Application of miRNA-454 in the treatment of atherosclerosis
By targeting the IRF1 protein with miRNA-454, inhibiting the IRF1-NF-κB pathway, suppressing M1 macrophage polarization and promoting M2 macrophage polarization, the problem of plaque inflammation and insufficient phagocytic capacity in atherosclerosis was solved, achieving the effect of slowing disease progression and reducing cardiovascular risk.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- FUWAI HOSPITAL CHINESE ACAD OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
- Filing Date
- 2026-04-27
- Publication Date
- 2026-06-02
AI Technical Summary
Current treatments for atherosclerosis are unable to effectively suppress plaque inflammation and restore macrophage phagocytic capacity, leading to a high risk of cardiovascular disease.
By using miRNA-454 and a formulation that promotes its expression, the IRF1 protein is targeted, the IRF1-NF-κB pathway is inhibited, M1 macrophage polarization is suppressed and M2 macrophage polarization is promoted, and the phagocytic capacity of macrophages is restored.
It reduces plaque inflammation, promotes the clearance of apoptotic debris from lesions, slows the progression of atherosclerosis, and reduces the risk of cardiovascular disease.
Smart Images
Figure CN122124086A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cardiovascular diseases and discloses the application of miRNA-454 in the treatment of atherosclerosis. Background Technology
[0002] Atherosclerosis (AS) is one of the leading causes of disease-related morbidity and mortality worldwide, often leading to coronary atherosclerotic heart disease, stroke, and peripheral vascular disease. Although lipid-lowering treatments, including classic statins and novel PCSK9 inhibitors, control blood lipid levels, slow AS progression, and improve patient prognosis, the cardiovascular risk associated with AS remains high. Therefore, researchers place great emphasis on the study and treatment of cardiovascular and cerebrovascular diseases.
[0003] MicroRNAs (miRNAs) are a class of conserved, endogenous, small (20-24 nt) single-stranded non-coding RNAs that function within cells and play an irreplaceable role in post-transcriptional regulation. In recent years, the regulatory role of miRNAs in atherosclerosis and their potential as therapeutic targets have received widespread attention. miRNAs can influence the proliferation, migration, apoptosis, and inflammatory responses of vascular smooth muscle cells by regulating the expression of related genes, thereby participating in the formation and development of atherosclerosis. Previous studies have reported that miRNA-221 / 222 downregulates p27 in an ApoE- / - atherosclerosis model, thereby inhibiting vascular smooth muscle cell (VSMC) proliferation and exerting a cardiovascular protective effect [Kothapalli D, Castagnino P, Rader DJ, et al. Apolipoprotein E-mediated cell cycle arrest linked to p27 and the Cox2-dependent repression of miR221 / 222 [J]. Atheroselerosis, 2013,227:65-71]. Highly differentiated VSMCs are rich in miRNA-143 and miRNA-145, which can reduce atherosclerotic plaques and vascular intimal hyperplasia [Albinsson S, Sward K. Targeting smooth muscle microRNAs for therapeutic benefit in vascular disease[J]. Pharmacol Res,2013,75:28-36]. Therefore, identifying the miRNAs specifically expressed in diseases such as atherosclerosis and elucidating their mechanisms of action is of great significance for disease treatment. Summary of the Invention
[0004] The purpose of this invention is to overcome the shortcomings of existing treatments and provide a miRNA, namely miRNA-454, that plays a key role in AS disease, thus providing a new, highly effective, and stable therapeutic agent for the treatment of AS.
[0005] To achieve the above objectives, the first aspect of the present invention provides the use of miRNA-454 and / or preparations that promote miRNA-454 expression in the preparation of a medicament for treating atherosclerosis.
[0006] A second aspect of the present invention provides the use of miRNA-454 and / or formulations that promote miRNA-454 expression in the preparation of a medicament for inhibiting macrophage polarization toward pro-inflammatory M1 macrophages.
[0007] A third aspect of the present invention provides the use of miRNA-454-3p and / or a formulation that promotes miRNA-454 expression in the preparation of a drug for promoting the polarization of macrophages into anti-inflammatory M2 macrophages.
[0008] The fourth aspect of the present invention provides the use of miRNA-454 and / or formulations that promote miRNA-454 expression in the preparation of a medicament for inhibiting IRF1 protein activity.
[0009] The fifth aspect of the present invention provides the use of miRNA-454 and / or formulations that promote miRNA-454 expression in the preparation of drugs for inhibiting the IRF1-NF-κB pathway.
[0010] The sixth aspect of the present invention provides the use of miRNA-454 and / or formulations that promote miRNA-454 expression in the preparation of a medicament for promoting the restoration of macrophage phagocytic capacity.
[0011] Through the above technical solution, this invention has for the first time discovered that miRNA-454 and / or preparations that promote miRNA-454 expression can inhibit the IRF1-NF-κB pathway by inhibiting IRF1 protein activity, thereby further inhibiting macrophage polarization towards pro-inflammatory M1 macrophages and reprogramming them towards anti-inflammatory M2 macrophage polarization, thus reducing plaque inflammation; at the same time, it can also restore the phagocytic capacity of phagocytes, promote the clearance of apoptotic debris from lesions, and ultimately slow down the progression of AS. Attached Figure Description
[0012] Figure 1A-1DThis study demonstrates the targeting relationship between hsa-miRNA-454-3p and IRF1, and confirms the activation of the IRF1-NF-κB pathway. Specifically, A: Dual-luciferase reporter assay quantifies the interaction between hsa-miRNA-454-3p and IRF1 (n = 3 per group); B: qPCR analysis of IRF1 and IL-12p35 mRNA expression levels in M0 and M1 macrophages (n = 4 per group); CD: Western blot analysis of representative images and quantitative analysis of IRF1 protein and nuclear NF-κB expression levels (n = 3-4 per group). P < 0.05, P < 0.01, P < 0.001, P < 0.0001.
[0013] Figure 2A-2G This diagram illustrates the mechanism by which hsa-miRNA-454-3p inhibits M1 macrophage polarization by targeting IRF1 and inhibiting the IRF1-NF-κB pathway. Specifically: A. A schematic workflow for verifying macrophage polarization after transfection with hsa-miRNA-454-3p mimics or hsa-miRNA-454-3p mimic NC; B. qPCR quantitative analysis of macrophage polarization marker mRNA expression levels after macrophage transfection with hsa-miRNA-454-3p mimics or hsa-miRNA-454-3p mimic NC (n = 3 per group); C. Immunofluorescence staining (C) and quantitative analysis (D) of macrophage polarization marker protein expression after macrophage transfection (n = 3 per group); E. qPCR analysis of IRF1, IFN-β, and IL-12p35 mRNA expression levels (n = 3 per group); FG. Representative immunoblot images (F) and quantitative analysis (G) of IRF1 protein levels and nuclear NF-κB in treated macrophages (n = 3 per group) P < 0.05, P < 0.01, P < 0.001, P < 0.0001.
[0014] Figure 3A-3JThe following images illustrate in vivo experimental results of hsa-miRNA-454-3p agonist treatment for AS; A. Schematic diagram illustrating the treatment regimens of hsa-miRNA-454-3p agonist and agonist NC in an ApoE- / - atherosclerotic mouse model; BD. Representative Oil Red O staining images showing the aorta (B) and aortic root (C), with corresponding quantitative analyses (D) (n = 6 per group); EF. Representative immunofluorescence staining images and quantitative analyses of CD68 and CD206 in the aortic root (n = 6 per group); GH. Representative immunofluorescence staining images and quantitative analyses of CD47, p-SHP-1, and free apoptotic bodies in the aortic root (n = 4 per group); IJ. Representative immunohistochemical images and quantitative analysis of IRF1 expression in the aortic root (n = 6 per group). P < 0.05, P < 0.01, P < 0.001, P < 0.0001. Detailed Implementation
[0015] The endpoints and any values of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.
[0016] The first aspect of the present invention provides the use of miRNA-454 and / or formulations that promote miRNA-454 expression in the preparation of a medicament for treating atherosclerosis.
[0017] In this invention, preferably, the miRNA-454 is hsa-miRNA-454-3p, and its sequence is shown in SEQ ID NO:1, UAGUGCAAUAUUGCUUAUAGGGU.
[0018] In this invention, preferably, the formulation that promotes miRNA-454 expression is an hsa-miRNA-454-3p agonist, the sequence of which is shown in SEQ ID NO:2.
[0019] In this invention, the disease is atherosclerosis, a common vascular disease characterized by thickening and hardening of the arterial walls, loss of elasticity, and narrowing of the lumen. Because the lipids accumulated on the arterial intima appear as a yellowish, porridge-like substance, it is called atherosclerosis. It mainly affects large and medium-sized arteries, and its clinical manifestations are primarily those of the affected organs. This invention uses miRNA-454 and / or preparations that promote miRNA-454 expression to treat atherosclerosis, which can effectively reduce plaque and alleviate symptoms.
[0020] A second aspect of the present invention provides the use of miRNA-454 and / or formulations that promote miRNA-454 expression in the preparation of a medicament for inhibiting macrophage polarization toward pro-inflammatory M1 macrophages.
[0021] A third aspect of the present invention provides the use of miRNA-454-3p and / or a formulation that promotes miRNA-454 expression in the preparation of a drug for promoting the polarization of macrophages into anti-inflammatory M2 macrophages.
[0022] The present invention also provides the application of miRNA-454 and / or formulations that promote miRNA-454 expression in the in vitro inhibition of macrophage polarization into pro-inflammatory M1 macrophages and promotion of anti-inflammatory M2 macrophage polarization.
[0023] In this invention, preferably, the miRNA-454 and / or the formulation that promotes miRNA-454 expression promotes the expression of M2 macrophage markers CD206, Fibronectin and CCL22.
[0024] In this invention, preferably, the miRNA-454 and / or the formulation that promotes miRNA-454 expression inhibits the expression of M1 macrophage markers CD80, IL-1β and TNF-α.
[0025] The fourth aspect of the present invention provides the use of miRNA-454 and / or formulations that promote miRNA-454 expression in the preparation of a medicament for inhibiting IRF1 protein activity.
[0026] This invention also provides the use of miRNA-454 and / or formulations that promote miRNA-454 expression in the in vitro inhibition of IRF1 protein activity.
[0027] The fifth aspect of the present invention provides the use of miRNA-454 and / or formulations that promote miRNA-454 expression in the preparation of drugs for inhibiting the IRF1-NF-κB pathway.
[0028] This invention also provides the application of miRNA-454 and / or formulations that promote miRNA-454 expression in the in vitro inhibition of the IRF1-NF-κB pathway.
[0029] In this invention, the miRNA-454 and / or the formulation that promotes miRNA-454 expression can specifically target IRF1, inhibit the IRF1-NF-κB pathway and M1 macrophage polarization, promote anti-inflammatory M2 macrophage polarization, reduce plaque inflammation, and thus slow down disease progression.
[0030] The sixth aspect of the present invention provides the use of miRNA-454 and / or formulations that promote miRNA-454 expression in the preparation of a medicament for promoting the restoration of macrophage phagocytic capacity.
[0031] Furthermore, this invention also provides the application of miRNA-454 and / or formulations that promote miRNA-454 expression in the in vitro promotion of macrophage phagocytic capacity recovery. By restoring macrophage phagocytic capacity and accelerating the clearance of apoptotic debris from lesions, the progression of atherosclerosis can be improved.
[0032] In this invention, the miRNA-454 is hsa-miRNA-454-3p, and its sequence is as described above and will not be repeated here.
[0033] In this invention, the agent that promotes miRNA-454 expression is an hsa-miRNA-454-3p agonist, the sequence of which is as described above and will not be repeated here.
[0034] In this invention, the miRNA-454 and / or the formulation that promotes miRNA-454 expression can be formulated into common dosage forms, such as injections, oral liquids, tablets, granules, capsules, and pills, etc., preferably, as injections.
[0035] In this invention, the drug may further include pharmaceutically acceptable excipients, which include at least one of excipients, fillers, binders, humectants, sustained-release agents, absorption enhancers, surfactants, and lubricants.
[0036] In this invention, the administration method of the miRNA-454 and / or the formulation that promotes miRNA-454 expression is injection, preferably intravenous injection.
[0037] In this invention, the animal model used is a rodent, preferably a mouse, to construct an atherosclerosis disease model.
[0038] The present invention will be described in detail below through embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available. In this invention, NC is an abbreviation for Negative Control.
[0039] hsa-miRNA-454-3p mimics and hsa-miRNA-454-3p mimic NC were purchased from Guangzhou RiboBioCo.,Ltd., product code MQPS0001461-1-200, name: Bulge-Loop hsa-miRNA-454-3p PrimerSet, 200T; ApoE- / - mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.
[0040] The specific sequences involved in this embodiment are as follows: Table 1
[0041] In the sequences shown in Table 1, the hsa-miRNA-454-3p agonist and agonist NC: the entire chain is modified with methoxy groups, with 2 and 4 thiocarbamates at the 5' and 3' ends, respectively, and cholesterol is modified at the 3' end. All of them were synthesized by Genen Biotechnology Co., Ltd.
[0042] Example 1 To verify the targeting relationship between hsa-miRNA-454-3p and IRF1, and the activation of the IRF1-NF-κB pathway.
[0043] To verify this prediction, a dual-luciferase reporter gene assay targeting the IRF1 3'UTR region was performed. 293T cells were grown in high-glucose DMEM medium (Gibco, USA) containing 10% FBS. 293T cells in logarithmic growth phase were digested with 0.25% EDTA-containing trypsin, digestion was terminated with complete medium, and the cells were centrifuged at 800 rpm for 5 minutes at 4°C. The cell pellet was then resuspended in antibiotic-free complete medium to form single cells and counted. The number of cells seeded in each well of a 96-well plate was 1.5 × 10⁶ cells. 40.525 μL of hsa-miRNA-454-3p mimic or hsa-miRNA-454-3p mimic NC (original concentration 20 μM) and 15 ng of wild-type or mutant dual-luciferase reporter gene plasmid were diluted in 5 μL of Opti-MEM (Gibco, USA) and gently mixed with an RNase A-free sterile pipette tip. 0.16 μL of Lipofactamine 3000 (Invitrogen, USA) was added to the above reaction system and gently mixed with an RNase A-free sterile pipette tip, and incubated at room temperature for 20 minutes. 105 μL of a mixture containing hsa-miRNA-454-3p mimic or hsa-miRNA-454-3p mimic NC, wild-type or mutant dual-luciferase reporter gene plasmid, and Lipofactamine was added to the mixture. Add the mixture of 3000 and Opti-MEM to the bottom of the 6-well plate and gently shake to mix. Then add 1.5 × 10⁻⁶ ppm of the mixture to each well. 4 Each cell was cultured in 100 μL of antibiotic-free complete medium, shaken well, and then incubated at 37°C with 5% CO2 for 6 hours. Cells were harvested and the presence of microRNAs that bound to and regulated the expression of target genes was detected using a dual-luciferase reporter gene system (Promega, USA).
[0044] like Figure 1A As shown, hsa-miRNA-454-3p reduced luciferase activity in the wild-type WT 3'UTR group of IRF1, but had no significant effect on luciferase activity in the mutant 3'-UTR-Mut group. Although there was no statistically significant difference between the hsa-miRNA-454-3p psiCHECK2-IRF1 3'UTR group and the hsa-miRNA-454-3p psiCHECK2-IRF1 3'UTR-Mut group, there was a clear decreasing trend.
[0045] IRF1 is an important molecule for the activation of M1 macrophages. Previous studies have reported that macrophages, under the stimulation of IFN-γ, form a complex by binding IRF1 to MyD88, which in turn activates the downstream TLR-MyD88 signaling pathway, mediating the production of iNOS, IFN-β, and IL-12p35, thereby playing a crucial role in initiating effective innate and adaptive immunity (Negishi H, Fujita Y, Yanai H, et al. Evidence for licensing of IFN-gamma-induced IFN regulatory factor 1 transcription factor by MyD88 in Toll-like receptor-dependent gene induction program[J]. Proc Natl Acad Sci USA, 2006,103(41): 15136-15141.).
[0046] To validate this pathway, adherent THP-1 cells (M1) were stimulated for 24 hours using 100 ng / mL LPS (Sigma-Aldrich, USA) and 20 ng / mL IFN-γ (PEPPROTECH, USA). Western blot (WB) and quantitative PCR (qPCR) (QuantStudio 5, Thermo, USA) were used to detect and analyze the expression levels of macrophage surface markers, as well as IRF1 and NF-κB proteins. WB and qPCR results showed that the levels of IRF1 protein and transcription were significantly upregulated in M1 macrophages. Figure 1B -D), while the level of NF-κB nuclear translocation increased significantly ( Figure 1D The transcriptional level of its downstream IL-12p35 was also significantly increased. Figure 1B ).
[0047] To clarify the functional role of hsa-miRNA-454-3p in the regulation of the IRF1-NF-κB pathway, unactivated monocytes (M0) and THP-1 cells (M1) stimulated with 100 ng / mL LPS and 20 ng / mL IFN-γ for 6 hours were used as negative and positive controls, respectively. Transfection was initiated when the transfected cell density reached 60%. 1.5 μL of lipofactamine 3000 (Invitrogen, USA) and 50 μL of Opti-MEM (Gibco, USA) were mixed and incubated at room temperature for 5 minutes. Then, 1 μL of 100 nM hsa-miRNA-454-3p mimic or hsa-miRNA-454-3p mimic NC was added to the mixture and incubated at room temperature for 30 minutes. The transfection mixture was then added to the cells and incubated in a cell culture incubator for 6-10 hours. Figure 2A ).
[0048] Immunofluorescence and qPCR results showed that, with the M1 group as the control, the levels of M1 macrophage markers CD80, IL-1β, and TNF-α protein and transcription were significantly decreased in the hsa-miRNA-454-3p mimic group, while the levels of M2 macrophage markers such as CD206, Fibronectin, and CCL22 protein and transcription were significantly upregulated. Figure 2B -D). Meanwhile, in the hsa-miRNA-454-3p mimicry group, the expression levels of IRF1 and NF-κB nuclear transposons were significantly downregulated, and the transcriptional levels of IRF1, IFN-β, and IL-12p35 were significantly decreased. Figure 2E In summary, hsa-miRNA-454-3p can inhibit macrophage M1 polarization and promote M2 activation by targeting and binding to IRF1 and inhibiting the expression and activation of its downstream IRF1-NF-κB pathway. This verifies the important mechanism by which hsa-miRNA-454-3p regulates macrophage polarity.
[0049] Example 2 A mouse model of atherosclerosis was constructed for testing.
[0050] Six-week-old ApoE- / - mice were fed a Western diet (containing 40% fat and 1.25% cholesterol) to induce atherosclerosis. After 12 weeks of continuous feeding, the model was established. Then, at 12 weeks (i.e., when the mice were 18 weeks old), the model mice were injected via tail vein with PBS (same volume as the agonist), 20 nmol hsa-miRNA-454-3p agonist NC, and 20 nmol hsa-miRNA-454-3p agonist, respectively, twice a week for 8 weeks (a total of 16 injections). At 20 weeks (i.e., when the mice were 26 weeks old), samples were collected from the mice, and the atherosclerotic plaques on the en-face surface of the aorta and the aortic root were analyzed by Oil Red O staining.
[0051] Oil Red O staining of the aorta revealed a significant reduction in plaque in the treated mouse group (hsa-miRNA-454-3p agonist NC: 23.02% ± 6.46% VS hsa-miRNA-454-3p agonist: 14.35% ± 3.22% VS PBS: 25.39% ± 5.64%, P<0.05-0.01), but plaque measurement at the aortic root showed no statistically significant difference (hsa-miRNA-454-3p agonist NC: 38.35% ± 5.97% VS hsa-miRNA-454-3p agonist: 38.50% ± 7.04% VS PBS: 41.96% ± 3.34%, P>0.05). Figure 3B -D).
[0052] To further clarify the effect of hsa-miRNA-454-3p agonist treatment on the polarization of macrophages infiltrating AS plaques, immunofluorescence was performed on aortic root plaques. Results showed that the number of CD68+ macrophages infiltrating the hsa-miRNA-454-3p agonist group was significantly lower than that in the PBS group and the hsa-miRNA-454-3p agonist NC group, while the number of CD206+ macrophages was significantly higher. Figure 3E -F). Additionally, the hsa-miRNA-454-3p agonist group significantly reduced CD47 and p-SHP-1 fluorescence intensity and significantly reduced free apoptotic bodies (F). Figure 3G Immunohistochemical analysis demonstrated that treatment with the hsa-miRNA-454-3p agonist significantly inhibited IRF1 expression, consistent with the hypothesized mechanism (H). Figure 3I -J).
[0053] In summary, hsa-miRNA-454-3p can directly target IRF1, inhibit the IRF1-NF-κB pathway, thereby suppressing M1 macrophage polarization and restoring phagocytosis by enhancing apoptosis clearance. This dual therapeutic effect effectively slows down the development of AS.
[0054] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.
Claims
1. The use of miRNA-454 and / or preparations that promote miRNA-454 expression in the preparation of drugs for the treatment of atherosclerosis.
2. The use of miRNA-454 and / or formulations that promote miRNA-454 expression in the preparation of drugs for inhibiting macrophage polarization into pro-inflammatory M1 macrophages.
3. The use of miRNA-454-3p and / or formulations that promote miRNA-454 expression in the preparation of drugs for promoting macrophage polarization into anti-inflammatory M2 macrophages.
4. The use of miRNA-454 and / or formulations that promote miRNA-454 expression in the preparation of drugs for inhibiting IRF1 protein activity.
5. The use of miRNA-454 and / or formulations that promote miRNA-454 expression in the preparation of drugs for inhibiting the IRF1-NF-κB pathway.
6. The use of miRNA-454 and / or formulations that promote miRNA-454 expression in the preparation of drugs for promoting the recovery of macrophage phagocytic capacity.
7. The application according to any one of claims 1-6, characterized in that, The miRNA-454 is hsa-miRNA-454-3p.
8. The application according to any one of claims 1-6, characterized in that, The formulation that promotes miRNA-454 expression is an hsa-miRNA-454-3p agonist.
9. The application according to any one of claims 1-6, characterized in that, The dosage form of the drug is at least one of the following: injection, oral liquid, tablet, granule, capsule, and pill.
10. The application according to any one of claims 1-6, characterized in that, The drug also includes pharmaceutically acceptable excipients, which are at least one of excipients, fillers, binders, humectants, sustained-release agents, absorption enhancers, surfactants, and lubricants.