Macrophage related gene KCNA3 and application thereof

Abstract

The invention is applicable to the technical field of molecular biology, and provides a macrophage related gene KCNA3 and application thereof. The invention discloses a macrophage related gene KCNA3 for the first time, and the gene is identified as a macrophage related differential expression gene of a patient with type 2 diabetes mellitus with carotid atherosclerosis, and shows high expression in the patient with type 2 diabetes mellitus with carotid atherosclerosis. Through analysis of a public database transcriptome chip and high-throughput sequencing data and further verification of CIBERSORTX and WGCNA algorithms, close association between the KCNA3 gene and the macrophages is confirmed. Based on differentiated expression of the KCNA3 gene and correlation of the KCNA3 gene and macrophages, the gene can be used as a potential treatment target for further treating patients with the diabetes mellitus with the carotid atherosclerotic plaque, so that a new mechanism participating in disease progression of the diabetes mellitus with the carotid atherosclerotic plaque is disclosed.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a macrophage-related gene KCNA3 and an application thereof. Background Art

[0002] Type 2 diabetes is a systemic metabolic disease characterized by abnormal glucose metabolism. Carotid atherosclerotic plaques are one of the most common pan-vascular complications of the disease and may lead to serious cerebrovascular diseases. Although the mechanism by which type 2 diabetes leads to carotid atherosclerotic plaques is not fully understood, it is currently believed that hyperglycemia can affect the progression of carotid atherosclerotic plaques by regulating the phenotype and function of macrophages.

[0003] Macrophages phagocytize lipids to form foam cells. Their excessive lipid metabolism leads to the production of inflammatory cytokines and induces cell death. These dead cells constitute the necrotic core of carotid atherosclerotic plaques. Macrophages not only have classic M1 and M2 phenotypes, but also have a variety of other phenotypes. The polarization of macrophages has an important influence on the progression of atherosclerotic plaques. In addition, macrophage activation, proliferation, migration, apoptosis, and cytokine secretion are all involved in and affect the development of atherosclerotic plaques, from the initial formation of atherosclerotic plaques to growth, and then to the final rupture and wound healing stage.

[0004] In a hyperglycemic environment, the infiltration of macrophages and the incidence of thrombosis in atherosclerotic plaques increase. Hyperglycemia can promote enhanced bone marrow production, thereby driving the production and activation of monocytes. In the context of diabetes, circulating monocytes and monocyte-derived macrophages exhibit a pro-inflammatory phenotype. For example, hyperglycemia promotes the production of pro-inflammatory cytokines by these cells, and when their surface receptors (RAGE) bind to advanced glycation end products (AGEs), they regulate gene expression by promoting a pro-inflammatory phenotype. Although studies have shown that hyperglycemia induces a well-trained immune phenotype in macrophages, thereby promoting persistent pro-atherosclerotic features. However, current research is not sufficient to fully explain the mechanism by which hyperglycemia triggers macrophage changes to cause atherosclerosis to progress, and therefore it is impossible to develop an effective therapeutic target. For this reason, the present invention proposes a macrophage-related gene KCNA3 and its application. Summary of the invention

[0005] The purpose of the present invention is to provide a macrophage-related gene KCNA3 and its application, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A therapeutic target for type 2 diabetes with carotid atherosclerotic plaques, wherein the therapeutic target is the macrophage-related gene KCNA3.

[0008] Application of a macrophage-related gene KCNA3 in the preparation of a drug for treating type 2 diabetes mellitus associated with carotid atherosclerotic plaques.

[0009] A preparation for detecting the expression amount of a macrophage-related gene KCNA3 is used in preparing a reagent for diagnosing or detecting type 2 diabetes mellitus associated with carotid atherosclerotic plaques.

[0010] Furthermore, the RT-qPCR method was used to detect the expression level of macrophage-related gene KCNA3 in the samples. The expression level of macrophage-related gene KCNA3 was increased in type 2 diabetes with carotid atherosclerotic plaques.

[0011] A reagent for diagnosing or detecting type 2 diabetes with carotid atherosclerotic plaques, the reagent comprising a preparation for detecting the expression amount of the macrophage-related gene KCNA3.

[0012] A kit for diagnosing or detecting type 2 diabetes mellitus associated with carotid atherosclerotic plaques, the kit comprising the reagents as described above.

[0013] Furthermore, the kit also includes a pair of primers for RT-qPCR detection, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.2.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention discloses for the first time a macrophage-related gene KCNA3, which is identified as a macrophage-related differentially expressed gene in patients with type 2 diabetes and carotid atherosclerosis, and is highly expressed in patients with type 2 diabetes and carotid atherosclerosis. The close association between the KCNA3 gene and macrophages was confirmed by the analysis of transcriptome chips and high-throughput sequencing data from public databases, as well as further verification by CIBERSORTX and WGCNA algorithms. Based on the differential expression of the KCNA3 gene and its correlation with macrophages, the present invention proposes that the gene and its related macrophages can be used as potential therapeutic targets for further treatment of patients with diabetes and carotid atherosclerotic plaques, thereby revealing a new mechanism involved in the progression of diabetes and carotid atherosclerotic plaques. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Four differentially expressed genes were obtained from the intersection of differentially expressed genes and macrophage-related genes.

[0017] Figure 2 The expression of KCNA3 gene is increased in diabetic carotid atherosclerotic plaques.

[0018] Figure 3 The expression level of KCNA3 gene was increased in diabetic carotid atherosclerotic plaques; * indicates P < 0.05.

[0019] Figure 4 The results of immunofluorescence are shown in Figure 1. The red fluorescence represents the KCNA3 gene, the green fluorescence represents macrophages, the blue fluorescence represents the cell nucleus, and the orange fluorescence represents the fusion of red and green fluorescence, that is, KCNA3 is co-expressed with macrophages. DETAILED DESCRIPTION

[0020] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0021] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0022] Example 1: In this example, samples from patients with carotid atherosclerotic plaques were obtained for transcriptomic analysis, and macrophage-related genes were further focused to explore the pathogenesis of hyperglycemia and carotid atherosclerotic plaque disease and to find potential therapeutic targets. We used transcriptomic data to identify differentially expressed genes between patients with type 2 diabetes and carotid atherosclerotic plaques and patients without type 2 diabetes and carotid atherosclerotic plaques. Then, macrophage-related genes were inferred by the WGCNA combined with CIBERSORTX method.

[0023] 1.1 Collect gene expression profile data from the GEO database;

[0024] The GSE118481 dataset (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE118481) was obtained from the Gene Expression Omnibus (GEO) database (http: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE118481), which includes carotid atherosclerotic plaque samples from 8 patients with type 2 diabetes and 16 patients without type 2 diabetes.

[0025] 1.2 Bioinformatics analysis;

[0026] 1.2.1 Differential expression analysis to determine differentially expressed genes;

[0027] The R software package “Limm package” was used to perform differential expression analysis on the GSE118481 dataset, and the threshold was selected as “FDR < 0.05 and |log2FC| > 0.5”, and a total of 690 differentially expressed genes were obtained.

[0028] 1.2.2 Immune infiltration analysis targets macrophage-related genes;

[0029] CIBERSORTX (https: / / cibersortx.stanford.edu / index.php) is commonly used to calculate the proportions of 22 immune cell types. We used this website to analyze the infiltration of immune cells in samples of the GSE118481 dataset.

[0030] 1.2.3 Weighted correlation network analysis (WGCNA) and selection of key modules identified the macrophage-related gene KCNA3 as a potential therapeutic target;

[0031] Outlier genes and samples were removed using the goodSamplesGenes method of the R package “WGCNA”. A scale-free network was constructed, and a height cutoff of 0.35 was set to merge modules with high similarity. The infiltration of immune cells in all samples in the GSE118481 dataset was used as the trait data for WGCNA. Module eigengenes (MEs), the first principal component of each gene module, representing the expression of all genes in a given module, were calculated. Pearson correlation analysis was performed to assess the correlation between traits and modules. The central genes were determined by calculating the module membership (MM) and the gene significance (GS) of the genes in the key modules. The central genes were screened out using the cutoff criteria (|MM|>0.8 and |GS|>0.4).

[0032] 1.2.4 Analysis results;

[0033] 690 differentially expressed genes were obtained by differential expression analysis, and 192 macrophage-related genes were obtained by WGCNA analysis. The intersection of the two obtained 4 macrophage-related differentially expressed genes, namely KCNA3, CAP1, IFIM1 and TTC14 ( Figure 1 ).

[0034] 1.3 The expression level of macrophage-related gene KCNA3 was increased in high-throughput sequencing data;

[0035] 1.3.1 Experimental methods;

[0036] Ten samples of carotid atherosclerotic plaques were collected in the clinic for high-throughput sequencing, including carotid plaque samples from 5 patients with type 2 diabetes and 5 patients without type 2 diabetes, and the project number in the NCBI database is PRJNA752896. After obtaining the expression profile data, the expression of the KCNA3 gene was compared between the two groups.

[0037] 1.3.2 Experimental results;

[0038] The macrophage-related gene KCNA3 was verified in the high-throughput expression profile data of carotid atherosclerotic plaques (P<0.05). That is, the expression level of the macrophage-related gene KCNA3 was increased in type 2 diabetes with carotid atherosclerotic plaques ( Figure 2 ).

[0039] Example 2: RT-qPCR experiments demonstrated the expression level of macrophage-related gene KCNA3;

[0040] 2.1 Experimental methods;

[0041] Twenty-four carotid atherosclerotic plaque samples were collected from the clinic, including carotid atherosclerotic plaque samples from 12 patients with type 2 diabetes and 12 patients without type 2 diabetes. Total RNA was extracted from them and reverse transcribed into cDNA using the RevertAid First-Strand cDNA Synthesis Kit from Thermo Scientific (Waltham, MA). Finally, the cDNA samples were mixed with FastStart Universal SYBR Green Master (Rox) from Roche (Mannheim, Germany) and added to the EppendorfAG 22331 PCR thermal cycler for amplification, with β-actin as the internal control. The primer sequences are shown in Table 1. All samples were repeated three times and analyzed using the 2(-ΔΔCt) method.

[0042] Table 1 Primer sequences

[0043]

[0044] 2.2 Experimental results;

[0045] RT-qPCR results showed that the expression level of KCNA3 gene was increased in type 2 diabetes with carotid atherosclerotic plaques (P<0.05) ( Figure 3 ).

[0046] Example 3: Co-expression of KCNA3 gene with macrophages in immunofluorescence experiments;

[0047] 3.1 Experimental methods;

[0048] Tissue sections from patients with type 2 diabetes and non-type 2 diabetes who underwent carotid endarterectomy were collected from the clinic, fixed with formalin and embedded in paraffin. The paraffin blocks were cut into thin sections.

[0049] The samples were immunofluorescently stained twice with CD68 antibody and KCNA3 antibody. The paraffin sections were first dewaxed and hydrated, and then antigen retrieval was performed. After blocking the samples with serum, the samples were incubated with anti-CD68 (Servicebio, GB113) and anti-KCNA3 antibodies at room temperature overnight. After that, the samples were stained with fluorescent secondary antibodies, and then the cell nuclei were stained with 4,6-diamino-2-phenylindole (DAPI). In order to eliminate autofluorescence, autofluorescence quencher B was added to the sections. Finally, the sections were sealed with anti-fluorescence quenching mounting medium. Images were acquired and scanned using an inverted fluorescence microscope (NikonEclipse C1) from Nikon and a scanner (Pannoramic MIDI) produced by 3DHISTECH.

[0050] 3.2 Experimental results;

[0051] Immunofluorescence results showed that KCNA3 gene was co-localized with macrophages in cells ( Figure 4 ).

[0052] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A therapeutic target for type 2 diabetes mellitus with carotid atherosclerotic plaques, characterized in that: The therapeutic target is the macrophage-related gene KCNA3.

2. Application of a macrophage-related gene KCNA3 in the preparation of a drug for treating type 2 diabetes mellitus associated with carotid atherosclerotic plaques.

3. Use of a preparation for detecting the expression level of the macrophage-related gene KCNA3 in the preparation of a reagent for diagnosing or detecting type 2 diabetes mellitus associated with carotid atherosclerotic plaques.

4. The use according to claim 3, characterized in that: The RT-qPCR method was used to detect the expression of macrophage-related gene KCNA3 in the samples. The expression of macrophage-related gene KCNA3 was increased in type 2 diabetes with carotid atherosclerotic plaques.

5. A reagent for diagnosing or detecting type 2 diabetes with carotid atherosclerotic plaques, characterized in that: The reagents include a preparation for detecting the expression amount of the macrophage-related gene KCNA3.

6. A kit for diagnosing or detecting type 2 diabetes with carotid atherosclerotic plaques, characterized in that: The kit comprises the reagent according to claim 5.

7. The kit according to claim 6, characterized in that The kit also includes a pair of primers for RT-qPCR detection, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.2.

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