TW202305146A - Method for detecting the risk of seronegative arthritis associated with psoriatic arthritis and ankylosing spondylitis and the risk of back pain caused by inflammatory bowel disease by microribonucleic acid - Google Patents

Abstract

The present invention is related to a method for detecting the risk of seronegative arthritis associated with psoriatic arthritis and ankylosing spondylitis and the risk of back pain caused by inflammatory bowel disease by microribonucleic acid(miRNA). The method includes measuring the expression level of at least one miRNA disclosed herein in a test sample of a subject, comparing the expression level of the at least one miRNA in the test sample with that of a seronegative arthritis-free sample or a back pain-free sample, and identifying the subject as having seronegative arthritis or back pain, or at risk of developing seronegative arthritis associated with psoriatic arthritis and ankylosing spondylitis or back pain caused by inflammatory bowel disease if the expression levels of the at least one miRNA in the test sample is higher relative to the expression level of the corresponding miRNA in the arthritis-free sample or a back pain-free sample.

Description

Method and application of using microRNA to detect the risk of seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis and the risk of associated back pain caused by inflammatory bowel disease

The present invention relates to a method for detecting the risk of seronegative arthritis using microRNA and its application, in particular to a method for detecting the risk and inflammation of seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis using microRNA in plasma The method and its application to the risk of associated back pain caused by enteritis.

Seronegative arthritis is a systemic disease that includes possible involvement of the skin, joints, nails, kidneys, vascular system, and heart, and may develop into associated cancers. Common features include the absence of rheumatoid factor (RF) and a strong genetic association with HLA-B27.

Compared with the normal control group, seronegative arthritis serum contained psoriatic arthritis, reactive arthritis, and ankylosing spondylitis, and patients with combined constitutions of psoriatic arthritis and ankylosing spondylitis had particularly worsening joints serious.

A recent study reported that the prevalence of psoriatic arthritis (PSA) was approximately 17% among patients with psoriasis in dermatology clinics. Psoriatic arthritis is a chronic inflammatory disease involving progressive arthritis associated with psoriasis. Approximately 30% of patients with psoriasis are estimated to have psoriatic arthritis. Typically, cutaneous manifestations precede the onset of PSA about 10 years. Haroon et al. (Annals of the rheumatic diseases 2015;74(6):1045-50) reported adverse radiographic findings and poor functional outcomes in patients with PSA due to delayed diagnosis of psoriatic arthritis over 6 months . Early diagnosis and timely intervention are the keys to avoid permanent joint deformation and destruction. Currently, the diagnosis of PSA is mainly based on clinical phenotype. So far there is no standard and reliable assessment method for early detection of PSA, resulting in many patients with psoriasis undiagnosed psoriatic arthritis leading to poor treatment outcomes (Veale DJ et al., Lancet 2018; 391(10136): 2273-84 ).

Ankylosing spondylitis (AS) is a chronic inflammatory disease of the spine joints, which occurs in young men aged 20 to 40, but women may also get this disease; typical symptoms of ankylosing spondylitis are: chronic Low back pain, morning spine stiffness, and limited range of motion. It is more obvious when you are resting, especially when you just wake up in the morning (usually more than one hour). In severe cases, the patient may even wake up in the middle of the night due to soreness and stiffness. will lighten. The diagnosis of ankylosing spondylitis is mainly based on detailed medical history inquiry, clinical symptoms, physical examination and X-ray photography (must see bilateral intestinal sacral arthritis), if necessary, blood test for HLA-B27 antigen in blood Ankylosing spondylitis is diagnosed by comprehensive judgment, so its diagnosis is particularly difficult.

Even with the aid of ultrasound, early detection and diagnosis of seronegative arthritis remains a challenge for rheumatologists. The sensitivity of the Madrid Ultrasound Seronegative Arthritis Index (MASEI) for seronegative arthritis is only 30%. Even if the patient has obvious symptoms, there is a delay of at least two years before a definitive diagnosis of seronegative arthritis is made. A Mayo Clinic study in 2020 also showed that such delayed diagnosis has not improved much from 2000 to 2017.

MicroRNAs (miRNAs) are evolutionarily conserved and non-coding RNA molecules, usually 21 to 25 nucleotides in length, and UTRs) to inhibit protein translation or promote mRNA degradation (see Griffiths-Jones S (2004). The microRNA Registry. Nucleic acids research 32: D109-111).

However, the current studies on miRNA biomarkers are rarely used in clinical diagnosis. Therefore, there remains an unmet need for the development of early detection and confirmation of seronegative arthritis, especially both psoriatic arthritis and ankylosing spondylitis, using miRNA, and the present invention satisfies this and other needs.

In view of the above problems, the purpose of the present invention is to provide a new method oriented by pathogenic mechanism to distinguish patients with seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis; Epigenetic regulation of the RNA detection network is a key factor controlling the physiological and pathological conditions of the immune system, and differential expression of certain microRNA detections in plasma may determine the clinical difference between those with and without seronegative arthritis The results were mixed; thus, specific microRNAs in plasma may be used as serological markers to improve the limited current available for early diagnosis of seronegative arthritis patients with concurrent psoriatic arthritis and ankylosing spondylitis. clinical testing.

In order to find out the microRNA in plasma that can be used to detect seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis, a study based on quantitative polymerase chain reaction (qPCR) was carried out; qPCR technology It uses a specific primer probe (primer probe) to generate fluorescence during the PCR process, and then uses a fluorescence detection system to detect the amount of fluorescence released in each cycle (cycle), and then calculates the amount of fluorescence in each cycle. The resulting product yield can achieve the purpose of real-time quantification.

Using qPCR technology, a prospective comparison was made between the detection characteristics of 149 microRNAs in plasma between patients with psoriatic arthritis and psoriasis, and 10 microRNAs in plasma with different differential expressions were found; The detection of 10 microRNAs in plasma with different differential expressions was compared with the detection of microRNAs in plasma from healthy individual samples as a control group. According to the Gene Ontology described by the detection of microRNAs in plasma with differential expression, the Several key inflammatory pathways are defined in patients with psoriatic arthritis disease.

Then, patients with ankylosing spondylitis and inflammatory bowel disease were searched, and their body fluids were extracted to perform qPCR on the microRNAs in their plasma to verify the detection of 5 microRNAs in plasma with differential expression, and three different plasma microRNAs were confirmed. Detection of microRNAs in microRNAs can represent the combined constitution of psoriatic arthritis and ankylosing spondylitis, which are three microRNAs in plasma, namely miRNA-140-5p, miRNA-192-5p, and miRNA-146a-5p, as follows Table 1 shows.

Table 1 miRNA name serial number miRNA-140-5p SEQ ID NO:1 miRNA-192-5p SEQ ID NO:2 miRNA-146a-5p SEQ ID NO:3

When the expression level of at least one of the above-mentioned 3 miRNAs in the test sample is higher than the corresponding miRNA in the sample without seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis, it means that the individual has combined seronegative arthritis with psoriatic arthritis and ankylosing spondylitis or at risk for seronegative arthritis with combined psoriatic arthritis and ankylosing spondylitis.

When the expression levels of the above three miRNAs in the test sample are higher than the corresponding three miRNAs in the samples without seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis, it means that the individual has combined psoriatic arthritis and ankylosing spondylitis. seronegative arthritis with arthritis and ankylosing spondylitis or at risk for seronegative arthritis with combined psoriatic arthritis and ankylosing spondylitis.

In summary, the differential expression of specific microRNA detection results can be used to detect seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis, or to predict the combination of psoriatic arthritis and ankylosing spondylitis The risk of seronegative arthritis of sexual spondylitis, and the detection of microRNA is very convenient, and can quickly screen out patients with the same inflammatory pathway, especially patients with psoriatic arthritis and ankylosing spondylitis combined constitution, for early risk analysis Diagnosis can prevent the deterioration of arthritis and prevent the golden opportunity of treatment from being missed.

Embodiments of the present invention are described by way of the following examples, which are not to be construed as limiting its scope in any way. On the contrary, it should be clearly understood that, without departing from the spirit of the present invention, other various embodiments, modifications and equivalents thereof can be conceived by those skilled in the art after reading this specification. . In the studies described in the following exemplars, conventional procedures were followed unless otherwise stated. Some of the procedures described below are for illustrative purposes.

The use of the word "or" in the claims means "and/or".

When the wording "comprising", "having", or "comprising" is used in this specification and claims, it is intended to be inclusive or open-ended and does not exclude additional, unlisted elements or method steps.

"Patient" or "subject" as used herein refers to a seronegative arthritis diagnosed with psoriatic arthritis and ankylosing spondylitis or suspected of developing psoriatic arthritis and ankylosing spondylitis Mammalian subjects with negative arthritis risk. Exemplary subjects are humans, apes, dogs, pigs, cows, cats, horses, sheep, sheep, rodents and other mammals that can develop seronegative arthritis of psoriatic arthritis and ankylosing spondylitis.

"MicroRNA (microRNA)", "microRNA (miR)" or "microRNA (miRNA)" as used interchangeably herein refers to unprocessed (e.g.: precursors), or processed Processed (eg, mature) RNA transcripts. MicroRNAs are endogenous non-coding single-stranded RNAs that negatively regulate gene expression in eukaryotes and constitute a novel class of gene regulators (Chua, et al. (2009) Curr. Opin. Mol. Ther. 11:189 -199). Individual miRNAs have been identified, sequenced and given names in different organisms. The names of miRNAs and their sequences are provided herein.

All numbers herein are to be understood as modified by "about" and are meant to cover a variation of ±10% of the specified value when, for example, these variations are appropriate to be indicative of the amount of miRNA.

"Higher" miRNAs appear in relative terms and can be compared in test samples with a referenced pool of seronegative arthritis subjects known to be free of psoriatic arthritis and ankylosing spondylitis Comparison of miRNA expression levels determines.

The term "sample" as used herein refers to a sample comprising miRNA. Samples can be used to detect the presence and/or expression of miRNAs of interest. Although predicted to contain miRNAs that behave differently compared to normal controls, biological fluids and organs are most suitable. In some embodiments, samples such as blood or components thereof are relatively easy to obtain. Non-limiting examples of samples include bodily fluids (e.g., peripheral blood), cells (e.g., blood cells) and tissues (e.g., biopsy specimens or joint fluid). Preferably, samples may be selected from biological samples, blood, plasma, serum, The group consisting of lymph, bone marrow, saliva and their combinations.

The measurement of miRNA expression refers to the quantification of the amount of miRNA present in a sample. The measurement of the expression level of specific or any miRNA can be performed using any method known to those having ordinary knowledge in the technical field of the present invention or such as by real-time polymerase chain reaction (real-time PCR), northern blot method (Northern blot) analysis achieved by the methods described herein. Measuring the expression of the miRNA comprises measuring the expression of a mature form of the miRNA or a precursor form associated with the expression of the miRNA.

In a specific embodiment, the expression level of at least one miRNA is quantified using northern blot analysis. For example, total cellular RNA can be purified from cells by homogenization in the presence of nucleic acid extraction buffer followed by centrifugation. Nucleic acids are precipitated, and DNA is removed by treatment with DNase and precipitation. RNA molecules were then separated by gel electrophoresis on agarose gels and transferred to nitrocellulose filters according to standard techniques. Afterwards, the RNA was immobilized on filter paper by heating. Detection and quantification of a specific RNA is accomplished using appropriate labeled DNA or RNA probes complementary to the RNA in question. See, eg, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7, the entire disclosure of which is hereby incorporated by reference.

In some embodiments, the use of microarrays is feasible. Microarrays are microscopic, ordered arrays of nucleic acids, proteins, small molecules, cells, or other substances that can be analyzed in parallel on complex biological samples. The technology provides many oligonucleotides (oligonucleotides) or polynucleotides (polynucleotides) with known sequence information as probes to find and hybridize with complementary strands (complementary strands) in the sample, so by Selective binding captures complementary strands. Probes comprise oligonucleotide or polynucleotide sequences that are complementary or substantially complementary to at least a portion of a target miRNA sequence. "Complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid strands. It is known that if the residue is thymine or uracil, the adenine residue of the first nucleic acid region can be aligned with the second nucleic acid region antiparallel to the first region. residues form specific hydrogen bonds ("base pairing"). Similarly, it is known that if the residue is guanine, the cytosine residue of the first nucleic acid region can be compared with the residue base of the second nucleic acid strand antiparallel to the first strand. base pairing. A first region of nucleic acid is complementary to the same or different nucleic acid when at least one nucleotide residue of the first region can base pair with a residue of the second region if the two regions are arranged in antiparallel fashion of the second area. Preferably, the first region comprises a first portion and the second region comprises a second portion such that when the first portion and the second portion are arranged in an antiparallel fashion, at least about 50 of the nucleotide residues in the first portion % and preferably at least about 75%, at least about 90%, or at least about 95% can base pair with the nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are base pairable with nucleotide residues in the second portion.

For example, microarray analysis of miRNAs can be accomplished according to any method known in the art. In one embodiment, RNA is extracted from the cells of the sample, and small RNAs (18-26 nucleotides in length) are size-selected from all RNAs using denaturing polyacrylamide gel electrophoresis. ). Oligonucleotide linkers were attached to the 5' and 3' ends of the small RNAs, and the resulting ligation products served as templates for RT-PCR reactions for 10 cycles of amplification. The sense strand PCR primer has a fluorophore attached to its 5' end, thus fluorescently labeling the sense strand of the PCR product. The PCR products are denatured and then hybridized to a microarray. The PCR product refers to the target nucleic acid that is complementary to the corresponding miRNA capture probe sequence on the array, which will hybridize via base pairing to the spot where the capture probe is immobilized. The spots will then fluoresce when excited using a microarray laser scanner. The fluorescence intensity of each spot is then assessed using several positive and negative control groups and array data normalization methods to assess the copy number of a particular miRNA, which will yield an estimate of the expression level of a particular miRNA. With respect to the miRNAs disclosed herein, the probes may be 100% complementary to the target miRNA or polynucleotide sequence. However, since the probe can specifically bind and capture the target polynucleotide from the sample, the probe need not necessarily be fully complementary to the target polynucleotide along its entire length. In some embodiments, the probe is complementary to a polynucleotide that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 3.

In some embodiments, the use of quantitative RT-PCR is available. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a modification of polymerase chain reaction used to rapidly determine the amount of product of the polymerase chain reaction. The purpose of qRT-PCR is generally to determine whether a gene sequence such as a miRNA is present in a sample, and if so, how much is replicated in the sample. Any method of PCR that can measure the expression of nucleic acid molecules comprising miRNAs is within the scope of the present disclosure. Several variations of the qRT-PCR method known in the art include, but are not limited to, analysis via agarose gel electrophoresis, use of SYBR Green (double-stranded DNA stain), and fluorescent reporter detection. Use of fluorescent reporter probes.

In one embodiment, the present invention provides a method for detecting seronegative arthritis with psoriatic arthritis and ankylosing spondylitis or predicting seronegative arthritis with psoriatic arthritis and ankylosing spondylitis in a subject A method for risk of arthritis, comprising: (a) obtaining a test sample from an individual suspected of having seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis, and measuring at least one miRNA in the test sample in vitro The expression level of said at least one miRNA is selected from the group consisting of miRNA-140-5p, miRNA-192-5p, and (b) if the expression level of said at least one miRNA in said test sample The individual is identified as having combined psoriatic arthritis and ankylosing spondylitis when the expression level of the corresponding miRNA is higher in the seronegative arthritis test sample relative to the absence of combined psoriatic arthritis and ankylosing spondylitis seronegative arthritis or at risk for seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis.

In another embodiment, the present invention provides a serum for detecting seronegative arthritis combined psoriatic arthritis and ankylosing spondylitis or predicting suffering from combined psoriatic arthritis and ankylosing spondylitis in a subject The method for negative arthritis risk, the method is the same as above, but at least one miRNA determined in vitro is selected from the group consisting of miRNA-140-5p, miRNA-192-5p, miRNA-146a-5p and the group thereof.

In yet another embodiment, the present invention provides a serum for detecting seronegative arthritis combined psoriatic arthritis and ankylosing spondylitis or predicting suffering from combined psoriatic arthritis and ankylosing spondylitis in a subject A method for negative arthritis risk, wherein the combination of psoriatic arthritis and ankylosing spondylitis is non-radiography axial spondyarthropathy (nr-axSpA).

Example 1

The study recruited 30 subjects as the confirmation group, including 20 subjects with seronegative arthritis (SPA_DSA) combined with psoriatic arthritis and ankylosing spondylitis (SPA_DSA), and 10 subjects without combined psoriatic arthritis Healthy subjects with seronegative arthritis (SPA_DSN) and ankylosing spondylitis (SPA_DSN) served as controls; a literature review was performed and various RNA targets of interest were selected to be tested in the validation set. Here, selected miRNA targets were tested for each subject to identify any specific target miRNAs that could differentiate SPA_DSA subjects from SPA_DSN subjects.

clinical assessment

Complement levels and anti-double-stranded DNA levels were collected for 30 subjects at recruitment and periodically thereafter. In addition, various clinical biomarkers, such as demographic data, biochemical data, leukocyte count, and erythrocyte sedimentation rate, were also collected for further analysis. Referring to Figure 1, Figure 1 is a seronegative arthritis subject with combined psoriatic arthritis and ankylosing spondylitis and a seronegative arthritis subject without combined psoriatic arthritis and ankylosing spondylitis Schematic diagram of the analysis of various physiological data of the subjects; in the first figure, the comparison of various clinical biomarkers between the SPA_DSA test group (20 persons) and the SPA_DSN test group (10 persons) was performed using SPSS version 22 by Mann Whitney U The test (Mann-Whitney U test) was completed, and the obtained P value was set as a threshold value below 0.05, which was considered to have a significant difference.

sample collection

Whole blood was separated into plasma and hemocytes (ie white blood cells and red blood cells) by centrifugation at 2,500 rpm (150 xg) for 20 minutes. Leukocytes were separated from red blood cells (RBC) by sedimentation with 4.5% dextran at a ratio of 1:5. Next, leukocytes were separated into polymorphonuclear cells (olymorphonuclear cells, PMNs) and mononuclear cells (MNCs) by density gradient centrifugation using Ficoll-Paque (Amersham Pharmacia Biotech) at a ratio of 2:1 at 1500 rpm for 30 minutes. Further, the collected mononuclear cells were treated with Direct-zol ^TM RNA MiniPrep kit (ZYMO RESEARCH) to extract total RNA, followed by next-generation sequencing (NGS) and/or qPCR analysis.

Confirmation of selected microRNA levels

Real-time qPCR verification of microRNA expression abundance (abundances)

cDNA was prepared using TaqMan ^™ MicroRNA Reverse Transcription Kit (Applied Biosystems). Each reaction requires 20 ng of total RNA from peripheral blood mononuclear cells (PBMC). Reverse transcription reactions were performed using a Veriti 96-well thermal cycler (Applied Biosystems) following the manufacturer's instructions. Next, qPCR reactions were performed using the reverse transcription products, and quantitative RT-PCR was performed using the 7500 Real-Time PCR System (Applied Biosystems) and TaqMan Universal PCR Master Mix II (Applied Biosystems) without UNG. The real-time PCR cycle conditions were 95° C. for 10 minutes, followed by 40 cycles of 95° C. for 15 seconds, and finally 60° C. for 1 minute. MicroRNA expression abundance was determined using the ΔCt (cycle threshold) value.

Statistical Analysis

All data in this study are presented as mean ± standard error (SD) or median (interquartile range). Categorical variables were compared using Chi-square test or Fisher's exact test when necessary. Continuous variables were compared between two groups using Student's t-test. Variables were considered statistically significant if p ≤ 0.05. All statistical analysis calculations were performed using the SAS package software, version 9.1 (2002, SAS Statistical Institute, North Carolina).

miRNA Profiling by NGS

The RNA samples collected from the MNCs of 6 SPA_DSA individuals and 6 SPA_DSN individuals (sex and age matched) were averagely combined with 3 SPA_DSAs and 3 SPA_DSNs to generate two SPA_DSA and SPA_DSN combined RNA gene pools; thereafter, Find 20 SPA_DSA individuals and 10 SPA_DSN individuals for verification (refer to Figure 1). We prepared pooled gene libraries following the TruSeq® Small RNA (Illumina) sample preparation protocol and sequenced using the Illumina MiSeq platform. Such as Kuo HC, Hsieh KS, Ming-Huey Guo M, Weng KP, Ger LP, Chan WC, Li SC. Next-generation sequencing identifies micro-RNA-based biomarker panel for Kawasaki disease. J Allergy Clin Immunol. 2016; 138: 1227-30. doi: 10.1016/j.jaci.2016.04.050, the generated raw data were analyzed using miRSeq analysis to assess overall sequencing quality and determine microRNA expression profiles. We maintained the quotient between the SPA_DSA group and the SPA_DSN control group at 0.8 to 1.2, which identified 3 target miRNAs, namely miRNA-140-5p, miRNA-192-5p, miRNA-146a-5p , these three miRNAs were significantly different between the SPA_DSA group and the SPA_DSN control group (refer to Figure 1, all p ≤ 0.05)

Example 2

receiver operating characteristic curve analysis

Receiver operating characteristic curve (ROC curve), which graphically presents the performance of a binary classifier system under a specific classification or threshold (discrimination threshold). The vertical axis (y-axis) of the graph is the true positive rate (true positive rate; TPR), also known as sensitivity (sensitivity); the horizontal axis (x-axis) is the false positive rate (false-positive rate; FPR), with 1-specificity (1-specificity) indicates that the sensitivity is the probability of correctly judging the result as positive, and the specificity is the probability of correctly judging the result as negative or negative. When specifying a cut-point (cut-point) to distinguish between positive and negative tests, this cut-point will affect the sensitivity (sensitivity) and specificity (specificity) of the diagnostic tool. In medicine, sensitivity indicates the probability that a patient with the disease is judged positive, while specificity indicates the probability that an undiagnosed person is judged negative.

It can be seen from Example 1 that miRNA-140-5p, miRNA-192-5p, miRNA-146a-5p and other three miRNAs have significant differences between the SPA_DSA group and the SPA_DSN control group, so ROC is performed on the three miRNAs Curve analysis. Referring to Figure 2, Figure 2 is a schematic diagram of the ROC curves of miRNA-140-5p, miRNA-192-5p, and miR-146a-5p. It can be seen from Figure 2 that the area under curve (AUC ) can reach 0.85, 0.69 and 0.74 respectively, and the larger the AUC value, the higher the detection accuracy.

In addition, the sensitivities of miRNA-140-5p, miRNA-192-5p, and miRNA-146a-5p were 93.8%, 87.5%, and 62.5% in sequence, while the differences were all 70%; although miRNA-140-5p, miRNA Both miRNAs of -192-5p can achieve a sensitivity of more than 85% and a specificity of 70%, that is, it can be identified that the individual suffers from seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis or has a combined Risk of seronegative arthritis of psoriatic arthritis and ankylosing spondylitis; simultaneous detection of three miRNAs to identify individuals with seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis or with combined psoriasis The positive predictive value of the risk of seronegative arthritis for acute arthritis and ankylosing spondylitis was 86%, and the negative predictive value was 50-70%.

Example 3

Analysis of nonradiographically confirmed axial spondyloarthritis

Non-radiography axial spondyarthropathy (nr-axSpA), which is an inflammatory arthritis that causes inflammation and other symptoms of the spine for which no significant damage is seen on radiographs , so it is called non-radiographically confirmed axial spondyloarthritis; but because it cannot be distinguished from X-ray images, it can be judged whether it may be axial spondyloarthritis by the following symptoms: inflammatory back pain, heel pain , Inflammatory back pain caused by arthritis, inflammatory bowel disease, psoriasis, etc.

For the detection of patients with inflammatory back pain caused by inflammatory bowel disease (inflammatory bowel disease, IBD), whether miRNA-140-5p, miRNA-192 -5p, miRNA-146a-5p and other three miRNAs also appear in patients with inflammatory back pain caused by inflammatory bowel disease; This method can be used when there is a difference in the maternal median between multiple groups of samples.

Refer to Figure 3 to Figure 5, which are the schematic diagrams of the single-factor multi-sample median difference test of miRNA-140-5p, miRNA-192-5p and miRNA-146a-5p in sequence, from Figure 3 to Figure 5 It can be seen that in patients with back pain associated with inflammatory bowel disease (IBD), the above three miRNAs can be detected as in psoriatic arthritis (PSA) and ankylosing spondylitis (AS). It can be seen that miRNA-140-5p, miRNA-192-5p and miRNA-146a-5p do have the ability to detect seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis, or to predict the combination of psoriasis Potential for the risk of seronegative arthritis in osteoarthritis and ankylosing spondylitis.

The above-mentioned embodiments are only illustrative to illustrate the principles and functions of the present invention, and are not intended to limit the present invention. Anyone skilled in the art can make modifications and changes to the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the rights of the present invention should be listed in the patent scope of the present invention.

none

The following collocation drawings further illustrate the present invention:

Figure 1 is a schematic diagram of analyzing various physiological data of subjects with arthritic spondyloarthritis disease spectrum and subjects without arthritic spondyloarthritis disease spectrum; Figure 2 is a schematic diagram of the ROC curves of miRNA-140-5p, miRNA-192-5p and miR-146a-5p; Figure 3 is a schematic diagram of the single-factor multi-sample median difference test for miRNA-140-5p; Figure 4 is a schematic diagram of the single-factor multi-sample median difference test for miRNA-192-5p; Figure 5 is a schematic diagram of the single-factor multi-sample median difference test for miR-146a-5p.

Claims (10)

A method for detecting seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis or predicting the risk of suffering from seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis, comprising the following steps: (a) Obtain a test sample from an individual suspected of having seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis, and determine the expression level of at least one miRNA in the test sample in vitro, the at least one miRNA is selected from the group consisting of miRNA-140-5p, miRNA-192-5p, and (b) if the expression level of the at least one miRNA in the test sample is higher than the expression level of the corresponding miRNA in a seronegative arthritis test sample without combined psoriatic arthritis and ankylosing spondylitis, then The individual is identified as having or at risk of developing seronegative arthritis combining psoriatic arthritis and ankylosing spondylitis. The method according to claim 1, wherein the step (a) further comprises measuring the expression level of miRNA-146a-5p in vitro. The method according to claim 1, wherein the seronegative arthritis combined with psoriatic arthritis and ankylosing spondylitis is nonradiographically confirmed axial spondyloarthritis. The method according to claim 1, wherein the expression level of the at least one miRNA is determined by real-time PCR. The method according to claim 1, wherein the test sample is selected from the group consisting of biological specimen, blood, plasma, serum, lymph, bone marrow, saliva and combinations thereof. A method for detecting back pain associated with inflammatory bowel disease or predicting the risk of suffering from back pain associated with inflammatory bowel disease, comprising the following steps: (a) Obtain a test sample from one of the individuals suspected of having back pain associated with inflammatory bowel disease, and measure the expression level of at least one miRNA in the test sample in vitro, the at least one miRNA is selected from miRNA-140-5p , miRNA-192-5p, and groups thereof; and (b) identifying that the individual has back pain associated with inflammatory bowel disease or has Risk of back pain associated with inflammatory bowel disease. The method according to claim 6, wherein the step (a) further comprises measuring the expression level of miRNA-146a-5p in vitro. The method according to claim 6, wherein the associated back pain caused by the inflammatory bowel disease is non-radiographically confirmed axial spondyloarthritis. The method according to claim 6, wherein the expression level of the at least one miRNA is determined by real-time PCR. The method according to claim 6, wherein the test sample is selected from the group consisting of biological specimen, blood, plasma, serum, lymph, bone marrow, saliva and combinations thereof.

Images