris, and then centrifuged at 1000 × g for 10 minutes to pellet the cells. The cell pellet was resuspended in DMEM containing 10% FBS and seeded in 6 cm culture dishes. After culturing at 37°C and 5% CO2 for 1.5 hours, the cells attached to the bottom of the culture dish were NMCFs.

[0054] miRNA transfection: hsa-miR-4324 mimic negative control (NC), hsa-miR-4324 inhibitor negative control, hsa-miR-4324 mimic, and hsa-miR-4324 inhibitor were all purchased from Sangon Biotech. NMCFs cells were cultured in culture plates and transfected with the corresponding miRNAs using Lipofectamine RNAiMAX reagent (Thermo) according to the manufacturer's instructions. The culture medium was changed after 6 hours, and cells were collected after 24 hours for subsequent experiments.

[0055] Western Blot (WB) experiments: Whole-cell protein concentration was determined using a biuret acid (BCA) protein quantification kit (Applygen Technologies) according to the manufacturer's instructions. Subsequently, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinyl glycol membrane (PVDF, IPVH00010, Millipore), and blocked with blocking buffer at room temperature for 2 hours. The membrane was then incubated with the following primary antibodies: anti-SKP1 (Proteintech, cat#67745-1-Ig) and anti-GAPDH Rabbit polyclonal antibody (Abcam, cat#ab9485). After washing, the membrane was incubated with secondary antibodies (anti-rabbit IgG, Abcam, catalog number ab6721; anti-mouse IgG, Abcam, catalog number 6728). Protein expression was detected using an enhanced chemiluminescence system (Millipore). The intensity of each band was measured using ImageJ software.

[0056] The quantitative PCR (qPCR) experimental method is the same as described above. The primer information used in this experiment is as follows:

[0057]

[0058] Dual-luciferase reporter assay: 3000 293T cells were seeded per well in a 96-well plate. After 24 hours, hsa-miR-4324 NC or hsa-miR-4324 and 30 ng of SKP1 3'-UTR plasmid, mutant plasmid, or empty vector control plasmid (psiCheck-2 plasmid, Promega, USA) were simultaneously transfected. A Lipo3000 (Thermo) was used during transfection, and the procedure was performed according to the manufacturer's instructions. Luciferase activity was measured 48 hours later using a dual-luciferase assay system (Promega) according to the manufacturer's protocol. Fluorescence intensity was measured using a BioTek Cytation5 automated microplate reader (Agilent, USA).

[0059] Test results:

[0060] We isolated neonatal cardiac fibroblasts (NMCFs) from 1-2 day old C57BL / 6J newborn mice to further investigate the mechanism of hsa-miR-4324 in cardiac surgery-induced collagen deposition. We added an hsa-miR-4324 mimic to the culture medium of NMCFs to simulate the upregulation of hsa-miR-4324 expression after cardiac surgery and observed its effect on SKP1. qPCR and Western blotting showed a significant decrease in the relative expression of SKP1, which could be reversed by an hsa-miR-4324 inhibitor (qPCR results were...). Figure 8 WB and statistical charts are Figure 9 and Figure 10 The above results indicate that hsa-miR-4324 promotes collagen deposition and the formation of proarrhythmic atrial matrix by targeting the SKP1 (S-phase kinase associated protein 1) gene. Conversely, hsa-miR-4324 inhibitors can block these effects.

[0061] To verify that SKP1 is a direct target of hsa-miR-4324, a dual-luciferase reporter gene assay was used. hsa-miR-4324 significantly inhibited luciferase activity. Conversely, mutations in the predicted hsa-miR-4324 binding sequence in the SKP1 3′-UTR attenuated this effect, while the empty control group showed the same results. Figure 11 ).

[0062] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of miRNA for the preparation of a biomarker for the diagnosis of postoperative atrial fibrillation, characterized in that, The method comprises detecting the expression level of the miRNA in the biological sample, and comparing the expression level of the miRNA in the biological sample with that of a control group, and determining the risk of postoperative atrial fibrillation disease when the expression level of the miRNA in the biological sample is higher; The miRNA is at least one of hsa-miR-1304-3p, hsa-miR-4324 and hsa-miR-329-3p; and the biological sample is an extracellular vesicle in pericardial drainage fluid.

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