阅读说明：本技术 一种风湿性关节炎的诊断标志物 (Diagnosis marker for rheumatic arthritis ) 是由 卢华定 徐祥赫 陈宇峰 于 2020-12-22 设计创作，主要内容包括：本发明公开了一种风湿性关节炎的诊断标志物,所述标志物含有环状RNA hsa-circ-0140271,所述环状RNA hsa-circ-0140271靶向X染色体上的MED14基因。本发明解决了现有技术中,RA的诊断仅能够依靠临床症状、实验室指标和影像学检查等综合判断,无单一的指标可用于明确的诊断的技术缺陷,而且相对于现有的anti-CCP,特异性更高,尤其是对女性RA有很好的特异性,误诊率更低。本发明中的标志物还对RA相关的炎症因子(IL-6、IL-8、TNF-α)的表达具有一定的影响。(The invention discloses a diagnostic marker for rheumatoid arthritis, which comprises circular RNA hsa _ circ _0140271, wherein the circular RNA hsa _ circ _0140271 targets MED14 gene on X chromosome. The invention overcomes the technical defect that RA diagnosis in the prior art can only depend on clinical symptoms, laboratory indexes, imaging examination and other comprehensive judgment, no single index can be used for definite diagnosis, and compared with the existing anti-CCP, the specificity is higher, especially good specificity is provided for female RA, and the misdiagnosis rate is lower. The markers of the invention also have some effect on the expression of the inflammatory factors associated with RA (IL-6, IL-8, TNF-. alpha.).)

1. A marker which is a circular RNA targeting MED14 gene on the X chromosome.

2. The marker of claim 1, wherein the circular RNA is hsa _ circ _ 0140271.

3. A specific primer set, wherein the specific primer set specifically amplifies a circular RNA targeting MED14 gene on the X chromosome.

4. The specific primer set according to claim 3, wherein the circular RNA is hsa _ circ _ 0140271.

5. The specific primer set as claimed in claim 4, wherein the nucleotide sequence of the specific primer set is:

an upstream primer: 5'-ATCGGCTTGTAACCACAGAT-3' (SEQ ID NO. 2);

a downstream primer: 5'-CTCATAAAGTTGTTCTCTCCTG-3' (SEQ ID NO. 3).

6. A detection reagent comprising the primer set according to any one of claims 3 to 5.

7. A test kit comprising the test reagent according to claim 6.

8. The detection kit according to claim 7, wherein the detection kit further comprises RT-PCR reaction reagents and enzymes.

9. Use of a reagent or material for detecting a marker according to claim 1 or 2 in the preparation of a rheumatoid arthritis detection formulation.

Technical Field

The invention belongs to the field of disease diagnosis markers, and particularly relates to a diagnosis marker for rheumatic arthritis.

Background

Rheumatoid Arthritis (RA) is a common chronic autoimmune disease with erosive arthritis as the major clinical manifestation. The global incidence of RA is 0.5-1%. Early diagnosis of RA is of great significance for treatment and prognosis. Statistically, the median time from onset of typical symptoms (morning stiffness) to diagnosis is 6 months for RA patients, while more than one year of illness is diagnosed in 25% of patients. At present, the early diagnosis of RA is comprehensively judged by combining laboratory detection and imaging detection results according to clinical manifestations of patients, so that no clear and effective single index is available for early diagnosis of RA.

In addition, RA has obvious sex difference in the sick population, female patients account for most of the patients, the male and female disease ratio is 1:4, and researches show that certain genes on the X chromosome have high expression phenomena in female RA patients, but no specific diagnostic index aiming at female RA exists at present.

The circular RNA (circRNA) has longer half-life period and higher stability compared with linear RNA because of insensitivity to nuclease, and the circular RNA has high expression abundance in cells, stronger tissue specificity and extremely high research value.

Therefore, in order to effectively ensure the early detection rate of RA, the development of a stable diagnostic marker for female RA patients has extremely high medical value and research value.

Disclosure of Invention

The object of the present invention is to provide a marker;

it is another object of the present invention to provide a specific primer set;

it is another object of the present invention to provide a detection reagent;

it is another object of the present invention to provide a detection kit;

the invention also aims to provide application of the reagent or the material for detecting the marker in preparing a rheumatoid arthritis detection preparation.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

a marker which is a circular RNA targeting the MED14 gene on the X chromosome.

The existence of more than 400 circular RNAs (circRNAs) can be detected in human saliva, and thousands of circRNAs can be detected in blood, which indicates that the circRNAs can exist stably in clinical samples such as blood, saliva and the like, so the circRNAs can become an ideal molecular marker type.

Further, the circular RNA is hsa _ circ _ 0140271.

The inventor discovers that hsa _ circ _0140271 generated by MED14 gene of X chromosome is specifically and highly expressed in RA female patients (the specificity is better than that of the existing RA diagnosis index-anti-cyclic citrullinated polypeptide antibody (anti-CCP)) by carrying out high-throughput sequencing on RNA in Peripheral Blood Mononuclear Cells (PBMC) of RA patients, and discovers that the specificity of the marker in the invention is 1 in comparison with a normal female group, a osteoarthritis female group and a ankylosing spondylitis female group through ROC curve analysis, thereby indicating that the marker in the invention can be used as a potential specificity index for diagnosing RA of females.

Further, the nucleotide sequence of the above circular RNAhsa _ circ _0140271 is:

5’-AGAACAACTTTATGAGATTGGGAATCATGATCACCTCCATTTTACAGTTGAAAAAAGCTGAGGCACAAAGAGGTTAAGTTACTTGCCCAAGGTTACAAGACTGGTAAGTAGCAGAGTTAAAATGGAGAACAAGGCAGTCTAGGTCTAGGAGTCCATGCTTTTAACTCCTTCATTACCAACCAACTCTGGAAGTCTGTAAGAGGAAAAAACCATTTTATTCTACTTATTTTTCCTAACTTTGATATTATGTGTGTGTAATGTTAAGGATGGAGAAATGAATTAGAAAGCTTTTACTTAAAGTGACAGAAAATTATTGCTCTTAATATCAAATACTAGTGTATTAAATATTTCTTCTTCAAGTTACTGGAGAATATTTGAAGAACTACTTTGAAATATGGAAAACTGATACTAATTCCCCTTGAAGTTTACTTTGGAATGATTTAACAATATTTAGTGTGAAAAGGAGATAGTTTTCAGGGATGCTTAATACATTTACCCTTTACTTTAAAGATGATTTCAAGCTTTTTAGATCAGCAAGCCATCCTGTTTGTGGACACTGCTGATCGCCTGGCCTCGTTAGCTAGAGATGCTCTGGTCCATGCACGCCTGCCTAGTTTTGCCATCCCATATGCCATTGATGTACTAACTACTGGATCTTACCCACGGCTGCCAACCTGCATTAGGGACAAAATTATTCCTCCAGACCCAATTACCAAAATTGAAAAACAAGCCACACTCCATCAGCTGAATCAGATTCTTAGACATCGGCTTGTAACCACAGATCTTCCTCCTCAGTTAGCAAATCTTACAGTTGCAAATGGCCGGGTGAAGTTTCGTGTTGAAGGAGAATTTGAAGCCACCTTGACTGTGATGGGAGATGACCCTGATGTTCCATGGCGTCTTCTCAAGCTAGAAATTCTAGTTGAGGATAAGGAAACAGGAG-3’(SEQ ID NO.1)。

the inventor predicts 74 and 50 mi RNAs aimed at by hsa _ circ _0140271 through circBANK and circinteactor databases respectively, wherein the circBANK and circinteactor databases predict that the number of the same miRNA is 8, which indicates that the 8 miRNAs are the most likely target sites of hsa _ circ _ 0140271.

In a second aspect of the present invention, there is provided:

a specific primer set that specifically amplifies a circular RNA that targets the MED14 gene on the X chromosome.

Further, the circular RNA is hsa _ circ _ 0140271.

The specific primer group is designed based on the nucleotide sequence of the marker and is used for the targeted amplification of the marker.

Further, the nucleotide sequence of the specific primer group is as follows:

an upstream primer: 5'-ATCGGCTTGTAACCACAGAT-3' (SEQ ID NO. 2);

a downstream primer: 5'-CTCATAAAGTTGTTCTCTCCTG-3' (SEQ ID NO. 3).

The detection method of the marker comprises the following steps:

(1) extracting RNA from a sample to be detected, and carrying out reverse transcription to obtain cDNA;

(2) amplifying the cDNA obtained in the step (1) by using a primer group, and calculating a Ct value;

(3) setting an internal reference gene, detecting the expression quantity of the internal reference gene, and calculating a Ct value;

(4) use 2^–ΔCTAnd (3) analyzing the Ct value obtained in the steps (2) and (3) by a method, and judging whether the sample to be detected contains the marker or not.

Further, the primer set in the step (2) includes the primer set.

Further, the standard for determining the sample to be tested in the step (4) is as follows:

if 2^–ΔCTAnalysis of the resulting product 2^–ΔCTIf the value is more than 0.110, the marker is contained in the sample to be tested, and the sample to be tested is a rheumatoid arthritis sample.

Further, the reference gene in step (3) includes GAPDH.

Further, the Omega Bio-tek is used for extracting RNA from a sample to be testedTotal RNA kit. Of course, other conventional methods in the art can be used for extraction according to actual use requirements.

Furthermore, when detecting the expression level of the internal reference gene GAPDH and amplifying the RNA obtained in the step (1), a Sybr Green method is adopted;

wherein, the specific primer group of the reference gene GAPDH is as follows:

GAPDH-F：5'-AGCCACATCGCTCAGACAC-3'(SEQ ID NO.4)；

GAPDH-R：5'-GCCCAATACGACCAAATCC-3'(SEQ ID NO.5)；

the reaction system of the Sybr Green method is as follows:

cDNA (1:20 dilution)	5.0μL
		10 μ M corresponding upstream primer	0.5μL
10 μ M of the corresponding downstream primer	0.5μL
		2x SYBR Green PCR Master Mix	10μL
dH2O	4.0μL
		Total volume	20μL

The reaction conditions are as follows:

95 ℃ for 5 min; 95 ℃ for 15 s; 60 ℃ for 15 s; 72 ℃ for 32 s; the cycle is repeated 40 times.

Fluorescence signals were collected at 72 ℃ and melting curves were plotted (temperature range 60 ℃ -95 ℃).

Further, 2 above^–ΔCTThe calculation method comprises the following specific steps:

calculating the-Delta CT value, namely subtracting the Ct value of GAPDH of the corresponding sample from the Ct value of hsa _ circ _0140271, and taking the negative value as the-Delta CT value. Then, taking the-Delta CT value as a power value, calculating 2^–ΔCTThe value is obtained.

Further, the sample to be tested is derived from a female animal.

In a third aspect of the present invention, there is provided:

a detection reagent comprising the primer set.

In a fourth aspect of the present invention, there is provided:

a detection kit contains the detection reagent.

Furthermore, the detection kit also contains an RT-PCR reaction reagent and an enzyme.

In a fifth aspect of the present invention, there is provided:

the application of the reagent or the material for detecting the marker in the preparation of a rheumatoid arthritis detection preparation.

The invention has the beneficial effects that:

1. the invention overcomes the technical defect that in the prior art, RA diagnosis can only depend on comprehensive judgment such as clinical symptoms, laboratory indexes, imaging examination and the like, and no single index can be used for definite diagnosis, and provides an effective single RA early diagnosis marker.

2. Compared with the existing anti-CCP, the marker of the invention has higher specificity, especially has good specificity to female RA, and the sensitivity of the marker of the invention is lower than that of the anti-CCP, namely the misdiagnosis probability of female RA diagnosis according to the marker of the invention is lower than that of the anti-CCP, which indicates that the marker of the invention is a better marker for early RA diagnosis.

3. The markers of the present invention all have an effect on the expression of RA-associated inflammatory factors (IL-6, IL-8, TNF-. alpha.), and IL-6, IL-8, TNF-. alpha.are differentially expressed in the positive and negative limits of the markers of the present invention (the presence of RA in a range greater than the positive limit and the absence of RA in a range less than the negative limit can be diagnosed).

Drawings

FIG. 1 is a graph of the difference in expression of hsa _ circ _0140271 in different populations, where A is the difference in expression of hsa _ circ _0140271 in the normal (HC) and RA groups; b is the difference of hsa _ circ _0140271 expression in normal male group (HC-M), normal female group (HC-F), RA male group (RA-M) and RA female group (RA-F);

FIG. 2 is a diagram showing the amounts of miRNA target sequences predicted by hsa _ circ _0140271 through circBANK and circintenectome databases, respectively.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Design of markers

Hsa _ circ _0140271 produced by the MED14 gene of the X chromosome was found to be specifically highly expressed in RA female patients by high throughput sequencing of RNA in PBMCs of RA patients, and hsa _ circ _0140271 was sequenced and found to have the nucleotide sequence:

5’-AGAACAACTTTATGAGATTGGGAATCATGATCACCTCCATTTTACAGTTGAAAAAAGCTGAGGCACAAAGAGGTTAAGTTACTTGCCCAAGGTTACAAGACTGGTAAGTAGCAGAGTTAAAATGGAGAACAAGGCAGTCTAGGTCTAGGAGTCCATGCTTTTAACTCCTTCATTACCAACCAACTCTGGAAGTCTGTAAGAGGAAAAAACCATTTTATTCTACTTATTTTTCCTAACTTTGATATTATGTGTGTGTAATGTTAAGGATGGAGAAATGAATTAGAAAGCTTTTACTTAAAGTGACAGAAAATTATTGCTCTTAATATCAAATACTAGTGTATTAAATATTTCTTCTTCAAGTTACTGGAGAATATTTGAAGAACTACTTTGAAATATGGAAAACTGATACTAATTCCCCTTGAAGTTTACTTTGGAATGATTTAACAATATTTAGTGTGAAAAGGAGATAGTTTTCAGGGATGCTTAATACATTTACCCTTTACTTTAAAGATGATTTCAAGCTTTTTAGATCAGCAAGCCATCCTGTTTGTGGACACTGCTGATCGCCTGGCCTCGTTAGCTAGAGATGCTCTGGTCCATGCACGCCTGCCTAGTTTTGCCATCCCATATGCCATTGATGTACTAACTACTGGATCTTACCCACGGCTGCCAACCTGCATTAGGGACAAAATTATTCCTCCAGACCCAATTACCAAAATTGAAAAACAAGCCACACTCCATCAGCTGAATCAGATTCTTAGACATCGGCTTGTAACCACAGATCTTCCTCCTCAGTTAGCAAATCTTACAGTTGCAAATGGCCGGGTGAAGTTTCGTGTTGAAGGAGAATTTGAAGCCACCTTGACTGTGATGGGAGATGACCCTGATGTTCCATGGCGTCTTCTCAAGCTAGAAATTCTAGTTGAGGATAAGGAAACAGGAG-3’(SEQ ID NO.1)。

detection of markers

The specific steps for detecting the expression of hsa _ circ _0140271 are as follows:

taking the peripheral venous blood of the patient as a detection sample.

1. And extracting PBMC and RNA in the PBMC.

10mL of peripheral venous blood was drawn from the patient and placed into a centrifuge tube previously containing an equal volume of mononuclear lymphocyte isolate (Sigma Hitopaque 1077).

After centrifugation at 400g for 30 minutes at room temperature, the middle PBMC were extracted and placed in a new centrifuge tube. 10mL of 1 XPBS was added and centrifuged at 250g for 10 minutes at room temperature. After the upper PBS layer was decanted, 5mL of 1 XPBS was added and centrifuged at 250g for 5 minutes at room temperature for 2 repetitions. After the upper PBS layer was poured out, the lysate was added to RNA extraction according to kit (b)Total RNA kit, Omega Bio-tek) instructions, the RNA is put into the environment of-80 ℃ for storage or RT-qPCR (the extracted RNA needs to be subjected to reverse transcription or the operation according to the instructions of the RT-qPCR kit) after being extracted.

2. And performing RT-qPCR amplification on the extracted RNA.

The nucleotide sequence of the specific primer group of the circRNAhsa _ circ _0140271 is as follows:

an upstream primer: 5'-ATCGGCTTGTAACCACAGAT-3' (SEQ ID NO. 2);

a downstream primer: 5'-CTCATAAAGTTGTTCTCTCCTG-3' (SEQ ID NO. 3).

The internal reference gene is GAPDH, and the specific primer set of the internal reference gene GAPDH is as follows:

GAPDH-F：5'-AGCCACATCGCTCAGACAC-3'(SEQ ID NO.4)；

GAPDH-R：5'-GCCCAATACGACCAAATCC-3'(SEQ ID NO.5)。

the detection is carried out by adopting a Sybr Green method, and a Real-time PCR instrument is7500 Sequence Detection System。

The reaction system of the Sybr Green method is as follows:

TABLE 1 reaction System of Sybr Green Process

cDNA (1:20 dilution)	5.0μL
		10 μ M corresponding upstream primer	0.5μL
10 μ M of the corresponding downstream primer	0.5μL
		2x SYBR Green PCR Master Mix	10μL
dH2O	4.0μL
		Total volume	20μL

The reaction conditions are as follows:

95 ℃ for 5 min; 95 ℃ for 15 s; 60 ℃ for 15 s; 72 ℃ for 32 s; the cycle is repeated 40 times.

Fluorescence signals were collected at 72 ℃ and melting curves were plotted (temperature range 60 ℃ -95 ℃).

Further, 2 above^–ΔCTThe calculation method comprises the following specific steps:

calculating the-Delta CT value, namely subtracting the Ct value of GAPDH of the corresponding sample from the Ct value of hsa _ circ _0140271, and taking the negative value as the-Delta CT value. Then, taking the-Delta CT value as a power value, calculating 2^–ΔCTThe value is obtained.

3. And (5) judging a sample.

According to 2^–ΔCTThe value is determined, if 2^–ΔCTIf the value is greater than 0.110, the sample is deemed to contain circRNAhsa _ circ _0140271 and the sample is deemed to be RA sample.

Verification of markers

PBMC samples were collected in 41 each of normal (HC) and RA groups, with 10 men and 31 women in each group. The detection method of the marker is adopted for detection so as to verify the accuracy of the marker.

As shown in FIG. 1, by comparing the actual expression levels of hsa _ circ _0140271 in the normal male group (HC-M), the normal female group (HC-F), the RA male group (RA-M) and the RA female group (RA-F), hsa _ circ _0140271 was found to be specifically highly expressed in female RA, indicating that hsa _ circ _0140271 can be used as an effective detection marker for female RA patients.

Comparison of the Effect of the markers of the present invention with that of the existing RA markers

PBMC samples of female RA patients and normal females are collected and tested by the above marker test method, and anti-CCP is used as a control, and the anti-CCP test method is an anti-CCP test method which is conventional in the art.

The expression values of hsa _ circ _0140271 and anti-CCP of female RA patients and normal females were input to SPSS software for ROC curve analysis. .

The results are shown in Table 2.

TABLE 2 Difference between the above markers and the anti-CCP assay

As a result, it was found that the anti-CCP specificity was lower than that of hsa _ circ _0140271, and that the anti-CCP specificity was still lower, only 0.4-0.89, during practical use. While specificity of hsa _ circ _0140271 was 1, indicating good specificity for diagnosing RA, especially female RA. Furthermore, the sensitivity of hsa _ circ _0140271 is lower than that of anti-ccp, i.e., the probability of misdiagnosis is lower when a female RA can be diagnosed according to the value of hsa _ circ _0140271 than that of anti-ccp.

When the influence of the cut-off value of hsa _ circ _0140271 on the inflammatory factors associated with RA (IL-1. alpha., IL-1. beta., IL-6, IL-8, TNF-. alpha., IFN-. gamma., etc.) was further analyzed, it was also found that IL-6, IL-8, and TNF-. alpha.were differentially expressed in the positive and negative cut-offs of the markers of the present invention (greater than the cut-off value allows diagnosis of RA in the positive and negative cut-offs, and less than the negative cut-off value).

Prediction of target sequences (miRNAs) for markers of the invention

miRNA related to hsa _ circ _0140271 is predicted by using a circBank database (http:// www.circbank.cn /) and a circulant RNA interactome database (https:// circulant. nia. nih. gov /), and miRNA common to the two databases is selected by further taking intersection of the predicted miRNA of the two databases.

The results are shown in FIG. 2 and Table 3.

TABLE 3 prediction of target sequences (miRNA) for markers of the invention

From the results, it is predicted from circBANK and circinteactor databases that hsa _ circ _0140271 targets 74 and 50 mirnas, respectively. And performing intersection analysis on the predicted miRNAs to obtain 8 miRNAs which are most closely related to hsa _ circ _ 0140271.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> secondary fifth Hospital of Zhongshan university

SUN YAT-SEN University

<120> a diagnostic marker for rheumatoid arthritis

<130>

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<170> PatentIn version 3.5

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