Application of XBP1 in prognosis of ankylosing spondylitis

Abstract

The invention discloses application of XBP1 in prognosis evaluation of ankylosing spondylitis, belongs to the technical field of biological medicine, and particularly relates to a method for providing XBP1 as a prediction biomarker of ankylosing spondylitis, and discovery of the biomarker provides a new possibility for prognosis evaluation of ankylosing spondylitis at present. The invention finds that the expression quantity of the neutrophil XBP1 of bone marrow blood of an ankylosing spondylitis patient is obviously reduced, and proves that the XBP1 is an important biomarker for judging prognosis of the ankylosing spondylitis. Based on the application of the XBP1 serving as a specific marker for the treatment effect and prognosis of the ankylosing spondylitis, clinical individualized precise treatment is further guided through a prognosis model.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of XBP1 in the preparation of ankylosing spondylitis prognosis assessment products. Background Art

[0002] Ankylosing spondylitis (AS) is an autoimmune disease characterized by chronic inflammation and pathological new bone formation. AS usually develops between the ages of 20 and 40, and is more common in men than in women. Ankylosing spondylitis mainly affects the sacroiliac joints and axial spinal joints, and is often accompanied by damage to tissues and organs such as the heart, eyes, lungs, kidneys, and nervous system. The bony ankylosis and deformity of the axial spinal joints caused by pathological new bone formation are the main causes of disability in patients with ankylosing spondylitis. Early symptoms include chronic low back pain and stiffness, especially in the morning or after long periods of inactivity. As the disease progresses, symptoms may extend to the entire spine, resulting in limited mobility. The diagnosis and treatment of this disease is a complex process. At present, the prognosis assessment of ankylosing spondylitis involves multiple aspects, including disease activity, joint function, imaging progression, patient-reported outcomes, and quality of life. There is currently no clear indicator to assess patient prognosis.

[0003] Therefore, the biomarker XBP1 for prognosis assessment of ankylosing spondylitis disclosed in the present invention has great clinical significance in the prognosis assessment of ankylosing spondylitis. Summary of the invention

[0004] The present invention aims at the deficiencies of the existing prognosis assessment technology for ankylosing spondylitis, and provides an ankylosing spondylitis prognosis marker. The present invention uses bone marrow blood obtained from the spine during clinical surgery as a research sample, confirms the significant correlation between the expression of XBP1 in bone marrow blood and the onset of ankylosing spondylitis, and thus determines that XBP1 in bone marrow blood can be used as an important indicator for the prognosis assessment of ankylosing spondylitis.

[0005] The present invention provides a novel kit for evaluating the prognosis of ankylosing spondylitis. The kit evaluates the prognosis of ankylosing spondylitis by detecting the expression level of the neutrophil specific marker XBP1 gene in a patient sample.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: A biomarker for the prognosis assessment of ankylosing spondylitis, wherein the biomarker is XBP1 significantly expressed by neutrophils in bone marrow blood, and the mRNA nucleotide sequence of the XBP1 is shown in GenBank accession number NM_001079539.2.

[0007] XBP1 is a transcription factor that plays a key role in endoplasmic reticulum stress and unfolded protein response. It works through the activation of the IRE1α pathway, which is one of the three main signaling pathways of the UPR. XBP1 plays an important role in a variety of biological processes, including protein folding, lipid metabolism, inflammatory response and immune regulation. The present invention found that the expression of XBP1 in bone marrow blood is significantly correlated with the onset of ankylosing spondylitis, and is very suitable as a biomarker for the prognosis assessment of ankylosing spondylitis.

[0008] Another object of the present invention is to provide a reagent or product for detecting the above-mentioned biomarkers for use in preparing a preparation for evaluating the prognosis of ankylosing spondylitis.

[0009] Preferably, the prognosis assessment preparation is a kit.

[0010] Preferably, the kit comprises specific primers for XBP1, and the nucleotide sequences thereof are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0011] Primer sequences: -CCTGGTTGCTGAAGAGGAGG-, SEQ ID NO: 1; -TGAGATACCCAGCTCCGGAA-, SEQ ID NO: 2.

[0012] Preferably, the kit further comprises reagents for extracting total RNA from bone marrow blood, reagents for reverse transcribing XBP1 into cDNA using total RNA as a template, and reagents for real-time quantitative PCR of cDNA.

[0013] Another object of the present invention is to provide a prognosis assessment kit for ankylosing spondylitis, comprising detection primers for detecting the above-mentioned biomarkers.

[0014] Preferably, the nucleotide sequences of the detection primers are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0015] Another object of the present invention is to provide a primer for detecting biomarkers for prognosis assessment of ankylosing spondylitis, wherein the primer is used to detect the expression level of the above biomarkers, and the nucleotide sequence thereof is shown in SEQ ID NO:1 and SEQ ID NO:2.

[0016] Another object of the present invention is to provide the use of the above-mentioned primers for detecting biomarkers for prognosis assessment of ankylosing spondylitis in the preparation of ankylosing spondylitis prognosis assessment preparations. Beneficial Effects

[0017] The present invention discovered and confirmed for the first time that XBP1 is a very important biomarker for assessing the prognosis of ankylosing spondylitis. The present invention found that the expression level of XBP1 in ankylosing spondylitis tissues was significantly decreased. By analyzing scRNA-seq data, the molecular mechanism of metabolic changes in neutrophils expressing XBP1 in the pathogenesis of ankylosing spondylitis was clarified, and it was found that XBP1 exerted its effects through the activation of the IRE1α pathway. Therefore, XBP1 can guide the clinical individualized treatment of ankylosing spondylitis through a prognostic model, which has important practical application value for assessing the prognosis of patients and guiding the individualized treatment of patients, and improves the subsequent treatment effect of ankylosing spondylitis patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0019] Figure 1 This UMAP image shows the expression of XBP1 in bone marrow blood cells of patients with ankylosing spondylitis.

[0020] Figure 2 Immunohistochemical expression of XBP1 in patients with ankylosing spondylitis.

[0021] Figure 3 Immunohistochemical analysis of neutrophils in ankylosing spondylitis samples and the expression of XBP1 in neutrophils. The expression of XBP1 in neutrophils was significant in ankylosing spondylitis samples. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially. Example 1

[0023] Detection of XBP1 in bone marrow blood 1. Extraction of total RNA from bone marrow blood cells 1) After adding Trizol to the bone marrow blood at a ratio of 1:1, leave it at room temperature for 5 minutes to allow it to fully lyse; centrifuge at 12000 rpm for 5 minutes and discard the precipitate; 2) Add 200ul chloroform / 1ml Trizol, mix well, place at room temperature for 15min, and centrifuge at 4°C, 12000rpm for 15min; 3) Pipette the upper aqueous phase into another centrifuge tube, add isopropanol at a ratio of 0.5 ml isopropanol / 1 ml Trizol, mix well, and leave at room temperature for 5-10 minutes. Centrifuge at 4°C, 12,000 rpm for 10 minutes and discard the supernatant; 4) Add ethanol at the ratio of 1 ml 75% ethanol / 1 ml Trizol, centrifuge at 4°C, 8000 rpm for 5 min, discard the supernatant, and air-dry at room temperature for 5-10 min; 5) Dissolve RNA samples with H2O, TE buffer or 0.5% SDS for 5-10 min; measure OD value to quantify RNA concentration. For human samples, OD260 / 280=1.74-1.95, OD260 / 230=1.02-2.19; 2. Reverse Transcription Reverse transcription was performed using PrimeScriptTM RT reagent Kit (TaKaRa Code No.RR037A) 1) Prepare RT reaction solution: 5× PrimeScript Buffer (for Real Time) 2μl, PrimeScript RT Enzyme MixⅠ 0.5ul, Oligo dTPrimer (50μM) 0.5μl, Random 6mers (100μM) 0.5μl, add Total RNA according to the reaction system (a maximum of 500ng of Total RNA can be used in a 10μl reaction system), and make up the rest with RNase Free dH2O to obtain a 10μl reaction system; 2) Set the reverse transcription reaction conditions: set the conditions to 37°C, 15 minutes, 85°C, 5 seconds, and 4°C to obtain cDNA; 3. Real-time fluorescence quantitative PCR Real-time fluorescence quantitative PCR using 2×FastHS SYBR QPCR mixture (ALLMEEK Cat#: AM2101) kit 1) Prepare PCR reaction solution: 2×QPCR Mixture 10μl, Forward Primer, 10μM 0.4μl, Reverse Primer, 10μM 0.4μl, calculate the amount of cDNA according to the concentration, and add RNase-free H2O to 20μl. After shaking and mixing, centrifuge briefly and put it into the qPCR instrument.

[0024] Human XBP1 mRNA nucleotide sequence (GenBank accession number NM_001079539.2) The primer sequences are as follows: -CCTGGTTGCTGAAGAGGAGG-, SEQ ID NO: 1, -TGAGATACCCAGCTCCGGAA-, SEQ ID NO: 2, 2) The operation program is pre-denaturation at 95℃ for 30s; denaturation at 95℃ for 10s; annealing / extension at 60℃ for 30s; 35-45 cycles. To reduce the operation error, make three replicate wells for each sample, and prepare the mixed solution and then divide it into three replicate tubes. Example 2

[0025] Specific testing of XBP1 levels in human bone marrow blood cells in patients with ankylosing spondylitis Bone marrow blood cells were collected from patients with ankylosing spondylitis diagnosed by imaging and laboratory tests and healthy volunteers from the First Affiliated Hospital of Guangxi Medical University. Total RNA of the samples was extracted according to the method of Example 1, reverse transcribed to obtain cDNA, and quantitatively detected by real-time fluorescence quantitative PCR. Example 3

[0026] Immunohistochemistry to determine the expression of XBP1 According to the method of Example 2, human spinal ligament samples were collected, properly processed and fixed, and the expression of XBP1 was measured according to conventional immunohistochemistry. The results are as follows: Figure 2 As shown, the expression of XBP1 in the ankylosing spondylitis group was significantly lower than that in the control group.

[0027] The results are attached Figure 2 , 3 As shown in the figure, compared with the control group, the expression level of XBP1 in the ankylosing spondylitis group was significantly reduced. Therefore, the expression of XBP1 in bone marrow blood cells can be used as a specific molecular biomarker for the prognosis evaluation of ankylosing spondylitis, which has important practical application value in evaluating the prognosis of patients and guiding the individualized treatment of patients.

[0028] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of XBP1 in the preparation of a product for the prognosis of ankylosing spondylitis.

2. The use according to claim 1, characterized in that The prognosis product for ankylosing spondylitis uses the XBP1 gene as a specific biomarker and determines the prognosis of ankylosing spondylitis by detecting the expression level of the XBP1 gene in the subject's sample.

3. The use according to claim 2, characterized in that: The subject sample is bone marrow blood from ankylosing spondylitis patients.

4. The use according to claim 2, characterized in that: The prognostic product is a kit.

5. The kit according to claim 4, characterized in that The kit contains detection substances and control substances for detecting the XBP1 gene.

6. The detection reagent according to claim 5, characterized in that The reagents include specific detection primers for XBP1.

7. The use according to claim 3, characterized in that: The prognostic product determines the prognosis of ankylosing spondylitis patients by detecting whether the expression level of the XBP1 gene in the neutrophils of the bone marrow blood of ankylosing spondylitis patients is decreased compared with normal tissues.

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