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

Abstract

The invention relates to the technical field of pharmaceutical compositions, and particularly discloses application of miRNA (micro Ribonucleic Acid) in prevention or treatment of postoperative atrial fibrillation diseases, and the miRNA is at least one of hsa-miR-1304-3p, hsa-miR-4324 and hsa-miR-329-3p. Also disclosed are compositions for the diagnosis, prevention or treatment of postoperative atrial fibrillation disease using miRNA inhibitors. The invention further discloses a kit for diagnosing the postoperative atrial fibrillation disease. The kit comprises a reagent capable of detecting miRNA. Also disclosed is an information providing method for diagnosing a postoperative atrial fibrillation disease, comprising detecting the expression level of miRNA in a biological sample and comparing it to a control group, and determining the risk of the presence of a postoperative atrial fibrillation disease when the expression level of miRNA in the biological sample is higher. The miRNA is applied to prevention or treatment of the postoperative atrial fibrillation disease, and the miRNA can be effectively used for prevention, treatment and the like of the postoperative atrial fibrillation disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical compositions, and in particular, to the application of miRNA in preventing or treating postoperative atrial fibrillation disease. BACKGROUND

[0002] Postoperative atrial fibrillation (POAF) has a morbidity of about 30% after coronary artery bypass grafting (CABG). This arrhythmia is not a temporary phenomenon, and it has a significant impact on the short-term and long-term prognosis of patients after surgery, but its complex pathogenesis is still unknown. POAF usually peaks on the 2nd to 4th day after surgery, and is strongly associated with CABG. The current mainstream hypothesis is that the combination of the transient surgical manipulation of the heart during surgery and the fragile atrial matrix before surgery interact to cause postoperative cardiac remodeling, which is characterized by early atrial collagen deposition, and further leads to the occurrence of POAF.

[0003] Pericardial fluid (PCF) provides a unique non-invasive and direct window to observe the heart, which is rich in bioactive factors from the surgical site and reflects the underlying cardiac pathological conditions. Extracellular vesicles (EVs) play a key role in intercellular and intertissue communication, especially in the occurrence of arrhythmia and fibrosis. Among EVs, microRNAs (miRNAs) stand out due to their high stability and potential effects on upstream gene transcription. EVs from PCF of patients with chronic atrial fibrillation carry specific miRNA markers associated with cardiac fibrosis. Plasma-derived exosomal miRNAs such as hsa-miR-184 have been shown to be diagnostic biomarkers of acute kidney injury associated with cardiac surgery; therapeutic EVs derived from human cardiac cells effectively help resist POAF in aseptic pericarditis rat models through anti-inflammatory and anti-fibrotic effects. Although these studies have demonstrated the role of EV-miRNA in chronic atrial fibrillation and the therapeutic potential for POAF, the role of EV-miRNA derived from PCF in the pathogenesis of POAF has never been studied. SUMMARY

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

[0005] The present application is achieved by the following technical solution: The application of miRNA in preventing or treating postoperative atrial fibrillation disease, 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 application also provides a pharmaceutical composition for preventing or treating postoperative atrial fibrillation, comprising an miRNA inhibitor as an active ingredient; the miRNA is at least one of hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p. The database code of hsa-miR-4324 is MIMAT0016876, and the sequence is 5'-CCCUGAGACCCUAACCUUAA-3'; the database code of hsa-miR-1304-3p is MIMAT0022720, and the sequence is 5'-UCUCACUGUAGCCUCGAACCCC-3'; and the database code of hsa-miR-329-3p is MIMAT0001629, and the sequence is 5'-AACACACCUGGUUAACCUCUUU-3'.

[0007] Further, the miRNA inhibitor regulates postoperative atrial fibrillation by inhibiting the up-regulated miRNA in pericardial effusion.

[0008] Further, the miRNA inhibitor is any one selected from siRNA, aptamer, antisense oligonucleotide, ribozyme, and compound specific to miRNA.

[0009] The present application also provides a kit for diagnosing postoperative atrial fibrillation, comprising a reagent capable of detecting miRNA.

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

[0011] Further, the kit is selected from a microarray, an aptamer chip kit, an enzyme-linked immunosorbent assay kit, a serial analysis of gene expression kit, a quantitative real-time PCR kit, and a combination thereof.

[0012] The present application also provides an information providing method for diagnosing postoperative atrial fibrillation, comprising 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] Further, the miRNA is at least one of hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p.

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

[0015] The application has at least the following advantages and beneficial effects: The application of the miRNA in preventing or treating postoperative atrial fibrillation disease shows that the expression of hsa-miR-1304-3p, hsa-miR-4324 and hsa-miR-329-3p, especially hsa-miR-4324, has a clear correlation with postoperative atrial fibrillation disease (POAF), and high expression of the miRNA means a high probability of POAF, so these miRNAs can be used in the clinic for POAF disease, and can be further used for preventing or treating POAF. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The expression of multiple miRNAs at two postoperative time points is shown; Figure 2 The role of multiple miRNAs in the TGF-β signaling pathway is shown; Figure 3 The incidence of POAF after injection of three miRNAs is shown; Figure 4 The induction rate of POAF by different concentrations of hsa-miR-4324 is shown; Figure 5 The onset time of POAF after injection of hsa-miR-4324 is shown; Figure 6 The expression of hsa-miR-4324 in PCF-EVs of POAF patients and non-POAF patients is shown; Figure 7 The prediction ability of the three miRNAs for POAF is shown; Figure 8 The effect of hsa-miR-4324 on the SKP1 gene level is shown; Figure 9 The effect of hsa-miR-4324 on the SKP1 protein level is shown; Figure 10 The effect of hsa-miR-4324 on the SKP1 protein level after normalization with the internal reference protein GAPDH is shown; Figure 11 The role of hsa-miR-4324 in the SKP1 gene targeting inhibition in the dual luciferase reporter gene experiment is shown. DETAILED DESCRIPTION

[0017] Example 1 hsa-miR-4324 was significantly upregulated in POAF group and was closely related to the pro-fibrotic pathway.

[0018] Detection method: The miRNA mass in PCF exosomes from individuals with POAF and non-POAF at 0.5 hours and 6 hours after surgery was sequenced.

[0019] Isolation of PCF-EVs: The isolation method of PCF-EVs followed the guidelines of the International Society for Extracellular Vesicles (MISE 2018). PCF-EVs were isolated using differential ultracentrifugation. Specifically, PCF samples were thawed on ice and subjected to centrifugation at 3000 g for 30 minutes followed by 10000 g for 1 hour at 4°C. The supernatant was filtered through a 0.45 pm filter (CORNING). Subsequently, the supernatant was transferred to an ultracentrifuge tube (part number: 331372, BECKMAN) and centrifuged at 100000 g for 70 minutes at 4°C. The pellet was washed with phosphate-buffered saline (PBS) and the ultracentrifugation step was repeated. The supernatant was discarded and the extracellular vesicle pellet was resuspended in PBS and stored at -80°C for further analysis.

[0020] miRNA extraction and library preparation: Total RNA (including miRNA) was purified from exosomes by miRNeasy Micro Kit (217084, QIAGEN, ) according to its manual. The gel-free workflow provided by QIAseq miRNA Library Kit (331505, QIAGEN, ) was used for miRNA-seq library preparation. Briefly, adapters were sequentially ligated to the 3' and 5' ends of miRNA in a non-biased reaction. Subsequently, the steps of universal cDNA synthesis with unique molecular identifier (UMI), cDNA purification, library amplification and library purification were performed.

[0021] miRNA-seq data analysis: Adapters were removed using Cutadapt (version 2.7). Reads with low quality, less than 16 bp in length, or less than 12 bp in UMI length were removed. Clean reads were mapped to human mature miRNA sequences downloaded from miRBase (version 22) by bowtie (version 1.2.2) with parameters -v 0 (no allowance for mismatches), -m 5 (allow up to 5 multi-mapping events), --best --strata (return only one 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 by 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).

[0022] Differential expression analysis: The trimmed mean of M-values (TMM) of each pair of sample pairs was normalized by 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. The mRNA with an absolute fold change greater than 1.5 and a p-value less than 0.05 was considered to be differentially expressed miRNA, and visualization was performed using ggplot2 (version 2.2.1).

[0023] 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. In order to obtain high-reliability target genes regulated by differentially expressed miRNAs, only miRNA-target gene regulatory relationships supported by both experimental and predictive evidence were retained.

[0024] Functional enrichment analysis: The potential target genes of miRNAs were input into the R package clusterProfiler (version 4.6.2) to calculate the enrichment degree (P-value) of the corresponding biological processes and KEGG pathways.

[0025] Detection results: The top ten miRNAs that continuously increased in expression at two time points (0.5 hours and 6 hours after surgery) were investigated, as shown in Table 1. Figure 1hsa-miR-4324 was shown to be significantly overexpressed at 0.5h and 6h post- surgery compared to other miRNAs. After removing the redundant miRNAs, we found that a total of 24 miRNAs were upregulated in POAF group, among which 20 had more than 20 highly credible potential target genes, which were further analyzed for functional enrichment. The results showed that, as shown in Fig. 2, these miRNAs played a key role in transforming growth factor-β (TGF-β) signaling pathway, which could accelerate the process of fibrosis by promoting fibroblast proliferation, collagen secretion and extracellular matrix deposition, and hsa-miR-4324 was the most significant. Figure 2

[0026] Example 2 The incidence of POAF in mice injected with hsa-miR-4324 was significantly increased and showed a dose-dependent effect.

[0027] Detection method: Intracardiac miRNAs injection: Mice were anesthetized, intubated, and the heart was exposed. To evaluate the proarrhythmic potential of the 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 of a previously prepared mixture of miRNA mimic (20 μL, 10 μM) and lipids (Lipofectamine RNAiMAX, 1:1 volume ratio) at the left ventricular anterior wall using a 1 mL syringe with a 34-gauge needle, following the manufacturer's protocol. Control mice received injections of a non-targeting sequence (NC) mimic. After injection, the intercostal space, muscle and skin were sutured. Subsequently, mice were implanted with a wireless telemetry device for continuous monitoring, as previously described, and the occurrence of sPOAF was observed within 5 days. Postoperative infection was prevented by intramuscular injection of penicillin (20000 units daily) for 3 days. Mice were extubated to restore normal breathing and placed on a heating pad until they woke up, then transferred to a cage.

[0028] ​Three miRNAs, hsa-miR-1304-3p, hsa-miR-4324 and hsa-miR-329-3p, were consistently upregulated at both time points (0.5h and 6h post-surgery) and had the largest fold change and abundance. Notably, hsa-miR-4324 was the only consistently upregulated miRNA with the largest fold change and abundance at both time points. Meanwhile, hsa-miR-1304-3p was the most variable miRNA at 6h post-surgery. Therefore, these three miRNAs were further functionally validated. The three miRNAs were injected into the left ventricular (LV) anterior wall of 6-month-old male mice, with null control mimics as controls. As shown in FIG. 8, 5-day continuous heart rate monitoring showed 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, and no POAF was observed in the control group. Figure 3

[0029] The induction of POAF by hsa-miR-4324 showed a clear dose-dependent effect. The proportion of mice developing POAF after injection of 5, 10 and 20 μΜ hsa-miR-4324 was 50%, 83.3% and 100%, respectively (as shown in FIG. 9). Figure 4

[0030] In mice injected with 10 μΜ miR-4324, the time distribution of POAF peaked at 2-3 days after injection (as shown in FIG. 10), which is closely related to the clinical manifestations of POAF after cardiac surgery. Figure 5

[0031] Example 3 hsa-miR-4324 can effectively predict POAF.

[0032] ​​​RNA Extraction and Real-Time Quantitative PCR: Total RNA (including miRNA) was extracted using Trizol reagent (Invitrogen). The 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 the One-Step gDNA Removal and cDNA Synthesis Supermix (TransGen Biotech), followed by real-time qPCR using the PerfectStart Green qPCR Supermix (TransGen Biotech). Data were analyzed according to the 2-ΔΔCt method. Primer information is as follows:

[0033] ROC Curve Analysis: In SPSS software (version 20), select "Analyze" > "Binary Logistic Regression" > "ROC Curve," 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; discriminatory ability). The ROC curve was used to determine the optimal cutoff value for miR-4324 to optimize sensitivity and specificity.

[0034] Quantitative PCR (qPCR) analysis showed that Figure 6 As shown in Figure 2, the level of hsa-miR-4324 in PCF-EVs of POAF patients was significantly increased compared with that of non-POAF patients. Figure 7 As shown, hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p effectively predicted POAF. The area under the curve (AUC) for each miRNA individually was 0.784 for hsa-miR-1304-3p, 0.853 for hsa-miR-4324, and 0.774 for hsa-miR-329-3p. The AUC for the combined prediction of the three miRNAs was 0.903. All three miRNAs demonstrated good predictive ability for POAF individually, with hsa-miR-4324 showing the best prediction. The combined prediction of the three miRNAs was even better than that of hsa-miR-4324.

[0035] Example 4 hsa-miR-4324 plays a role in promoting fibrosis by targeting the SKP1 gene.

[0036] Detection method: Isolation and culture of primary cardiac fibroblasts: Neonatal mouse cardiac fibroblasts (NMCFs) were isolated from 1-2 day old neonatal C57BL / 6J mice. After the ventricle was rinsed with cold phosphate buffer solution (PBS), it was cut into 1 mm³ size pieces and digested with 0.0625% trypsin (Sigma-Aldrich) at room temperature for 5 minutes, followed by continuous shaking digestion with collagenase type II (1 mg / mL, Solarbio) at 37°C for 5 minutes. After precipitation, the supernatant was collected and added with Dulbecco's modified Eagle medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin / streptomycin (Hyclone). The 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 the cells were precipitated by centrifugation at 1000 × g for 10 minutes. The cell pellet was resuspended with DMEM containing 10% FBS and inoculated in a 6 cm culture dish. After incubation at 37°C, 5% CO2 for 1.5 hours, the cells adhering to the bottom of the culture dish were NMCFs.

[0037] miRNA transfection: hsa-miR-4324 mimic negative control (N.C.), hsa-miR-4324 inhibitor negative control, hsa-miR-4324 mimic and hsa-miR-4324 inhibitor were purchased from Sangon Biotech. NMCF cells were cultured in culture plates, and the corresponding miRNA transfection was performed using Lipofectamine RNAiMAX reagent (Thermo) according to the instructions. The medium was replaced after 6 hours, and the cells were collected after 24 hours for subsequent experiments.

[0038] Western Blot (WB) experiment: The whole cell protein concentration was determined by bicinchoninic acid (BCA) protein assay kit (Applygen Technologies) according to the instruction. Then, the protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (PVDF, IPVH00010, Millipore) and blocked with blocking buffer at room temperature for 2 hours. Then, the membrane was incubated with the following primary antibody: anti-SKP1 (Proteintech, cat#67745-1-Ig), anti-GAPDH Rabbit Polyclonal antibody (Abcam, cat#ab9485). After washing the membrane, it was incubated with secondary antibody (anti-rabbit IgG, Abeam, cat# ab6721; anti-mouse IgG, Abeam, cat# 6728). Protein expression was detected by enhanced chemiluminescence system (Millipore). The intensity of each band was measured using Image J software.

[0039] Quantitative PCR (qPCR) experiment method was the same as above, and the primer information used in this experiment was as follows:

[0040] Dual-luciferase reporter assay: 3000 293T cells were seeded in each well of a 96-well plate, and after 24 hours, hsa-miR-4324 N.C. or hsa-miR-4324 and 30 ng SKP1 3'-UTR plasmid or mutant plasmid or empty vector control plasmid (psiCheck-2 plasmid, Promega, USA) were transfected simultaneously. Lipo3000 (Thermo) was used during the transfection process and the operation was carried out according to the manufacturer's instructions. After 48 hours, the dual-luciferase detection system (Promega) was used and the luciferase activity was determined according to the manufacturer's protocol. The fluorescence intensity was determined by BioTek Cytation5 automatic microplate reader (Agilent, USA).

[0041] Detection results: We isolated neonatal mouse cardiac fibroblasts (NMCFs) from 1-2 day old C57BL / 6J neonatal mice to further investigate the mechanism of hsa-miR-4324 in cardiac collagen deposition induced by cardiac surgery. We added hsa-miR-4324 mimics to the culture medium of NMCFs to simulate the upregulation of hsa-miR-4324 expression after cardiac surgery and observe its effect on SKP1. qPCR and WB detection showed that the relative expression of SKP1 was significantly decreased, while these phenomena could be reversed by hsa-miR-4324 inhibitors (qPCR results are Figure 8 ; WB and statistical graphs are Figure 9 and Figure 10 ). The above results show that hsa-miR-4324 promotes collagen deposition and forms proarrhythmia atrial substrate by targeting the SKP1 (S-phase kinase associated protein 1) gene. Conversely, hsa-miR-4324 inhibitors can block these effects.

[0042] To verify that SKP1 is a direct target of hsa-miR-4324, a dual luciferase reporter gene experiment was used. hsa-miR-4324 significantly inhibited luciferase activity. Conversely, mutation of 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 ).

[0043] The above is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. The use of miRNA in preventing or treating postoperative atrial fibrillation, characterized in that: The miRNA is at least one of hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p.

2. The use of the miRNA according to claim 1 in preventing or treating postoperative atrial fibrillation, characterized in that: The miRNA is preferably hsa-miR-4324.

3. A pharmaceutical composition for preventing or treating postoperative atrial fibrillation, characterized in that: The pharmaceutical composition comprises a miRNA inhibitor as an active ingredient; The miRNA is at least one of hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p.

4. The pharmaceutical composition for preventing or treating postoperative atrial fibrillation according to claim 3, characterized in that: The miRNA inhibitor regulates postoperative atrial fibrillation by inhibiting the upregulated expression of miRNA in the pericardial sac fluid.

5. The pharmaceutical composition for preventing or treating postoperative atrial fibrillation according to claim 3, characterized in that: The miRNA inhibitor is any one selected from siRNA, aptamer, antisense oligonucleotide, ribozyme and compound specific to miRNA.

6. A kit for diagnosing postoperative atrial fibrillation, characterized in that: The kit comprises a reagent capable of detecting miRNA; The miRNA is at least one of hsa-miR-1304-3p, hsa-miR-4324, and hsa-miR-329-3p.

7. The kit for diagnosing postoperative atrial fibrillation according to claim 6, characterized in that: The kit is selected from the group consisting of a microarray, an aptamer chip kit, an enzyme-linked immunosorbent assay kit, a gene expression serial analysis kit, a quantitative real-time PCR kit, and a combination thereof.

8. A method for providing information for diagnosing postoperative atrial fibrillation, characterized in that: The method includes detecting the expression level of miRNA in the biological sample and comparing it with 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.

9. The method for providing information for diagnosing postoperative atrial fibrillation according to claim 8, wherein: The biological sample is extracellular vesicles in pericardial drainage fluid.

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