Application of CCL3-AS1 in regulating and controlling stability of carotid plaque

Abstract

The invention discloses an application of CCL3-AS1 in regulating and controlling the stability of carotid plaques, which is characterized in that the CCL3-AS1 is overexpressed to induce the inflammatory activity of macrophages and MMP9 secretion and promote plaque rupture and thrombosis; on the contrary, the CCL3-AS1 is knocked down to inhibit the inflammatory activity of macrophages and MMP9 secretion, and plaque rupture and thrombosis are reduced; in addition, the invention also discloses that CCL3-AS1 is combined with hnRNP-K to enhance the mRNA stability of MMP9, so that the expression and secretion of MMP9 are increased, and the fibrocap collagen is degraded. The invention discloses an important function of CCL3-AS1 in carotid plaque stability and a molecular mechanism of CCL3-AS1, provides a novel diagnostic marker and a novel therapeutic target, not only provides a novel direction for research of atherosclerosis related diseases, but also provides a novel tool and means for clinical diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of CCL3-AS1 in regulating the stability of carotid artery plaques. Background Art

[0002] In recent years, atherosclerotic diseases (such as stroke and coronary heart disease) have caused serious consequences due to their high mortality and disability rates. Growing evidence indicates that extracranial carotid artery plaque instability, rather than the degree of stenosis, is associated with an increased risk of stroke. Carotid endarterectomy is the primary treatment for carotid stenosis and the prevention of ischemic stroke. Its core goal is to restore vascular patency by removing the thickened intima and atherosclerotic plaques. Some patients cannot tolerate surgery due to advanced age or diffuse vascular disease. Therefore, in-depth analysis of the molecular mechanisms of carotid plaque instability and the identification of non-invasive early warning molecular markers will help risk stratify patients with carotid atherosclerosis, accurately perform carotid endarterectomy, and reduce the risk of stroke.

[0003] Long noncoding RNAs (lncRNAs) are a class of single-stranded RNA molecules exceeding 200 nucleotides in length. They do not encode proteins but instead play a variety of important regulatory roles within cells. lncRNAs play a key role in numerous biological processes, including gene expression regulation, chromatin modification, cell cycle control, cell differentiation, and apoptosis. As key epigenetic regulators of gene expression, lncRNAs are gaining increasing attention in atherosclerosis. However, research on lncRNAs specific for vulnerable carotid plaques and their mechanisms of action remains scarce, requiring further investigation. Summary of the Invention

[0004] The purpose of the present invention is to address the deficiencies of the existing technology and provide an application of CCL3-AS1 (CCL3 Antisense RNA 1) in regulating the stability of carotid artery plaques.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a use of CCL3-AS1 in regulating carotid plaque stability. The sequence of CCL3-AS1 is shown below: 5'--3' (SEQ ID NO. 1).

[0006] Furthermore, overexpression of CCL3-AS1 induced macrophage inflammatory activity and MMP9 secretion, promoting plaque rupture and thrombosis; conversely, knockdown of CCL3-AS1 inhibited macrophage inflammatory activity and MMP9 secretion, reducing plaque rupture and thrombosis.

[0007] Furthermore, CCL3-AS1 enhances MMP9 mRNA stability by binding to hnRNP-K, thereby increasing MMP9 expression and secretion and degrading fibrous cap collagen.

[0008] In a second aspect, the present invention provides the use of CCL3-AS1 as a biomarker in the preparation of a product for predicting diseases related to carotid plaque rupture.

[0009] Furthermore, the product is a kit or a probe.

[0010] In a third aspect, the present invention provides a product for predicting diseases related to carotid plaque rupture, wherein the product comprises a reagent for detecting the expression of CCL3-AS1.

[0011] In a fourth aspect, the present invention provides use of CCL3-AS1 in the preparation of a macrophage regulator, wherein the macrophage regulator is used to regulate macrophage inflammatory activity and MMP9 secretion.

[0012] In a fifth aspect, the present invention provides a method for predicting the risk of carotid artery plaque rupture, comprising the following steps: Total RNA is extracted from the subject's carotid plaque tissue or blood sample, and the expression level of CCL3-AS1 is detected and compared with that of the normal control group. If the CCL3-AS1 expression level is significantly higher than that of the normal control group, the subject is judged to have a higher risk of carotid plaque rupture.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides the application of CCL3-AS1 as a biomarker in predicting carotid plaque rupture, revealing 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, filling the research gap in this field, providing a new perspective for understanding the molecular basis of atherosclerotic plaque instability, and laying a solid theoretical foundation for the development of new diagnostic markers and therapeutic targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 for Figure 1 Schematic diagram of the high expression of lncRNA CCL3-AS1 in unstable carotid plaques of human carotid arteries, where A: Whole transcriptome sequencing of human carotid artery stable plaques (n=5) and unstable plaque tissues (n=5), heat map of differential lncRNAs (FC>1, FPKM>1); B: Real-time PCR verification of CCL3-AS1 expression levels in sequencing samples; C: Genomic structure of CCL3-AS1.

[0015] Figure 2 Schematic diagram showing that CCL3-AS1 is highly enriched in macrophages in unstable carotid plaques of human carotid artery, where A: HE and Sirius red staining of unstable carotid plaques of human carotid artery; B: RNA FISH showing the in situ expression of CCL3-AS1 in human carotid artery plaques; C: CCL3-AS1 is enriched in plaque macrophages; D: Expression levels of CCL3-AS1 in different vascular cells; E: RNA FISH showing the in situ expression of CCL3-AS1 in monocytes and macrophages.

[0016] Figure 3 Schematic diagram of CCL3-AS1 promoting macrophage inflammatory response and MMP9 expression, where A: Real-time PCR detection of inflammatory factor and MMPs gene expression levels in THP1-derived macrophages with overexpression or knockdown of CCL3-AS1; B: Western blot detection of MMP9 protein level.

[0017] Figure 4 The results of CCL3-AS1 promoting carotid plaque instability in mice, where A: Flowchart of the construction of the mouse carotid plaque instability model; B: Masson staining of segment II of the mouse carotid artery; C: Immunofluorescence staining of the mouse carotid artery, F4 / 80 represents macrophages, and iNOS represents inflammatory macrophages.

[0018] Figure 5 Figure 3: CCL3-AS1 enhances MMP9 mRNA stability by binding to hnRNP-K protein. A: Effect of CCL3-AS1 on MMP9 mRNA stability; B: Prediction of CCL3-AS1 interacting proteins using catRAPID omics v2.1; C: Schematic diagram of hnRNP-K protein structure and its binding to CCL3-AS1; D: RIP assay confirms interaction between CCL3-AS1, hnRNP-K, and MMP9 mRNA; E: Immunofluorescence staining of hnRNP-K and CCL3-AS1 RNA in human carotid artery plaques. DETAILED DESCRIPTION

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The examples are only used to explain the present invention but not to limit the scope of the present invention.

[0020] The following experimental materials are involved in the embodiments of the present invention: ApoE – / –Mice (Weitong Lihua, China), high-fat diet (Research Diets, #D12492, USA), lentivirus (Gene Gene, China), synthetic oligonucleotide ASO (RiboBio, China), hnRNP-K antibody (#ab39975), CD68 antibody (#ab213363), F4 / 80 antibody (#ab6640), MMP9 antibody (#ab283575), and iNOS antibody (#ab178945) were all from Abcam, cleaved casepase 3 antibody (CST, #9661, USA), Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, #17-700, USA), Fluorescent InSitu Hybridization Kit (FISH Kit) (RiboBio, #C10910, China), Masson staining kit, Sirius red staining kit, and phorbol esters (Sigma-Aldrich, USA).

[0021] In the examples of the present invention, SPSS 20.0 software was used to perform statistical processing on the results. Measurement data that conformed to normal distribution or measurement data with sample size ≤ 6 in each group were expressed as mean ± standard deviation (SD). The comparison between the two groups was performed using the independent sample t test. P A value < 0.05 was considered statistically significant.

[0022] Example 1: Histological staining of human carotid artery plaques Plaque tissue was collected from patients undergoing carotid endarterectomy and was stained with hematoxylin and eosin (HE) and iris red to characterize plaque structure and stability. Plaque instability was characterized by a large lipid core, a thin fibrous cap, and a reduced proportion of type III collagen. Macrophage infiltration in plaques was assessed using CD68 immunofluorescence staining, and the distribution of CCL3-AS1 and its colocalization with CD68 macrophages and hnRNP-K proteins were assessed using RNA FISH.

[0023] 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 Figure 1). CCL3-AS1 (Gene ID: 102724850) is transcribed in the opposite direction to the coding gene CCL3 and is located on chromosome 17. It is 646 bp long and currently has no functional annotation for this lncRNA ( Figure 1 C in ).

[0024] Histological staining showed that unstable carotid plaques in humans have vulnerable plaque characteristics such as a larger lipid core, a thinner fibrous cap, decreased type III collagen, and increased inflammatory cell infiltration ( Figure 2 RNA FISH staining results showed that CCL3-AS1 was mainly enriched in macrophages of unstable plaques ( Figure 2 In cultured vascular cells, CCL3-AS1 was highly expressed in THP1 monocytes and monocyte-derived macrophages ( Figure 2 Subcellular localization showed that CCL3-AS1 was mainly expressed in the cytoplasm of macrophages with active inflammatory response ( Figure 2 These results suggest that CCL3-AS1 is enriched in macrophages in plaques and may affect the stability of carotid artery plaques by regulating macrophage inflammation.

[0025] Example 2: Construction of unstable carotid artery plaque model in mice and histological staining In ApoE – / – In the mouse carotid artery unstable plaque model, the carotid artery plaques showed unstable atherosclerotic lesions, similar to the vulnerable and rupture-prone carotid artery plaques in humans. – / – Thirty mice, fed a high-fat diet for 6 weeks, were anesthetized with ketamine (100 mg / kg) and xylazine (10 mg / kg) and underwent right carotid artery tandem ligation. A 160 µm diameter needle was introduced into the right carotid bifurcation and ligated with 6-0 black polyester suture 1 mm distal to the bifurcation and 3 mm proximal to the distal stenosis. The needle was then removed. The mice were infected with a 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 specimen collection.

[0026] Frozen sections of the proximal carotid artery segment II of the right carotid artery of mice were prepared to analyze the effect of CCL3-AS1 on plaque stability. Masson staining was used to assess collagen content in the plaque fibrous cap. Thrombosis type (intra-plaque thrombus, luminal thrombus), fibrous cap integrity, and the presence of rupture were analyzed. Immunofluorescence staining was used to assess 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 macrophage secretion of matrix metalloproteinases.

[0027] Masson staining results showed that the control group (Lv-NC, lentivirus overexpressing a meaningless sequence) showed vascular remodeling with thickening of the tunica media after serial ligation, while overexpression of CCL3-AS1 promoted the rupture of the fibrous cap of the carotid artery plaque in mice, forming a lumen-occluding thrombus ( Figure 4 B). Immunofluorescence staining results showed that overexpression of CCL3-AS1 significantly promoted the infiltration of macrophages (F4 / 80 marker), the proportion of inflammatory macrophages (iNOS marker) and apoptotic macrophages (cleaved caspase 3 marker) increased significantly, and the expression of macrophage MMP9 increased ( Figure 4 These results indicate that CCL3-AS1 promotes macrophage infiltration and inflammatory activity in mouse plaques, accelerating plaque instability and rupture.

[0028] Example 3: Cell culture Human THP1 monocyte / macrophage cell line culture: Cultured in RPMI 1640 medium supplemented with 10% FBS. THP1 cells were differentiated into M0 macrophages after stimulation with 100 ng / mL phorbol ester for 48 h.

[0029] THP1 cells were infected with lentivirus (MOI = 50) to generate a cell line stably overexpressing CCL3-AS1. ASO (50 nM) was administered to macrophages to reduce CCL3-AS1 expression. The regulatory role of CCL3-AS1 in macrophage inflammatory activity was determined by observing the expression of inflammatory factors and MMPs, subtype differentiation, lipid phagocytosis, and apoptosis in macrophages.

[0030] In vitro studies have shown that lentivirus-mediated overexpression of CCL3-AS1 significantly upregulated the expression levels of macrophage inflammatory genes (MCP1, TNFα, IL1β, iNOS, and CCL3); ASO knockdown of CCL3-AS1 significantly downregulated the expression levels of MCP1 and iNOS genes. Since macrophages can secrete a variety of MMPs that affect the content and stability of plaque collagen, the effects of CCL3-AS1 on the expression levels of MMP1, MMP3, and MMP9 were tested. The results showed that CCL3-AS1 had no significant effect on the RNA levels of MMP1 and MMP3, while overexpression of CCL3-AS1 significantly increased the expression of MMP9 mRNA by approximately 1.8 times ( P= 0.002), whereas, knockdown of CCL3-AS1 decreased the expression of CCL3-AS1 by 34% ( P= 0.001) of MMP9 expression ( Figure 3 At the protein level, overexpression of CCL3-AS1 increased the expression level of MMP9 protein by approximately 2.2 times ( P= 0.004), knockdown of CCL3-AS1 significantly reduced the MMP9 protein level by 60% (P< 0.001, Figure 3 These results suggest that CCL3-AS1 mediates plaque instability by promoting macrophage inflammatory activity and MMP9 expression.

[0031] Example 4: RNA stability experiment The THP1 cell line stably overexpressing CCL3-AS1 (Lv-CCL3AS1) and the control cell line (Lv-NC) were seeded in 12-well plates. Monocytes were stimulated with phorbol ester for 48 h to induce differentiation into macrophages. 10 μg / mL actinomycin D was added and cells were harvested at 0 h, 4 h, 8 h, 12 h, and 24 h. Real-time qPCR was used to detect the expression level of MMP9 mRNA.

[0032] Example 5: RNA-binding protein immunoprecipitation Macrophages from the Lv-CCL3AS1 and Lv-NC groups were collected and immunoprecipitated with the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit for RNA-protein complexes. Briefly, cell lysates were incubated overnight with 5 μg of hnRNP-K antibody and incubated with protein A / G magnetic beads for 8 hours. The RNA complexes bound to hnRNP-K were collected using a magnetic stand, and real-time qPCR was used to measure CCL3-AS1 and MMP9 levels to verify their interaction.

[0033] 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 ).

[0034] To explore the molecular mechanism by which CCL3-AS1 maintains MMP9 mRNA stability, we used the catRAPID omics v2.1 website (http: / / s.tartaglialab.com / page / catrapid_omics2_group) to predict proteins that interact with CCL3-AS1. Bioinformatics prediction results showed that CCL3-AS1 may bind to heterogeneous nuclear ribonucleoprotein K (hnRNP-K), as evidenced by the high affinity between amino acids 389-453 (KH3 domain) of hnRNP-K and the base fragment 26-77 of CCL3-AS1 ( Figure 5 RNA binding protein immunoprecipitation experiments confirmed that CCL3-AS1, hnRNP-K protein and MMP9 mRNA interact with each other ( Figure 5 D). Immunofluorescence staining and RNA FISH results of human carotid artery plaques showed that CCL3-AS1 colocalized with hnRNP-K ( Figure 5 These results suggest that CCL3-AS1 enhances MMP9 mRNA stability by binding to hnRNP-K protein.

[0035] The above embodiments are not limitations of the present invention, and the present invention is not limited to the above embodiments. As long as the requirements of the present invention are met, they belong to the protection scope of the present invention.

Claims

1. Application of CCL3-AS1 in regulating carotid artery plaque stability, the sequence of CCL3-AS1 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that Overexpression of CCL3-AS1 induces macrophage inflammatory activity and MMP9 secretion, promoting plaque rupture and thrombosis; conversely, knockdown of CCL3-AS1 inhibits macrophage inflammatory activity and MMP9 secretion, reducing plaque rupture and thrombosis.

3. The use according to claim 1, characterized in that CCL3-AS1 enhances MMP9 mRNA stability by binding to hnRNP-K, thereby increasing MMP9 expression and secretion and degrading fibrous cap collagen.

4. Application of CCL3-AS1 as a biomarker in the preparation of products for predicting diseases related to carotid artery plaque rupture, wherein the sequence of CCL3-AS1 is shown in SEQ ID NO.

1.

5. The use according to claim 4, characterized in that The product is a kit or a probe.

6. A product for predicting diseases related to carotid artery plaque rupture, characterized in that: The product includes a reagent for detecting the expression of CCL3-AS1, and the sequence of CCL3-AS1 is shown as SEQ ID NO.

1.

7. Use of CCL3-AS1 in the preparation of a macrophage regulator, wherein the sequence of CCL3-AS1 is shown in SEQ ID NO. 1; characterized in that, The macrophage regulator is used to regulate the inflammatory activity of macrophages and simultaneously regulate the secretion of MMP9.

8. A method for predicting the risk of carotid plaque rupture, characterized in that: The following steps are involved: Total RNA is extracted from the subject's carotid plaque tissue or blood sample, and the expression level of CCL3-AS1 is detected and compared with that of the normal control group. If the CCL3-AS1 expression level is significantly higher than that of the normal control group, the subject is judged to have a higher risk of carotid plaque rupture. The sequence of the CCL3-AS1 is shown in SEQ ID NO. 1.

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