Application of intron retention transcript as marker in diagnosis of atherosclerosis

Abstract

The invention provides application of an intron retention transcript as a marker in diagnosis of atherosclerosis. The research finds that intron retention transcripts HIPK < 2. Ir > and COPE.ir of the two genes are remarkably and highly expressed in peripheral blood of a patient with atherosclerosis, meanwhile, the higher the expression level of the HIPK < 2. Ir > and the COPE.ir is, the higher the atherosclerosis degree is, and the HIPK < 2. Ir > and the COPE.ir can be used as diagnostic markers of the atherosclerosis and the atherosclerosis degree. The atherosclerosis or the degree of the atherosclerosis can be diagnosed by detecting the expression levels of the HIPK < 2. Ir > and the COPE.ir. A new marker is provided for diagnosis of atherosclerosis, and a new means is provided for diagnosis and treatment of atherosclerosis.

Description

Technical Field

[0001] The present invention relates to the application of intron-retained transcripts as markers in the diagnosis of atherosclerosis, and belongs to the field of biomedical technology. Background Art

[0002] Atherosclerosis (AS) is a chronic inflammatory disease characterized by lipid deposition, infiltration of inflammatory cells, and fibrosis in the arterial wall, ultimately leading to vascular stenosis or occlusion. The pathogenesis of atherosclerosis is complex and involves multiple factors, including dyslipidemia, inflammatory response, endothelial dysfunction, and activation of immune cells. With the development of molecular biology techniques, more and more studies have begun to focus on molecular markers of atherosclerosis in order to improve the accuracy of early diagnosis and the specificity of treatment.

[0003] Regarding the mechanism study of AS, it has been found that LDL penetrates into endothelial cells and remains, and after being oxidized and modified by endothelial cells into oxidized low-density lipoprotein (oxLDL), it becomes a harmful substance to the body and is phagocytosed by monocytes. Monocytes that have phagocytosed oxLDL become macrophages, and under the stimulation of endotoxin, heat shock protein, etc., secrete inflammatory cytokines, proteases, peroxyanions, etc. to cause an inflammatory reaction, resulting in inflammatory damage to tissues.

[0004] Traditional markers of atherosclerosis are mainly abnormal blood lipid levels, especially the elevation of low-density lipoprotein cholesterol (LDL-C) level. Oxidized low-density lipoprotein (Ox-LDL) is the main lipid in atherosclerotic lesions, and the elevation of its plasma level is a key risk factor for atherosclerosis. In addition, the plasma levels of inflammatory markers such as C-reactive protein (CRP) and lipoprotein-associated phospholipase A2 (Lp-PLA2) may reflect the degree of oxidative stress within atherosclerotic plaques. Metalloproteinases, such as matrix metalloproteinases (MMP) 1, 2, 9, pregnancy-associated plasma protein (PAPP-A), and inflammation within plaques can also cause MMP activation, decomposing extracellular matrix components such as collagen and elastic fibers in the fibrous cap, degrading the fibrous cap, and thus making stable plaques become unstable. The elevation of platelet activation and aggregation markers, such as soluble CD40 ligand (sCD40L), soluble P-selectin (sP-selectin) levels, combined with the rupture and / or erosion of unstable plaques, can lead to thrombosis.

[0005] In recent years, there have also been successive studies exploring new markers, such as ceramide, which is related to the occurrence of atherosclerosis, showing a significantly higher concentration of ceramide in aortic plaques. In addition, in addition to participating in reverse cholesterol transport, high-density lipoprotein (HDL) also participates in processes such as anti-inflammation, antioxidant, anti-platelet aggregation, anticoagulation, protecting the integrity of endothelial cells, anti-apoptosis, and vasodilation.

[0006] In the study of atherosclerosis, intron retention (IR), as an important RNA splicing event, has been confirmed to be related to the occurrence and development of various diseases. Intron retention refers to the phenomenon that introns fail to be removed and remain in mature mRNA during the RNA splicing process, which may lead to changes in protein sequence or function. RBM15 is an RNA-binding protein known to be involved in the regulation of m6A modification and alternative splicing (AS), but its specific regulatory mechanism is still unclear. The latest research found that RBM15 is related to atherosclerosis, can form punctate structures, and is in close contact with or even embedded in nuclear speckles, which are rich in splicing factors, suggesting that RBM15 may be involved in the alternative splicing of RNA. Through RNA-seq analysis, it was found that knockdown of RBM15 (RBM15-KD) could lead to 1,279 differential alternative splicing events in 1,111 transcripts, among which 191 transcripts were directly bound by RBM15, indicating that these transcripts may be the direct targets of RBM15. In addition, RBM15 promoted intron or exon retention in the neighboring regions near its binding sites in 121 transcripts.

[0007] The discovery and study of these markers provide important biological information for the early diagnosis, treatment, and prognosis evaluation of atherosclerosis, but further research is still needed to clarify the specific correlation between these markers and the occurrence and development of atherosclerosis, and to develop new diagnostic and treatment strategies. Summary of the Invention

[0008] The embodiments of the present invention provide the application of intron-retained transcripts as markers in the diagnosis of atherosclerosis to at least solve one of the problems existing in the related art.

[0009] To achieve this purpose, the present invention is realized through the following technical solutions.

[0010] A marker for diagnosing atherosclerosis and / or the degree of atherosclerosis, wherein the marker is an intron-retained transcript, and the intron-retained transcript includes HIPK2.ir and / or COPE.ir. The nucleotide sequence of HIPK2.ir is as shown in SEQ ID NO.1, and the nucleotide sequence of COPE.ir is as shown in SEQ ID NO.3. HIPK2.ir is a transcript generated by intron retention of the HIPK2 gene; COPE.ir is a transcript generated by intron retention of the COPE gene.

[0011] Studies on the COPE gene have shown that it plays an important role in lipid metabolism and inflammatory responses, and may affect the development of atherosclerosis by regulating intracellular lipid levels and the expression of inflammatory factors. The homeodomain-interacting protein kinase 2 gene (HIPK2 gene) is related to cell cycle regulation, apoptosis, and stress responses. Existing studies have pointed out its role in atherosclerosis, which may affect the development of atherosclerosis by regulating endothelial cell function and inflammatory responses. However, the relationship between the alternative splice variants of the COPE gene or HIPK2 gene and atherosclerosis is unclear. The inventors of this application established a screening and analysis process based on the characteristics of intron retention (IR) in the early stage. After focusing on the analysis of the transcriptome of peripheral blood leukocytes in atherosclerosis, it was found that the expression levels of the intron retention transcripts of two genes (COPE.ir and HIPK2.ir) were increased in the peripheral blood leukocytes of atherosclerosis patients. COPE.ir and HIPK2.ir can be used as diagnostic markers for atherosclerosis. Using multiple parameter Logistic regression analysis, it was found that combining HIPK2.ir and COPE.ir as a predictive index for atherosclerosis, the AUC reached 0.8062, which was significantly higher than other common clinical risk indicators for atherosclerosis, such as CK, lipoprotein a, apolipoprotein A1, A2, B, CII, and E, etc. Using the combined expression levels of COPE.ir and HIPK2.ir as a diagnostic index, the predictive ability for atherosclerosis was significantly better.

[0012] Meanwhile, the higher the expression levels of COPE.ir and HIPK2.ir, the higher the degree of atherosclerosis, especially the degree of coronary atherosclerosis. It indicates that COPE.ir and HIPK2.ir can be used as diagnostic markers for the degree of atherosclerosis.

[0013] Preferably, the amino acid sequence of the protein encoded by HIPK2.ir is as shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by COPE.ir is as shown in SEQ ID NO.4.

[0014] A combined marker, comprising the said marker.

[0015] Preferably, the combined biomarker further comprises one or more of oxidized low-density lipoprotein (oxLDL), C-reactive protein (CRP), apolipoprotein A1 (apoA1), apolipoprotein A2 (apoA2), apolipoprotein B (apoB), apolipoprotein CII (apoCII), apolipoprotein E (apoE), lipoprotein-associated phospholipase A2 (Lp-PLA2), small dense low-density lipoprotein cholesterol (SdLd), creatine kinase (CK), soluble CD40 ligand (sCD40L), soluble P-selectin (sP-selectin), or lipoprotein(a) (Lipo_a).

[0016] Use of a reagent for detecting the expression level of intron-retained transcripts in the preparation of a product for diagnosing atherosclerosis and / or the degree of atherosclerosis, wherein the intron-retained transcripts include HIPK2.ir and / or COPE.ir, the nucleotide sequence of HIPK2.ir is as shown in SEQ ID NO.1, and the nucleotide sequence of COPE.ir is as shown in SEQ ID NO.3.

[0017] Through research, the present invention has found that the intron-retained transcripts HIPK2.ir and COPE.ir of two genes are significantly highly expressed in the peripheral blood of patients with atherosclerosis. At the same time, the higher the expression levels of HIPK2.ir and COPE.ir, the higher the degree of atherosclerosis. HIPK2.ir and COPE.ir can be used as diagnostic markers for atherosclerosis and the degree of atherosclerosis. By detecting the expression levels of HIPK2.ir and COPE.ir, atherosclerosis or the degree of atherosclerosis can be diagnosed.

[0018] Preferably, the amino acid sequence of the protein encoded by HIPK2.ir is as shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by COPE.ir is as shown in SEQ ID NO.4.

[0019] Preferably, the product includes a kit or a test strip.

[0020] Preferably, the reagent for detecting the expression level of intron-retained transcripts includes primers for specifically amplifying HIPK2.ir or primers for specifically amplifying COPE.ir. The sequences of the primers for specifically amplifying HIPK2.ir are as shown in SEQ ID NO.5-6, and the sequences of the primers for specifically amplifying COPE.ir are as shown in SEQ ID NO.7-8.

[0021] A kit for diagnosing atherosclerosis and / or the degree of atherosclerosis, comprising reagents for detecting the expression level of intron-retained transcripts, wherein the intron-retained transcripts include HIPK2.ir and / or COPE.ir, the nucleotide sequence of HIPK2.ir is as shown in SEQ ID NO.1, and the nucleotide sequence of COPE.ir is as shown in SEQ ID NO.3.

[0022] Preferably, the amino acid sequence of the protein encoded by HIPK2.ir is as shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by COPE.ir is as shown in SEQ ID NO.4.

[0023] Preferably, the reagents for detecting the expression level of intron-retained transcripts include primers for specifically amplifying HIPK2.ir or primers for specifically amplifying COPE.ir, the sequences of the primers for specifically amplifying HIPK2.ir are as shown in SEQ ID NO.5-6, and the sequences of the primers for specifically amplifying COPE.ir are as shown in SEQ ID NO.7-8.

[0024] The embodiments of the present invention have the following beneficial effects:

[0025] Through research, the present invention found that the intron-retained transcripts HIPK2.ir and COPE.ir of two genes were significantly highly expressed in the peripheral blood of patients with atherosclerosis. At the same time, the higher the expression levels of HIPK2.ir and COPE.ir, the higher the degree of atherosclerosis. HIPK2.ir and COPE.ir can be used as diagnostic markers for atherosclerosis and the degree of atherosclerosis. The present invention provides a new marker for the diagnosis of atherosclerosis and a new means for the diagnosis and treatment of atherosclerosis. Description of the Drawings

[0026] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0027] Figure 1 It shows the expression differences of the proteins corresponding to the TMT-labeled mass spectrometry IR transcripts in the peripheral plasma of patients with atherosclerosis in Example 1 of the present invention. The abscissa is the clinical grouping, the Control group is the healthy control group without atherosclerosis (n = 11), the SCAD is the group of patients with atherosclerosis (n = 11), and the ordinate is the TMT probe signal intensity.

[0028] Figure 2 It shows the specific bands of HIPK2.ir and COPE.ir in Example 2 of the present invention.

[0029] Figure 3 This is the result of the multivariate Logistic regression analysis in Example 2 of the present invention, where AUC is the area under the ROC curve.

[0030] Figure 4 This is the DNA expression difference between HIPK2.ir and COPE.ir in the peripheral plasma of patients with atherosclerosis in Example 3 of the present invention. In the figure, A is the CT value distribution map of HIPK2.ir and COPE.ir. The abscissa is the ΔCT value of HIPK2.ir, and the ordinate is the ΔCT value of COPE.ir. B is the result of the ΔCT value of COPE.ir, and C is the result of the ΔCT value of HIPK2.ir; The CAD classification represents the grouping of the degree of coronary atherosclerosis. 0 is the normal control group (n = 21), 1 is the mild stenosis group (n = 28), and 2 is the severe stenosis group (n = 28); The Peripheral classification represents the grouping of peripheral atherosclerosis. 0 is the group without peripheral atherosclerosis, and 1 is the group with peripheral atherosclerosis. Detailed implementation manners

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will elaborate on each implementation manner of the present invention with reference to the accompanying drawings. However, those of ordinary skill in the art can understand that in each implementation manner of the present invention, many technical details are provided for the convenience of readers to better understand the present application. However, even without these technical details and various changes and modifications based on the following implementation manners, the technical solutions claimed in the present application can still be achieved. The division of the following embodiments is for convenience of description and should not constitute any limitation to the specific implementation manner of the present invention. The various embodiments can be combined and cross-referenced with each other on the premise of no contradiction.

[0032] Example 1

[0033] Analysis of intron retention (IR) transcripts in atherosclerosis

[0034] 1. Method

[0035] Data source: The original data of TMT-labeled mass spectrometry of peripheral plasma from human patients with atherosclerosis is from the ProteomXchange database PXD028664 dataset.

[0036] Mass spectrometry data analysis process: First, import the original data into the Maxquant software to automatically complete peak detection and quantification. Then, based on the IR analysis target, use the existing toolkit combination to predict the candidate proteins corresponding to the atherosclerotic-related IR transcripts as a database (the database construction method is to use the IRfinder software (doi:10.1186 / s13059-021-02515-8) to predict all possible IR transcripts, and then use Transdecoder (doi:10.1038 / nprot.2013.084) to predict the corresponding open reading frame and encoded protein) to perform sequence matching and identification of peptides and proteins. Subsequently, Maxquant automatically performs visual analysis, and finally exports the analysis results for statistical analysis through Prism software into readable tables and charts.

[0037] 2. Results

[0038] Through Maxquant analysis, it was found that there were only 6 proteins corresponding to the atherosclerotic-related IR transcripts in peripheral blood, and their transcripts were ENST120691.p1 (IR transcript of the GDNF-AS1 gene), ENST100887.p1 (IR transcript of the CCAR1 gene), ENST100904.p3 (transcript of the HIPK2 gene), ENST71652.p1 (IR transcript of the PNKD gene), ENST72627.p1 (IR transcript of the COPE gene), and ENST90205.p2 (IR transcript of the ENTPD3 gene). The expression differences of the proteins corresponding to the TMT-labeled mass spectrometry IR transcripts in the peripheral plasma of atherosclerotic patients are as Figure 1 shown. The abscissa represents the clinical grouping, the Control group is the healthy control group without atherosclerosis (n = 11), and the SCAD is the atherosclerotic patient group (n = 11). The ordinate represents the TMT probe signal intensity. The results show that the highly expressed IR transcripts in the atherosclerotic patient group are ENST100904.p3 and ENST72627.p1, and the P value is less than 0.01. ENST100904.p3 is the transcript generated by intron retention of the HIPK2 gene, denoted as HIPK2.ir, and ENST72627.p1 is the transcript generated by intron retention of the COPE gene, denoted as COPE.ir. That is, the IR transcripts HIPK2.ir and COPE.ir are highly expressed in the peripheral plasma of atherosclerotic patients.

[0039] The nucleotide sequence of HIPK2.ir is shown in SEQ ID NO.1:

[0040] atgcgcagtccaatgtggggaccaaaggagacaaaggccaaagggggtgaagtgccaagcagaacactggcagctgaagacgcccagcagcaggcgatggggacaacggaaggttcattaaatagcaacaaggcaactcactccagcccccagcggtcactccttctgctgcccagaccggcaagtgtgactggtactctagaagcacgtagtagccagtacccagtctctagttgtgaggaaaggaataaactaaaatttttttttttttttgagacagggtcttgctctgttaccaggctggagtgcaatggtgcaaatcatggctcatggcaccctcaaactcctgggctcaagcagtcctcccacttcagcctcctgagtagctgggaccacaggtgcgcacctccatgcctggctttttttttttttctttttgcgacggactcttactctgtcacccaggctggagtacaatggcacgatcccggctcactgcagcctctgcctcctgggttcaagcgattctcatgcctcagcctcctgaatagctgggattacagacctgcgccaccacgcctggctaatttttgtatttttagtagagacagggtttcaccatgttga(SEQ ID NO.1);

[0041] The amino acid sequence of the protein encoded by HIPK2.ir is shown in SEQ ID NO.2:

[0042] MRSPMWGPKETKAKGGEVPSRTLAAEDAQQQAMGTTEGSLNSNKATHSSPQRSLLLLPRPASVTGTLEARSSQYPVSSCEERNKLKFFFFFETGSCSVTRLECNGANHGSWHPQTPGLKQSSHFSLLSSWDHRCAPPCLAFFFFLFATDSYSVTQAGVQWHDPGSLQPLPPGFKRFSCLSLLNSWDYRPAPPRLANFCIFSRDRVSPC(SEQ IDNO.2);

[0043] The nucleotide sequence of COPE.ir is shown in SEQ ID NO.3:

[0044] atgtctgctagccaaggagcaccacgagtggccagcagccaccagagctgggagagaggcctgggacggccttgccctccagcctccagctggagccagccctgccgccaccttgacttcagacttacggcctccagagctgtgaggaacgaatccctgttgtccttaactgcccgggctgtggtgctttgccacagcagctccaggacattgagacaggtgacctcccagggccactgtttctcccaccctgcacttacttcaccagctggagtgaaggcagggaaccctgggtcccccaggagcagcagctgctgtgagcatcacagaaaagcagccccggagagcaggcggtccaggcaggggcttgtggtccgttcatctggctgcacagccgcgacctcattggcaggacgccccggggacaaggagcatccattagtaattggttttggttttgattttgttttcttgagatacggtcttgctctgtcgttcagcctggcatacagtggcacaatcttggcttactgcagccttgatctcccaggctcaagtgatcatcccacctcagcctcccgaatagctgggactacaggcacgcatcaccatgcctggctaatttgtatattttttagagatggggttttgcctggccgggcgcagtggctcacacctgtaatcccagcactttgggaggccgaggcgggcggatcacgaggtcaggagatggagaccatcctggctaacacgtga(SEQ ID NO.3)

[0045] The amino acid sequence of the protein encoded by COPE.ir is shown in SEQ ID NO.4:

[0046] MSASQGAPRVASSHQSWERGLGRPCPPASSWSQPCRHLDFRLTASRAVRNESLLSLTARAVVLCHSSSRTLRQVTSQGHCFSHPALTSPAGVKAGNPGSPRSSSCCEHHRKAAPESRRSRQGLVVRSSGCTAATSLAGRPGDKEHPLVIGFGFDFVFLRYGLALSFSLAYSGTILAYCSLDLPGSSDHPTSASRIAGTTGTHHHAWLICIFFRDGVLPGRAQWLTPVIPALWEAEAGGSRGQEMETILANT(SEQ ID NO.4).

[0047] Example 2

[0048] Detection of the RNA expression levels of HIPK2.ir and COPE.ir in peripheral blood leukocytes of patients with atherosclerosis and determination of the predictive ability of HIPK2.ir and COPE.ir as atherosclerosis prediction indicators.

[0049] 1. Method

[0050] Peripheral blood specimens: The peripheral blood specimens were from the Dongfang Hospital affiliated to Tongji University, and whether there was atherosclerosis was determined by the results of coronary angiography and peripheral vascular ultrasound examination.

[0051] Isolation of leukocytes and RNA extraction: Add 1 ml of Trizol to the precipitated leukocytes obtained by isolation and lyse the cells thoroughly. Place the homogenized sample at 15 - 30 °C for 5 min to completely separate the nucleic acid - protein complex. Add 0.2 ml of chloroform for every 1 ml of Trizol used, cover the tube cap, shake vigorously for 15 s, and let it stand at room temperature for 3 min. Centrifuge at 12000 rpm at 4 °C for 10 min, and carefully aspirate the upper aqueous phase. Add an equal volume of isopropanol to the obtained aqueous solution, mix well, and place it at - 20 °C for 20 - 30 min. Wash the precipitate with 75% ethanol to remove all residual proteins and inorganic salts. Centrifuge at 12000 rpm at 4 °C for 10 min, carefully aspirate the supernatant, air - dry the RNA precipitate, and dissolve it with double - distilled water treated without DEPC. Store the extracted RNA in a - 80 °C refrigerator for later use.

[0052] RT-PCR experiment: In an RNase- and DNase-free EP tube, add RNA, Oligo(dT)15 primer, reverse transcriptase, and buffer for reverse transcription. First, mix the RNA with Oligo(dT)15 and heat at 65 °C for 5 minutes. Then add reverse transcriptase and other buffers, and incubate at 37 °C for 20 minutes to obtain cDNA by reverse transcription. Use the obtained cDNA as a template for RT-PCR amplification. The specific information of the amplification primers is shown in Table 1 below,

[0053] Table 1

[0054]

[0055] The reaction system includes: 1 μl of cDNA template, 0.5 μl of each upstream and downstream primer, 0.2 μl of Taq DNA polymerase, 0.5 μl of dNTP, 2 μl of Taq enzyme buffer, and 15.3 μl of nuclease-free water (total system 20 μl). The amplification program includes: pre-denaturation (95 °C for 5 minutes), denaturation (94 °C for 30 seconds), annealing (60 °C for 30 seconds), and extension (72 °C for 30 seconds), for a total of 26 - 30 cycles. After the amplification is completed, analyze the products by agarose gel electrophoresis to observe the specific bands of the target genes (HIPK2.ir and COPE.ir). The specific bands of HIPK2.ir and COPE.ir are as Figure 2 shown, where the amplified fragment corresponding to the specific band of HIPK2.ir (H.ir) is 306 bp; the amplified fragment corresponding to the specific band of COPE.ir (C.ir) is 151 bp.

[0056] Whether specific bands of COPE.ir and HIPK2.ir can be amplified by RT-PCR is used as a parameter for multivariate Logistic regression analysis to predict the occurrence of atherosclerosis. The main analysis steps are as follows: ① Define the dependent variable: Determine the binary dependent variable of the study as whether atherosclerosis occurs; ② Select independent variables: According to the literature, select independent variables that may affect the dependent variable, including apolipoprotein A1 (apoA1), apolipoprotein A2 (apoA2), apolipoprotein B (apoB), apolipoprotein CII (apoCII), and apolipoprotein E (apoE), small and dense low-density lipoprotein cholesterol (SdLd), creatine kinase (CK), and lipoprotein a (Lipo_a), as well as the target variables C.ir and H.ir (whether the target band is detected by RT-PCR results); ③ Multivariate model construction: Stepwise regression (two-way method), and pairwise combinations of key variables are modeled based on domain knowledge; ④ Parameter estimation: Use the maximum likelihood estimation method (MLE) to solve the regression coefficient (β); ⑤ Model performance evaluation: Use the goodness-of-fit index, the area under the ROC curve (AUC); ⑥ Plot the ROC curve to show the classification performance of different models. The pairwise combinations of the key variables specifically include: the combination of HIPK2.ir and COPE.ir, denoted as H.ir&C.ir; the combination of CK and Lipo_a, denoted as CK&Lipo_a; the combination of apoB and apoE, denoted as apoB&apoE; apoA1 and apoCII, denoted as apoA1&apoCII; apoA2 and SdLd, denoted as apoA2&SdLd; apoB and apoCII, denoted as apoB&apoCII.

[0057] Pairwise combinations of key variables are used as prediction indicators for multivariate Logistic regression analysis according to the analysis steps mentioned above.

[0058] 2. Results

[0059] The results of the multivariate Logistic regression analysis are as Figure 3 shown, where AUC is the area under the ROC curve. The results show that the AUC of COPE.ir and HIPK2.ir as prediction indicators is 0.8062, and the P value is 0.0004. Compared with pairwise combinations of other common clinical risk indicators for atherosclerosis (including CK, lipoprotein a, apolipoproteins A1, A2, B, CII, and E, etc.) as prediction indicators, the predictive ability of COPE.ir and HIPK2.ir as combined prediction indicators is significantly better.

[0060] Example 3

[0061] Detection of DNA expression levels of HIPK2.ir and COPE.ir in peripheral blood of patients with atherosclerosis

[0062] 1. Method

[0063] Peripheral blood specimens: The peripheral blood specimens were from the Dongfang Hospital Affiliated to Tongji University. Whether there was coronary atherosclerosis and the degree of atherosclerosis were determined by the results of coronary angiography and peripheral vascular ultrasound. The degree of coronary atherosclerosis was divided into a severe stenosis group with the stenosis degree of 1 or more branch arteries being greater than 50%, a mild stenosis group with the stenosis degree of all branch arteries being less than 50%, and a normal group without arterial stenosis.

[0064] Isolation of white blood cells, extraction of RNA, and acquisition of cDNA templates were performed in the same method as in Example 2.

[0065] qPCR experiment: The reaction system included qPCR Mix (SYBR Green), primers (the same primers as in Table 1 of Example 2), cDNA templates, and deionized water. Each component was added to the reaction tube according to the ratio (10 μl of qPCR Mix, 0.5 μl of each upstream and downstream primer, 1 μl of cDNA template, and 8 μl of deionized water). The amplification program included pre-denaturation (95°C for 5 minutes), denaturation (94°C for 30 seconds), annealing (60°C, 30 seconds), and extension (72°C, 30 seconds), and a total of 40 cycles were performed. Fluorescence signals were detected during the annealing / extension stage of each cycle. The qPCR instrument recorded the fluorescence signals to obtain Ct values. The relative expression levels of the target genes (HIPK2.ir and COPE.ir) were calculated using the ΔCt method, that is, the Ct value of the detected ir transcript minus the Ct value of the internal reference gene GAPDH in the same sample to represent its relative expression level. The lower the Ct value, the higher the expression level.

[0066] 2. Results

[0067] The DNA expression differences of HIPK2.ir and COPE.ir in the peripheral plasma of atherosclerotic patients were as follows Figure 4As shown in the figure, in Figure A, the CT value distribution diagrams of HIPK2.ir and COPE.ir are presented. The abscissa represents the ΔCT value of HIPK2.ir, and the ordinate represents the ΔCT value of COPE.ir. In Figure B, the results of the ΔCT value of COPE.ir are shown, and in Figure C, the results of the ΔCT value of HIPK2.ir are shown; the CAD classifications respectively represent the groupings of the degree of coronary atherosclerosis. 0 represents the normal control group (n = 21), 1 represents the mild stenosis group (n = 28), and 2 represents the severe stenosis group (n = 28); the Peripheral classifications respectively represent the groupings of peripheral atherosclerosis. 0 represents the group without peripheral atherosclerosis, and 1 represents the group with peripheral atherosclerosis. The results show that the expressions of both COPE.ir and HIPK2.ir are correlated with the presence or absence of coronary / peripheral atherosclerosis (A). As the degree of coronary atherosclerosis increases, the ΔCT values of both COPE.ir and HIPK2.ir decrease (B and C), that is, the higher the expression levels of COPE.ir and HIPK2.ir. This indicates that the expression levels of COPE.ir and HIPK2.ir in peripheral blood leukocytes are significantly correlated with the degree of atherosclerosis, especially coronary atherosclerosis. The higher the expression levels of COPE.ir and HIPK2.ir, the higher the degree of atherosclerosis.

[0068] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A marker for diagnosing atherosclerosis and / or the degree of atherosclerosis, characterized in that, The biomarker is an intron-retained transcript, and the intron-retained transcript includes HIPK2.ir and / or COPE.ir. The nucleotide sequence of HIPK2.ir is shown in SEQ ID NO.1, and the nucleotide sequence of COPE.ir is shown in SEQ ID NO.

3.

2. A combined marker, characterized in that It includes the biomarker described in claim 1.

3. The combined marker according to claim 2, wherein, The combined biomarker further includes one or more of oxidized low-density lipoprotein, C-reactive protein, apolipoprotein A1, apolipoprotein A2, apolipoprotein B, apolipoprotein CII, apolipoprotein E, lipoprotein-associated phospholipase A2, small dense low-density lipoprotein cholesterol, creatine kinase, soluble CD40 ligand, soluble P-selectin, or lipoprotein(a).

4. Use of a reagent for detecting the expression level of intron-retained transcripts in the preparation of a product for diagnosing atherosclerosis and / or the degree of atherosclerosis, characterized in that, The intron-retained transcript includes HIPK2.ir and / or COPE.ir. The nucleotide sequence of HIPK2.ir is shown in SEQ ID NO.1, and the nucleotide sequence of COPE.ir is shown in SEQ ID NO.

3.

5. The application according to claim 4, characterized in that The product includes a kit or a test strip.

6. A kit for diagnosing atherosclerosis and / or the degree of atherosclerosis, characterized in that, It includes a reagent for detecting the expression level of an intron-retained transcript, and the intron-retained transcript includes HIPK2.ir and / or COPE.ir. The nucleotide sequence of HIPK2.ir is shown in SEQ ID NO.1, and the nucleotide sequence of COPE.ir is shown in SEQ ID NO.

3.

7. The marker according to claim 1, or the application according to claim 4, or the kit according to claim 6, characterized in that The amino acid sequence of the protein encoded by HIPK2.ir is shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by COPE.ir is shown in SEQ ID NO.

4.

8. The application according to claim 4 or the kit according to claim 6, characterized in that, The reagent for detecting the expression level of an intron-retained transcript includes primers for specifically amplifying HIPK2.ir or primers for specifically amplifying COPE.ir. The sequences of the primers for specifically amplifying HIPK2.ir are shown in SEQ ID NO.5-6, and the sequences of the primers for specifically amplifying COPE.ir are shown in SEQ ID NO.7-8.

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