Application of CCL3-AS1 in regulating stability of carotid plaque
By regulating macrophage inflammatory activity and MMP9 secretion through CCL3-AS1, this study addresses the lack of research on the molecular mechanisms of carotid plaque instability, provides a predictive and therapeutic approach for carotid plaque rupture, and achieves effective regulation of plaque stability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- XIANGYA HOSPITAL CENT SOUTH UNIV
- Filing Date
- 2025-06-16
- Publication Date
- 2026-06-23
Smart Images
Figure CN120591395B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of CCL3-AS1 in regulating stability of carotid artery plaque. BACKGROUND
[0002] In recent years, atherosclerotic diseases (such as stroke, coronary heart disease, etc.) have caused serious influence due to high mortality and high disability. More and more evidence shows that the instability of extracranial carotid artery plaque, rather than the degree of stenosis, is associated with increased risk of stroke. Carotid endarterectomy is a main means for treating carotid stenosis and preventing ischemic stroke, and the core is to restore the patency of the blood vessel by removing the thickened intima and atherosclerotic plaque. Some patients cannot tolerate surgery due to aging or diffuse vascular lesions, therefore, in-depth analysis of the molecular mechanism of carotid artery plaque instability and finding of non-invasive early warning molecular markers are helpful for risk stratification of carotid atherosclerotic patients and precise implementation of carotid endarterectomy to reduce the risk of stroke.
[0003] Long non-coding RNA (lncRNA) is a single-stranded RNA molecule with a length of more than 200 nucleotides, which does not encode proteins but plays multiple important regulatory functions in cells. lncRNA plays a key role in gene expression regulation, chromatin modification, cell cycle regulation, cell differentiation, apoptosis and other biological processes. As a key epigenetic regulatory factor of gene expression, lncRNA has attracted increasing attention in atherosclerosis, however, the specific lncRNA molecules and their mechanisms of action for carotid vulnerable plaque are still relatively scarce and need to be further developed. SUMMARY
[0004] The purpose of the present application is to provide application of CCL3-AS1 (CCL3 Antisense RNA 1) in regulating stability of carotid artery plaque in view of the deficiencies in the prior art.
[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:
[0006] In the first aspect, the present application provides application of CCL3-AS1 in regulating stability of carotid artery plaque, and the sequence of the CCL3-AS1 is as follows:
[0007] 5'--3' (SEQ ID NO.1).
[0008] Furthermore, overexpression of CCL3-AS1 induces macrophage inflammatory activity and MMP9 secretion, promoting plaque rupture and thrombus formation; conversely, knockdown of CCL3-AS1 inhibits macrophage inflammatory activity and MMP9 secretion, reducing plaque rupture and thrombus formation.
[0009] Furthermore, CCL3-AS1 enhances MMP9 mRNA stability by binding to hnRNP-K, thereby increasing MMP9 expression and secretion and degrading fibrous cap collagen.
[0010] Secondly, the present invention provides the application of CCL3-AS1 as a biomarker in the preparation of products for predicting carotid plaque rupture-related diseases.
[0011] Furthermore, the product is a kit or a probe.
[0012] Thirdly, the present invention provides a product for predicting carotid plaque rupture-related diseases, the product comprising a reagent for detecting CCL3-AS1 expression.
[0013] Fourthly, the present invention provides the application of CCL3-AS1 in the preparation of macrophage regulators, which are used to regulate macrophage inflammatory activity and simultaneously regulate MMP9 secretion.
[0014] Fifthly, the present invention provides a method for predicting the risk of carotid plaque rupture, comprising the following steps:
[0015] Total RNA was extracted from carotid artery plaque tissue or blood samples of the subjects, and the expression level of CCL3-AS1 was detected and compared with that of the normal control group. If the expression level of CCL3-AS1 was significantly higher than that of the normal control group, the subjects were judged to have a higher risk of carotid artery plaque rupture.
[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0017] This invention provides the application of CCL3-AS1 as a biomarker in predicting carotid plaque rupture. It reveals for the first time the key role of long non-coding RNA CCL3-AS1 in carotid plaque stability and its molecular mechanism of regulating macrophage inflammatory response through the hnRNP-K / MMP9 pathway. This fills a research gap in this field, provides a new perspective for understanding the molecular basis of atherosclerotic plaque instability, and lays a solid theoretical foundation for the development of new diagnostic biomarkers and therapeutic targets. Attached Figure Description
[0018] Figure 1 for Figure 1 A schematic diagram of high expression of lncRNA CCL3-AS1 in unstable human carotid artery plaques, where A: whole transcriptome sequencing of stable and unstable human carotid artery plaques (n=5), heatmap of differential lncRNAs (FC>1, FPKM>1); B: verification of CCL3-AS1 expression level using real-time PCR in sequencing samples; C: CCL3-AS1 genome structure.
[0019] Figure 2This diagram illustrates the high enrichment of CCL3-AS1 in macrophages of unstable human carotid artery plaques. A: HE and Sirius red staining of unstable human carotid artery plaques; B: RNA FISH showing in situ expression of CCL3-AS1 in human carotid artery plaques; C: CCL3-AS1 enrichment in plaque macrophages; D: CCL3-AS1 expression levels in different vascular cells; E: RNA FISH showing in situ expression of CCL3-AS1 in monocytes and macrophages.
[0020] Figure 3 This diagram illustrates how CCL3-AS1 promotes macrophage inflammatory response and MMP9 expression. In A: Real-time PCR was used to detect the expression levels of inflammatory factors and MMP genes in THP1-derived macrophages that overexpress or knock down CCL3-AS1; B: Western blot was used to detect MMP9 protein levels.
[0021] Figure 4 The diagram shows the results of CCL3-AS1 promoting carotid plaque instability in mice. A: Flowchart of the mouse carotid plaque instability model construction; B: Masson staining of mouse carotid segment II; C: Immunofluorescence staining of mouse carotid artery, where F4 / 80 represents macrophages and iNOS represents inflammatory macrophages.
[0022] Figure 5 The diagram shows the results of CCL3-AS1 enhancing MMP9 mRNA stability by binding to the hnRNP-K protein. A: The effect of CCL3-AS1 on MMP9 mRNA stability; B: Prediction of CCL3-AS1 interacting proteins using catRAPID omics v2.1; C: Structure of the hnRNP-K protein and schematic diagram of its binding to CCL3-AS1; D: RIP experiment confirming the interaction between CCL3-AS1 and hnRNP-K and MMP9 mRNA; E: Immunofluorescence staining of human carotid artery plaques with hnRNP-K and CCL3-AS1 RNA FISH. Detailed Implementation
[0023] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention.
[0024] The following experimental materials are involved in the embodiments of this invention:
[0025] ApoE – / –Mice (Vitollife, China), high-fat diet (Research Diets, #D12492, USA), lentivirus (GeekGene, China), synthetic oligonucleotide ASO (Rabobio, China), hnRNP-K antibody (#ab39975), CD68 antibody (#ab213363), F4 / 80 antibody (#ab6640), MMP9 antibody (#ab283575), and iNOS antibody (#ab178945) were all from Abcam, cleaved casepase3 antibody (CST, #9661, USA), Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, #17-700, USA), Fluorescent InSitu Hybridization Kit (FISH kit) (Rabobio, #C10910, China), Masson staining kit, Sirius red staining kit, and phorbol ester (Sigma-Aldrich, USA).
[0026] In all embodiments of this invention, SPSS 20.0 software was used for statistical processing of the results. Normally distributed measurement data or measurement data with a sample size ≤ 6 in each group were expressed as mean ± standard deviation (SD). Independent samples t-tests were used for comparisons between two groups. P A difference of <0.05 is considered statistically significant.
[0027] Example 1: Histological staining of human carotid artery plaques
[0028] Plaque tissue was collected from patients who underwent carotid endarterectomy. Plaque structure and stability were identified using HE staining and Sirius red staining. A large lipid core, thin fibrous cap, and reduced type III collagen content were characteristics used to indicate plaque instability. CD68 immunofluorescence staining was used to identify macrophage infiltration in the plaque, and RNA FISH was used to detect the distribution of CCL3-AS1 and its co-localization with CD68 macrophages and hnRNP-K protein.
[0029] Whole transcriptome sequencing of stable and unstable carotid artery plaque tissues revealed that lncRNA CCL3-AS1 was significantly overexpressed (5.1-fold) in unstable plaques. P= 0.013, Figure 1 (A and B in the original text). CCL3-AS1 (Gene ID: 102724850) is transcribed in the opposite direction to its encoding gene CCL3, located on chromosome 17, and is 646 bp in length. Currently, this lncRNA lacks functional annotation. Figure 1 (C in the middle).
[0030] Histological staining revealed that unstable carotid artery plaques in humans exhibit characteristics of vulnerable plaques, including a large lipid core, a thin fibrous cap, reduced type III collagen, and increased inflammatory cell infiltration. Figure 2 (A) RNA FISH staining results showed that CCL3-AS1 was mainly enriched in macrophages of unstable plaques ( Figure 2 (B and C in the text). In cultured vascular cells, CCL3-AS1 is highly expressed in THP1 monocytes and monocyte-derived macrophages (B and C in the text). Figure 2 (D in the text). Subcellular localization showed that CCL3-AS1 was mainly expressed in the cytoplasm of macrophages with active inflammatory responses (D). Figure 2 (E in the text). The above results suggest that CCL3-AS1 is enriched in macrophages of plaques and may affect carotid plaque stability by regulating macrophage inflammation.
[0031] Example 2: Construction and histological staining of an unstable carotid plaque model in mice
[0032] In ApoE – / – In a mouse model of unstable carotid plaques, the carotid plaques exhibit unstable atherosclerotic lesions, similar to the vulnerable and easily ruptured carotid plaques in humans. Therefore, 6-week-old male ApoE mice were selected. – / – Thirty mice were fed a high-fat diet for 6 weeks and anesthetized with ketamine (100 mg / kg) and toluidine (10 mg / kg) for tandem ligation of the right carotid artery. A 160 µm needle was inserted at the bifurcation of the right carotid artery, and ligation was performed using 6-0 black polyester sutures at 1 mm distal to the bifurcation and 3 mm proximal to the distal stenosis. The needle was then removed. The mice were infected with CCL3-AS1 overexpressing lentivirus via local infiltration. The wound was sutured 10 minutes later, and the mice were fed a high-fat diet for another 4 weeks before tissue samples were collected.
[0033] Frozen sections of the proximal carotid artery (segment II) of the right carotid artery in mice were prepared to analyze the effect of CCL3-AS1 on plaque stability. Masson staining was used to detect the collagen content of the plaque fibrous cap and to analyze the thrombus formation type (intraplaque thrombosis, luminal thrombosis), fibrous cap integrity, and presence or absence of rupture. Immunofluorescence staining was used to identify macrophage infiltration: F4 / 80 antibody staining was used to identify the total number of macrophages within the plaque, iNOS antibody staining was used to identify inflammatory giant cells, cleaved casepase 3 (C-Cas3) antibody staining was used to identify apoptotic macrophages, and MMP9 antibody staining was used to detect the secretion of matrix metalloproteinases by macrophages.
[0034] Masson staining results showed that in the control group (Lv-NC, lentivirus overexpressing a meaningless sequence), the carotid artery mainly exhibited vascular remodeling characteristics of medial thickening after tandem ligation, while overexpression of CCL3-AS1 promoted the rupture of the fibrous cap of carotid plaques in mice, forming occlusive thrombi. Figure 4 (B in the text). Immunofluorescence staining results showed that overexpression of CCL3-AS1 significantly promoted macrophage infiltration (F4 / 80 label), and the proportions of inflammatory macrophages (iNOS label) and apoptotic macrophages (cleaved caspase 3 label) were significantly increased, and macrophage MMP9 expression was increased. Figure 4 (C in the text). The above results indicate that CCL3-AS1 promotes macrophage infiltration and inflammatory activity in mouse plaques, accelerating plaque instability and rupture.
[0035] Example 3: Cell Culture
[0036] Human THP1 mononuclear / macrophage cell line culture: THP1 cells were cultured in RPMI 1640 medium containing 10% FBS. THP1 cells differentiated into M0 macrophages after stimulation with 100 ng / mL phorbol ester for 48 h.
[0037] Lentiviral infection of THP1 cells (MOI=50) yielded a stable CCL3-AS1 overexpressing cell line. Macrophages were treated with ASO (50 nM) to reduce CCL3-AS1 expression. The regulatory role of CCL3-AS1 in macrophage inflammatory activity was clarified by observing macrophage inflammatory cytokines and MMP expression, subtype differentiation, lipid phagocytosis, and apoptosis.
[0038] In vitro studies have shown that lentivirus-mediated CCL3-AS1 overexpression significantly upregulates the expression levels of macrophage inflammatory genes (MCP1, TNFα, IL1β, iNOS, and CCL3); ASO knockdown of CCL3-AS1 significantly downregulates the expression levels of MCP1 and iNOS genes. Since macrophages can secrete various MMPs that affect plaque collagen content and stability, the effects of CCL3-AS1 on the expression levels of MMP1, MMP3, and MMP9 were examined. The results showed that CCL3-AS1 had no significant effect on the RNA levels of MMP1 and MMP3, but overexpression of CCL3-AS1 significantly increased MMP9 mRNA expression by approximately 1.8-fold. P= 0.002), conversely, knocking down CCL3-AS1 reduced it by 34% ( P= MMP9 expression of 0.001 Figure 3 (A) At the protein level, overexpression of CCL3-AS1 increases MMP9 protein expression level by approximately 2.2-fold. P=0.004), knocking down CCL3-AS1 significantly reduced MMP9 protein levels by 60% ( P< 0.001, Figure 3 (B in the text). The above results suggest that CCL3-AS1 mediates plaque instability by promoting macrophage inflammatory activity and MMP9 expression.
[0039] Example 4: RNA stability experiment
[0040] THP1 cell lines stably overexpressing CCL3-AS1 (Lv-CCL3AS1) and control cell lines (Lv-NC) were seeded in 12-well plates. Monocytes were induced to differentiate into macrophages by phorbol ester stimulation for 48 h. 10 μg / mL actinomycin D was added, and cells were collected at 0 h, 4 h, 8 h, 12 h and 24 h, respectively. The expression level of MMP9 mRNA was detected by Real-time qPCR.
[0041] Example 5: Immunoprecipitation of RNA-binding proteins
[0042] Macrophages from the Lv-CCL3AS1 and Lv-NC groups were collected, and RNA-protein complex immunoprecipitation was performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit. In short, cell lysates were incubated overnight with 5 μg of hnRNP-K antibody, followed by incubation with protein A / G magnetic beads for 8 h. RNA complexes bound to hnRNP-K were collected using a magnetic rack, and CCL3-AS1 and MMP9 levels were detected using real-time qPCR to verify the existence of any interaction among the three.
[0043] The results showed that CCL3-AS1 significantly affected the expression level of MMP9 in macrophages. Further RNA stability experiments revealed that CCL3-AS1 could delay the degradation of MMP9 mRNA and significantly promote its mRNA stability. P= 0.032, Figure 5 (A in the middle).
[0044] To explore the molecular mechanism by which CCL3-AS1 maintains the stability of MMP9 mRNA, the catRAPID omics v2.1 website (http: / / s.tartaglialab.com / page / catrapid_omics2_group) was used to predict proteins interacting with CCL3-AS1. Bioinformatics predictions showed that CCL3-AS1 may bind to heterogeneous nuclear ribonucleoprotein K (hnRNP-K), specifically through a high affinity between amino acids 389-453 (KH3 domain) of hnRNP-K and bases 26-77 of CCL3-AS1. Figure 5 (B and C in the text). RNA-binding protein immunoprecipitation assays confirmed the interaction between CCL3-AS1, hnRNP-K protein, and MMP9 mRNA. Figure 5 The results of immunofluorescence staining and RNA FISH analysis of human carotid artery plaques showed that CCL3-AS1 and hnRNP-K co-localized (D). Figure 5 (E in the text). The above results suggest that CCL3-AS1 enhances MMP9 mRNA stability by binding to the hnRNP-K protein.
[0045] The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above embodiments. Any embodiment that meets the requirements of the present invention is within the protection scope of the present invention.
Claims
1. The application of CCL3-AS1 as a biomarker in the preparation of products for predicting carotid plaque rupture-related diseases, wherein the sequence of CCL3-AS1 is shown in SEQ ID NO.
1.
2. The application according to claim 1, characterized in that, The product is a kit or probe.