CN109486942B

CN109486942B - Biomarker for rheumatoid arthritis diagnosis and application thereof

Info

Publication number: CN109486942B
Application number: CN201811599941.6A
Authority: CN
Inventors: 邓飞艳; 雷署丰; 何培
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2018-12-26
Filing date: 2018-12-26
Publication date: 2021-04-06
Anticipated expiration: 2038-12-26
Also published as: CN109486942A

Abstract

The invention discloses a biomarker for diagnosing rheumatoid arthritis and application thereof, belonging to the technical field of biological detection. The mRNA and protein of the gene RPN2 are used as biomarkers for diagnosing rheumatoid arthritis. The corresponding primer group, the probe and the protein provided by the invention can be used for preparing a kit, and have excellent sensitivity and specificity when being applied to diagnosis of rheumatoid arthritis by using single nuclear cells of peripheral blood and plasma samples. The AUC value of the RPN2mRNA can reach 0.911, and the diagnostic value is excellent; with 3.3 as the cut-off point, the sensitivity and specificity were 82.1% and 83.3%, respectively. The AUC value of the RPN2 protein is 0.672, the cut-off point is 14.29ng/ml, and the sensitivity and the specificity are 65.0 percent and 67.5 percent respectively. Therefore, the RPN2mRNA and the protein shown by the invention can be used as biomarkers of rheumatoid arthritis, and have application value for diagnosis and treatment of rheumatoid arthritis.

Description

Biomarker for rheumatoid arthritis diagnosis and application thereof

Technical Field

The invention relates to a biomarker for diagnosing rheumatoid arthritis and application thereof, belonging to the technical field of biological detection.

Background

Rheumatoid Arthritis (RA) is a chronic autoimmune disease with erosive joints as the major manifestation. The disease usually causes the incompetence of muscles and bones, the hypofunction of the body and the induction of symbiotic diseases by eroding cartilages and destroying skeletal joints, and has higher disability rate in the later period. If left untreated or unresponsive to treatment, inflammation and joint destruction can lead to loss of physical function, severely affecting the quality of life of the patient, with a concomitant heavy socio-economic burden. Therefore, screening of novel biomarkers of RA, revealing of molecular pathogenic mechanism, and establishment of early disease diagnosis models and prevention means are extremely important strategic measures for controlling RA immune inflammation, and preventing and relieving later skeletal injury or deformity.

RA is a complex disease which is jointly regulated by environmental factors and genetic factors, and the pathogenesis and mechanism of RA are not clear. Genetic factors are important influencing factors of the onset of RA, and the proportion of the genetic factors in the onset of RA is about 50-60%. Previous genome-wide association studies have found more than 100 susceptibility genes associated with RA, and many of them are immunologically relevant. However, the genetic susceptibility sites found combine to explain the small prevalence of RA (15%), and a large number of genetic factors remain to be found. Clinically, the traditional serum diagnostic indicators include autoantibodies anti-citrullinated protein antibody (ACPA) and Rheumatoid Factor (RF). The sensitivity of ACPA is about 67%, lower in early RA patients. The specificity of RF is 38% -85%. The two indexes have better diagnosis effect in combination than the single index. However, there are studies that show that diagnostic models of the combination of ACPA and RF can only correctly identify 54% -57% of RA patients. Therefore, biomarkers with superior performance have yet to be developed.

Peripheral blood mononuclear cells contain a series of immune-related cells, such as T cells, B cells, monocytes and the like, and therefore most of the current functional omics studies on RA use peripheral blood mononuclear cells as study target cells. The plasma contains abundant proteins, which reach all tissues of the body through blood circulation and participate in the life process. The gene which is abnormally expressed in RA is screened from peripheral blood mononuclear cells and plasma to be used as a biomarker, and a corresponding auxiliary diagnosis kit is developed, so that the early diagnosis, the prediction and the treatment, the recurrence monitoring and the like of RA in China are powerfully promoted, and the clinical application value is important.

Disclosure of Invention

In order to solve the technical problems, a peripheral blood mononuclear cell gene and a plasma protein biomarker for diagnosing rheumatoid arthritis are provided. Meanwhile, the rheumatoid arthritis detection kit can be used for early detection and has high sensitivity and high specificity in detection and application thereof.

The first object of the present invention is to provide a biomarker for the diagnosis of rheumatoid arthritis, wherein the biomarker is RPN2mRNA encoded by the gene RPN2 (fully known as ribophorin II) or RPN2 protein (protein name: dolichol-diphosphohologosaccharide-protein glycosyltransferase subnitrile 2; alias: dolichol-diphosphohologosharide-protein glycosyltransferase 63kDa subnitrile; oligosaccharyltransferase complex subnitrile (non-catalytic); ribophorin-2).

In one embodiment of the invention, the nucleotide sequence of the RPN2mRNA is shown in SEQ ID NO. 1.

In one embodiment of the invention, the amino acid sequence of the RPN2 protein is shown in SEQ ID No. 2.

In one embodiment of the invention, the RPN2mRNA and RPN2 proteins are derived from peripheral blood mononuclear cells and plasma, respectively.

The second purpose of the invention is to provide a combination of an RPN2mRNA primer group and a probe for diagnosing rheumatoid arthritis.

It is a third object of the present invention to provide an antibody for rheumatoid arthritis diagnosis, which can specifically recognize and bind to RPN2 protein.

The fourth purpose of the invention is to provide a kit for diagnosing rheumatoid arthritis, which comprises a combination of an mRNA primer group and an mRNA probe for detecting the expression level of RPN2mRNA in peripheral blood mononuclear cells; or antibodies that measure plasma RPN2 protein content.

In one embodiment of the invention, the mRNA primer set comprises a forward primer and a reverse primer for quantitative PCR reverse transcription of RPN2 mRNA.

In one embodiment of the invention, the nucleotide sequences of the reverse transcription forward primer and reverse primer of the quantitative PCR of the RPN2mRNA are shown in SEQ ID NO.3 and SEQ ID NO.4, or SEQ ID NO.5 and SEQ ID NO.6, respectively.

In one embodiment of the invention, the kit further comprises a component for qPCR amplification detection, wherein the component for qPCR amplification detection comprises reverse transcriptase, buffer solution, dNTPs, MgCl and the like₂、dd H₂O, fluorescent dye, Taq enzyme, standard and control.

In one embodiment of the invention, the antibody specifically recognizes RPN2 protein.

In one embodiment of the invention, the kit further comprises components for ELISA detection, including a detection plate, buffer, and RPN2 protein standard.

The fifth object of the present invention is to provide a chip for rheumatoid arthritis diagnosis comprising an mRNA probe for detecting RPN2 mRNA.

In one embodiment of the present invention, the nucleotide sequence of the mRNA probe is completely complementary to the full-length mature mRNA of the mRNA probe.

The sixth purpose of the invention is to provide the application of the mRNA biomarker in preparing products for detecting rheumatoid arthritis.

The application of the mRNA biomarker in the diagnosis of rheumatoid arthritis comprises the following steps: measuring the expression level of a biomarker derived from peripheral blood mononuclear cells of the subject, said biomarker being RPN2mRNA, to obtain a measured value; comparing said determined value to a reference value, and determining that the subject has rheumatoid arthritis if the determined value of RPN2mRNA is higher than the reference value.

In an embodiment of the present invention, the application specifically includes: extracting biomarkers in peripheral blood mononuclear cells of the subject; providing a primer set and a probe corresponding to the biomarker; and measuring the measurement value by a PCR detection method. Wherein, the sequence of the forward/reverse primer detected by PCR is shown as SEQ ID NO.3 (forward direction)/SEQ ID NO.4 (reverse direction), or SEQ ID NO.5 (forward direction)/SEQ ID NO.6 (reverse direction).

In an embodiment of the present invention, the application specifically includes: extracting mRNA biomarkers in peripheral blood mononuclear cells of the subject; providing a detection chip, wherein an mRNA probe of a sequence completely complementary to the full-length mature mRNA of the biomarker is loaded on the detection chip; and measuring the detection value by using the detection chip.

The invention has the beneficial effects that:

the RPN2 gene biomarker, the corresponding primer group and the probe thereof provided by the invention can be used for preparing a diagnostic kit, and have excellent sensitivity and specificity when being applied to the rheumatoid arthritis diagnosis of peripheral blood mononuclear cells and plasma samples. The AUC value of the RPN2mRNA can reach 0.911, the sensitivity and the specificity are 82.1 percent and 83.3 percent respectively by taking 3.3 as a cut-off point; the AUC value of the RPN2 protein can reach 0.672, the 14.29ng/ml is taken as a cut-off point, and the sensitivity and the specificity are 65.0 percent and 67.5 percent respectively. The gene can be used as a biomarker for diagnosing human rheumatoid arthritis peripheral blood mononuclear cells and blood plasma samples, and is favorable for promoting the development of early diagnosis, prediction treatment and relapse monitoring of rheumatoid arthritis in China.

Drawings

In fig. 1, a is the expression level of the RPN2mRNA of the present invention in rheumatoid arthritis patients and normal controls (N-43, FC-2.09, P < 0.05); b is an ROC curve chart of the RPN2mRNA used for distinguishing patients with rheumatoid arthritis from normal control;

fig. 2 is a graph showing the change in the expression level of RPN2mRNA of the present invention in 35 rheumatoid arthritis and 35 healthy human peripheral blood mononuclear cell samples (N70, FC 1.14, P < 0.05);

fig. 3 is a graph showing the expression levels of RPN2mRNA of the present invention in 6 rheumatoid arthritis and 3 samples of healthy human T cells (N-9, FC-1.79, P < 0.05);

fig. 4 is a graph showing the expression level of RPN2 protein in peripheral blood mononuclear cells of the present invention in 18 cases of rheumatoid arthritis and 10 cases of healthy persons (N-28, FC-1.29, P < 0.05);

FIG. 5 is a graph showing the mass spectrometric identification of the RPN2 protein in peripheral blood mononuclear cells of the present invention; wherein A is a patient suffering from rheumatoid arthritis; b is a healthy control individual;

in fig. 6, a is the expression level of RPN2 protein in 40 patients with rheumatoid arthritis and 40 normal control plasma (N80, FC 2.12, P < 0.05); b is ROC curve of the RPN2 protein for distinguishing rheumatoid arthritis patients from normal control;

in FIG. 7, A is the expression level of RPN2mRNA in Jurkat cells after PHA stimulation and in unstimulated control cells (P < 0.05); b is the effect of RPN2 overexpression on the cell cycle of Jurkat cells (P < 0.05); c is the effect of RPN2 overexpression on apoptosis of Jurkat cells (P < 0.05); d is the effect of RPN2 overexpression on cell activation of Jurkat cells (P < 0.05).

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Experiments in which specific conditions are not specified in the examples are generally performed under conventional conditions such as those described in the manufacturer's instructions, experimental guidelines, or the contents of textbooks.

Before describing the examples, it is necessary to note that: the reagent of different manufacturers and different batches can cause the difference of experimental results, and belongs to the normal phenomenon.

In small-scale experiments, in order to ensure the repeatability among parallel experiments, the reagent is recommended to be prepared, fully mixed and subpackaged so as to ensure the uniformity of the reagent in each experiment.

Example 1: chip screening of differentially expressed mRNA in peripheral blood mononuclear cells

1. After informed consent was obtained, 25 peripheral blood samples from patients diagnosed with rheumatoid arthritis and 18 peripheral blood samples from healthy persons as normal controls were collected in sodium citrate anticoagulation tubes.

2. Obtaining peripheral blood mononuclear cells of a rheumatoid arthritis patient and a normal person by a density gradient centrifugation method, which comprises the following specific steps:

(1) 15ml of density gradient separation medium was added to a Leucosep separator tube and centrifuged at 100g for 1 minute.

(2) 15ml of peripheral blood samples were centrifuged at 2000rpm and the supernatant after 2 min of separation was collected in 15ml centrifuge tubes and centrifuged at 2500g for 15 min. Then, the mixture was dispensed into 2ml centrifuge tubes and stored in a freezer at-80 ℃.

(3) The extracted supernatant was supplemented with an equal amount of PBS and the mixture was pooled into a 50ml centrifuge tube. An equal amount of mixed PBS was then added.

(4) The mixture was transferred to the leucosep tube in (1) and centrifuged at 500g for 20 minutes.

(5) The supernatant in the upper separation tube was discarded by suction. The middle layer leukocytes were extracted into a new 50ml centrifuge tube, PBS was added to a final volume of 15ml, and centrifuged at 400g for 10 min.

(6) The supernatant in the upper centrifuge tube was discarded. Adding 4ml PBS, mixing, sucking 10ul, counting cells, packaging the rest cell liquid into 2ml freezing tube, centrifuging at 2000rpm for 2 minutes.

(7) After draining the tube, 3 of the tubes were removed and 1ml of TRIzol was added to each tube to prevent RNA degradation. Blowing, beating, mixing, and storing in a refrigerator at-80 deg.C.

3. In the discovery experiment, Total RNA was extracted from peripheral blood mononuclear cells of patients with rheumatoid arthritis and healthy controls to which TRIzol was added, and then mRNA Expression was detected by a whole genome Expression profiling chip (lncRNA + mRNA Human Expression Microarray V4.0).

The main experimental steps are as follows:

3.1 after extracting RNA from RA and normal human peripheral blood mononuclear cells, further adopting

The RNA clean-up kit is used for purifying Total RNA, then the Total RNA is quantified by a spectrophotometer method, and the integrity of the Total RNA is detected by an agarose gel electrophoresis method.

3.2 Total RNA Synthesis of cDNA

(1) The First Strand cDNA was synthesized by reverse transcription. The following reagents were added sequentially to a 0.2ml nuclease-free centrifuge tube:

a.5ul Total RNA(100-500ng)；

b. adding into

Gene expression profiling exogenous gene (Cat. No.360030)1ul, while adding the corresponding volume of Agilent spike-in according to Table 1.

TABLE 1

c. Preparing a reverse transcription Master Mix on ice, gently mixing, performing short-time centrifugation, and placing on an ice bath, wherein the Master Mix reaction system is as follows: 4ul of IncRNA + mRNA First Strand Buffer Mix, 1ul of First Strand Enzyme Mix.

d. 5ul of the c is taken out and added into a centrifuge tube containing a Total RNA sample, and the final reaction volume of the reverse transcription is 10 ul.

e. Mixing with soft air, blowing and sucking for 2-3 times, centrifuging, and placing on ice.

f. Placing the reverse transcription centrifugal tube on a PCR instrument, reacting for 1h at 25 ℃, reacting for 1h at 42 ℃, and keeping for more than 5min at 4 ℃. The tubes were removed, centrifuged briefly, and placed on ice in preparation for Second Strand cDNA synthesis.

(2) Second Strand cDNA was synthesized.

a. Second Strand Master Mix was made up on ice, gently mixed, centrifuged briefly and then iced. The Second Strand Master Mix reaction system is: 32.5ul of nucleic-free Water, 12.5ul of Second Strand Buffer Mix, 5ul of Second Strand Enzyme Mix.

b. Add 50. mu.L of Second Strand Master Mix to the reaction tube in step f of the First Strand cDNA synthesized in the previous step, and Mix in a volume of 60. mu.L; blowing, sucking, mixing for 2-3 times, centrifuging instantaneously, and placing on ice.

c. And (3) placing the second chain synthesis centrifugal tube on a PCR instrument, reacting for 1h at 16 ℃ (closing the heating function of a cover of the PCR instrument), reacting for 10 minutes at 65 ℃, and keeping for more than 5 minutes at 4 ℃.

d. After the reaction is finished, the reaction tube is placed on ice to continue the synthesis reaction, or is quickly frozen and stored at-20 ℃.

3.3 in vitro transcription Synthesis of cRNA

(1) And (3) synthesizing cRNA.

a. Prepare in vitro transcription Master Mix, Mix gently, centrifuge briefly and collect the solution at the bottom of the tube. The Master Mix reaction system is as follows: 24ul of T7Buffer Mix; 6ul of T7Enzyme Mix.

b. And (3) taking 30 mu L of IVT Master Mix to a reaction tube in the cDNA synthesis step, uniformly blowing and sucking, and instantly centrifuging and placing on ice.

c. The in vitro transcription synthesis centrifugal tube is placed on a PCR instrument, reacted for 16h at 40 ℃ and kept at 4 ℃.

d. After the reaction is finished, the mixture is instantaneously centrifuged and used

The product was purified using an RNA clean-up kit (MN, 740.948.250), and the purified cRNA product was quantified using an ultraviolet spectrophotometer.

3.4 reverse transcription of cRNA to cDNA

a. And (3) taking 10 mu g of the cRNA purified product, adjusting the volume to 22 mu L, adding the cRNA purified product into a 0.2mL nuclease-free centrifuge tube, adding 2 mu L Random Primer, uniformly mixing, placing on a PCR instrument, performing instantaneous centrifugation to collect liquid to the bottom of the tube at 70 ℃ for 5 minutes, at 25 ℃ for 5 minutes and at 4 ℃ for 2 minutes, and placing on ice.

b. The cRNA reverse transcription Master Mix was prepared, mixed gently, centrifuged briefly and the solution collected at the bottom of the tube. The Master Mix reaction system is as follows: 8ul of 2nd-Cycle Buffer Mix, 8ul of 2nd-Cycle Enzyme Mix.

c. Adding 16 mu L of reverse transcription Master Mix into a centrifuge tube reacted in the 4.1.1 steps, controlling the total volume to be 40 mu L, blowing, sucking and mixing uniformly for 2-3 times, and performing instantaneous centrifugation.

d. Placing the cRNA reverse transcription centrifugal tube on a PCR instrument, reacting for 10 minutes at 25 ℃, reacting for 1.5 hours at 40 ℃, reacting for 10 minutes at 70 ℃, reacting for 5 minutes at 4 ℃, and placing the centrifugal tube on ice.

e. And (3) operating on ice, adding 2 mu L of RNase H into a cRNA reverse transcription centrifugal tube, uniformly mixing, performing instantaneous centrifugation, placing on a PCR instrument, reacting for 45 minutes at 37 ℃, reacting for 5 minutes at 95 ℃, and maintaining for 5 minutes at 4 ℃.

f. After the reaction is finished, the reaction mixture can be frozen at-20 ℃ overnight, or can be immediately subjected to purification operation. Use of

Extract II (MN, cat. No.740609.250) kit was used for cDNA purification, and the purified cRNA product was quantified using uv spectrophotometer.

3.5 fluorescent labeling

(1) Fluorescent dye labeling reaction

a. The volume of the cDNA product obtained after reverse transcription and purification is concentrated to 14 mu L, 4 mu L of Random Primer is added to be mixed evenly, the mixture is placed on a PCR instrument after short-time centrifugation, denaturation is carried out for 3 minutes at 95 ℃, and ice bath is carried out for 5 minutes.

b. 5ul of 5 XKlenow Buffer, 1ul of Cy5-dCTP (or Cy3-dCTP), and 1.2ul of Klenow Fragmen were added sequentially.

c. After a short centrifugation, the reaction mixture was placed on a PCR instrument and reacted at 37 ℃ for 1.5 hours and 70 ℃ for 5 minutes. Keeping at 4 ℃.

d. After the labeling reaction with the fluorescent dye is completed, the reaction mixture is used

Extract II (MN, Cat. No.740609.250) kit was used for cDNA purification, and fluorescence incorporation and nucleic acid quantification were performed on the purified fluorescently labeled product using an ultraviolet spectrophotometer.

3.6 chip hybridization

(1) Labeled product hybridization preparation

a. Warp beam

The elution volume of the purified labeled product from the Extract II kit was about 30. mu.L.

b. If single-channel chip hybridization is performed, the single-tube labeled (cy3-dCTP or cy5-dCTP) purified eluate is concentrated under vacuum or made up to a volume of 27.5. mu.L for use.

c. If the two-channel chip hybridization is carried out, 1 tube of cy3-dCTP labeled purified product is mixed with another 1 tube of cy3-dCTP labeled purified product, and the total amount is concentrated to 27.5. mu.L by vacuum pumping for standby after about 60. mu.L.

(2) Preparation of hybridization system and hybridization reaction

a. The labeled product prepared in the above step was mixed with 55ul of 2 XGEx Hyb Buffer, 27.5ul of Formamide, and 27.5ul of Sample.

b. And (3) adding 100 mu L of hybridization solution into the hybridization cover plate fence, slightly covering the fence with the Agilent label surface facing downwards, installing the Agilent hybridization box, screwing, slightly horizontally rotating the hybridization box, and checking whether the liquid in the hybridization cavity of each subarray flows or not.

c. The hybridization cassettes were mounted on the rotor of the hybridization oven, taking care of the symmetrical mounting, while after adding a suitable amount of ultrapure water to the trays, hybridization was carried out overnight at 45 ℃.

3.7 chip cleaning and scanning

a. After the end, the chip is taken out and cleaned in a Boo Slide Washer 8 chip Washer dryer, and the cleaning program is as follows:

washing liquor I: 0.2% SDS, 2 XSSC, 120S at 42 ℃ for 2 washes.

Washing liquid II: 0.2% SDS, 2 XSSC, 80S at 42 ℃ 3 times.

After the cleaning procedure is finished, centrifugally drying, and waiting for scanning

b. The washed chip was scanned using an Agilent chip scanner (G2565CA) to obtain a hybridization image.

3.8 chip data analysis

Fluorescence scanning image of chip is processed by AGCC software: (

Command

Software) converts the image signal into a digital signal, and obtains the fluorescence signal intensity of each probe. Then, a Robust Multi-array Average (RMA) module of Affimetrix Expression Console software is used for preprocessing data, including normalization of raw data, discrimination of whether a probe signal is significantly higher than a background signal or not, and integration of the probe signal into a probe set signal. The data were then normalized and analyzed for differences using Agilent GeneSpring software.

Differential mRNA expression analysis. The analysis mainly comprises the following steps: comparing the mRNA expression difference between the case group and the control group by a fold-change value (FC) method; the difference p-value between the case group and the control group was calculated using t-test. The screening conditions were p < 0.05.

4. Differential expression analysis of peripheral blood mononuclear cell gene expression was performed in 25 rheumatoid arthritis patients and 18 normal controls, and RPN2mRNA expression was upregulated compared to the normal controls. Differential expression of RPN2mRNA was significant (FIG. 1A, p <0.05, Fold Change (FC) ═ 2.09), and the mRNA had an area under the ROC curve (AUC) of 0.91(p < 0.05; 95% confidence interval: 0.83-0.99), and was of diagnostic value. When the cut-off value was 3.3, the sensitivity and specificity were 82.1% and 83.3%, respectively (FIG. 1B).

Example 2: qRT-PCR experiments of mRNA in peripheral blood mononuclear cells

1. And performing qRT-PCR verification on the RPN2mRNA according to the chip result. The qRT-PCR detection of RPN2mRNA was performed on peripheral blood mononuclear cells from rheumatoid arthritis patients and normal controls, with strict quality control performed throughout the study, with at least three consecutive detections per sample. The forward and reverse primers used are shown in SEQ ID NO.3 and SEQ ID NO. 4.

2. After informed consent was obtained, 35 peripheral blood samples from patients diagnosed with rheumatoid arthritis and 35 healthy persons as normal controls were collected in sodium citrate anticoagulation tubes.

3. The same density gradient centrifugation method as in example 1 was used to obtain peripheral blood mononuclear cells from rheumatoid arthritis patients and normal humans.

4. And (3) carrying out extraction of total RNA of the sample, reverse transcription of mRNA and quantitative PCR of cDNA on the peripheral blood mononuclear cells added with TRIzol. The specific experimental steps are as follows:

4.1 extraction of sample RNA

(1) Taking out the mononuclear cells soaked in the TRIzol from the refrigerator, thawing at room temperature, and mixing uniformly;

(2) adding chloroform according to the proportion of 200ul chloroform/1 mL TRIzol, rapidly mixing for 15 seconds (mixing without a vortex instrument to prevent genome DNA from breaking), and standing for 2-3 minutes at room temperature;

(3) centrifuging at 12000g for 15 min at 4 deg.C, and extracting upper water phase;

(4) isopropanol with the same volume as the water phase was added, mixed by inversion, left at room temperature for 10 minutes, and then centrifuged at 12000rpm at 4 ℃ for 10 minutes to precipitate RNA at the bottom of the tube.

(5) Sucking out supernatant, adding 75% alcohol pre-cooled at-20 deg.C into the tube with precipitate, and suspending the precipitate (the alcohol is prepared with RNase-free water);

(6) centrifuging at 7500rpm at 4 deg.C for 5min, discarding the supernatant, air drying for 5-10 min, and dissolving RNA precipitate with 30ul of water to remove RNase.

4.2 digestion of residual genomic DNA in RNA

Taking 30ul of RNA stock solution extracted in the step for DNA digestion reaction. The reaction program was 37 ℃ for 30 minutes. The reaction system was as follows (50 ul): 30ul of RNA stock; 0.5ul recombined RNase Inhibitor; 5ul DNaseI buffer; 13.5ul RNase Free H₂O；1ul Dnase I(Rnase free)。

To the RNA obtained by the reaction, 500ul TRIzol was added, RNA extraction was performed according to the procedure of 3.1 in example 1, and finally 30ul RNase-free water was added to dissolve the RNA.

4.3 reverse transcription was performed using the 1st-Strand cDNA Synthesis kit (environmental image organism) according to the operating manual.

4.4 qPCR reactions were performed using the SYBR Green qPCR Master Mix Kit (environmental biology) using Roche real-time fluorescent quantitative PCR instrument Roche light cycler 480II (Roche).

5. Data analysis

The PCR amplification result is expressed as Ct value, which is the number of cycles in the PCR reaction at which the fluorescence signal reaches the set threshold. Calculating the Delta Ct value of differentially expressed mRNA, normalizing with B2M as internal reference, and using relative quantification

The method calculates the difference of mRNA expression between the case and the control group. Comparison between groups Using t-test to compare differences, P<0.05 was considered statistically different.

6. Data analysis showed that differential expression of RPN2mRNA was significant (fig. 2, FC ═ 1.14, p < 0.05).

Example 3: qRT-PCR experiments for mRNA in human T cells

1. The qRT-PCR detection of RPN2mRNA was performed on rheumatoid arthritis patients and normal control T cells, with stringent quality control performed throughout the study, with at least three consecutive detections per sample. The forward and reverse primers used are shown in SEQ ID NO.5 and SEQ ID NO. 6.

2. After informed consent was obtained, 6 peripheral blood samples from patients diagnosed with rheumatoid arthritis and 3 peripheral blood samples from healthy persons as normal controls were collected in sodium citrate anticoagulation tubes.

3. Human T cell extraction

(1) Peripheral blood mononuclear cells were extracted as described in example 1.

(2) T cells from peripheral Blood mononuclear cells were extracted using the EasySep Human Blood CD3 Positive Selection Kit (STEMCELL Technologies, Canada).

a. Add 1ml PBS to resuspend peripheral blood mononuclear cells, transfer to a new round transparent tube.

b. CD3+ cells were labeled with 60ul of both EasySep Human CD3 Positive Selection Cocktail and EasySep magnetic nanoparticles and left to stand for 3 minutes.

c. The clear tube was shaken for 30 seconds, 45ul of Rapidsphere was added to the tube, and the tube was allowed to stand for 3 minutes.

d. The solution was placed in a magnet and allowed to stand for 3 minutes.

f. The magnet and the transparent tube were simultaneously inverted and the suspension was poured out. The clear tube was removed from the magnet, at which time the CD3+ cells were attached to the tube wall.

g. PBS was added to resuspend the cells, centrifuge, 300g, 5 min. The supernatant was discarded. And the sediment pellet at the bottom of the tube is the target cell.

4. And adding a proper amount of TRIzol to the extracted T cells to extract the total RNA of the sample. The specific experimental procedure was the same as that for the extraction of RNA in example 3.

5. 2ug of RNA was collected and used

First-Strand cDNA Synthesis kit (Promega, USA) according to the operating manual reverse transcription reaction.

6. Use of

qPCR Master Mix Kit (Promega, USA) for qPCR reaction, the instrument uses QuantStaudio 6 Flex (Life Technologies, USA).

7. Data analysis

The PCR amplification result is expressed as Ct value, which is the number of cycles in the PCR reaction at which the fluorescence signal reaches the set threshold. Calculating the delta Ct value of differentially expressed mRNA, normalizing by using GAPDH as internal reference, and using relative quantification

8. Data analysis showed that differential expression of RPN2mRNA was significant (fig. 3, FC ═ 1.79, p < 0.05).

Example 4: screening of differentially expressed proteins in peripheral blood mononuclear cells

1. After informed consent was obtained, 18 peripheral blood samples from patients diagnosed with rheumatoid arthritis and 10 healthy persons as normal controls were collected in sodium citrate anticoagulation tubes.

2. The same density gradient centrifugation method as in example 1 was used to obtain peripheral blood mononuclear cells from rheumatoid arthritis patients and normal humans.

3. Extraction, pretreatment and quantification of total protein of peripheral blood mononuclear cell

(1) To each tube of peripheral blood mononuclear cell sample, 300. mu.l of SDT buffer (4% SDS,1.0mM DTT,100mM Tris-HCl, pH 7.6) was added to lyse the cells, and incubated for 15 minutes in a boiling water bath.

(2) High speed centrifugation, 14000g and 40 minutes, and collecting supernatant to obtain the total protein of the mononuclear cells of the peripheral blood. Boiling water bath for 5min, and ultrasonic cracking.

(3) The protein samples were centrifuged at 13400rpm/min for 30 minutes and the supernatant was removed.

(6) Protein quantification was performed by BCA method.

4. SDS-PAGE experiments of proteins

Mu.g of each protein sample was taken at 20. mu.g of the protein sample at 5:1(v/v), 5-fold concentrated loading buffer was added, the mixture was subjected to a boiling water bath for 5 minutes, and 14000g of the mixture was centrifuged for 10 minutes to take the supernatant, which was subjected to 12.5% SDS-PAGE. Electrophoresis conditions: constant current: 15mA, electrophoresis time 60 minutes. Coomassie brilliant blue staining.

5. Mu.g of each sample was taken, DTT was added to a final concentration of 100mM, and the mixture was cooled to room temperature in a boiling water bath for 5 minutes. Add 200. mu.L of UA buffer (8M Urea, 150mM Tris-HCl pH8.0), mix well, transfer to 30kd ultrafiltration centrifuge tube, centrifuge 14000g for 15 minutes. 200 μ L of UA buffer was added and 14000g was centrifuged for 15 minutes, and the filtrate was discarded. Add 100. mu.L IAA (50mM IAA in UA), shake for 1 min at 600rpm, protect from light for 30 min at room temperature, and centrifuge for 14000g for 10 min. Add 100 u L UA buffer, centrifugal 14000g 10 minutes repeated 2 times. Add 100. mu.L NH4HCO3buffer and centrifuge 14000g for 10min for 2 replicates. 40 μ L of Trypsin buffer (2 μ g of Trypsin 40 μ L of NH4HCO3buffer) was added, shaken at 600rpm for 1 minute and at 37 ℃ for 16-18 h. The collection tube was replaced, the tube was centrifuged for 14000g for 10 minutes, the filtrate was collected, and the filtrate was desalted by C18-SD Extraction Disk Cartridge and quantified as OD 280.

6. LC-MS/MS analysis of enzymatic products

According to the quantitative results, 2. mu.g of each sample was analyzed by LC-MS/MS. Separation was carried out using a nano liter flow rate HPLC liquid phase system EASY-nLC 1000. The liquid A was 0.1% acetonitrile solution of formic acid (acetonitrile: 2%), and the liquid B was 0.1% acetonitrile solution of formic acid (acetonitrile: 84%). The column Thermo EASY column SC 200150 μm 100mm (RP-C18) was equilibrated with 100% solution A. Samples were applied by autosampler to Thermo EASY column SC001 taps 150 μm 20mm (RP-C18) (Thermo) and separated by chromatography at 400 nL/min. The relevant liquid phase gradients are as follows: 0 min-100 min, linear gradient of B liquid from 0% to 45%; 100 min-108 min, linear gradient of B liquid from 45% to 100%; 108 minutes to 120 minutes, and the B solution is maintained at 100 percent. The enzymolysis product is separated by capillary high performance liquid chromatography and then is subjected to mass spectrometry by using a Q-exact mass spectrometer (Thermo Finnigan). Analysis duration: 120min, detection mode: positive ion, parent ion scan range: 300-1800m/z, mass-to-charge ratios of polypeptides and fragments of polypeptides were collected as follows: 20 fragment patterns (MS2scan, HCD) were collected after each full scan (full scan). The resolution of MS1 at M/Z200 was 70,000, and the resolution of MS2 at M/Z200 was 17,500.

7. Unlabeled quantitative analysis of Maxquant

The LC-MS/MS raw files of 6 samples were imported into Maxquant software (version number 1.3.0.5) for database lookup and LFQ non-standard quantitative analysis. The database was uniprot _ human _151619_20160229.fasta (entry 151619, download 2016-02-29). The main parameters are as follows:

Main search ppm：6 Missed cleavage：2

MS/MS tolerance ppm：20 De-Isotopic：TRUE

enzyme：Trypsin

database：uniprot_human_151619_20160229.fasta

Fixed modification：Carbamidomethyl(C)

Variable modification:Oxidation(M)，Acetyl(Protein N-term)

Decoy database pattern:reverse

LFQ：TRUE

LFQ min.ratio count：1

Match between runs：2min

Peptide FDR：0.01

Protein FDR：0.01

(9) statistical analysis of Persecus

The data after Maxquant processing was statistically analyzed using Perseus 1.3.0.4 software to screen for proteins with dysregulated expression in the patient groups by comparing the proteome expression profiles in peripheral blood mononuclear cells of the rheumatoid arthritis patient groups and normal control groups. The analysis result of the difference among groups shows that: there was a significant difference in RPN2 protein expression levels between the patient group and the normal group (P < 0.05).

Fig. 4 shows that the expression level of RPN2 protein was significantly up-regulated in rheumatoid arthritis peripheral blood mononuclear cells (FC ═ 1.29; P <0.05) compared to the normal control group.

FIG. 5 is a graph of secondary mass spectrometric identification of the RPN2 protein in peripheral blood mononuclear cells. Wherein A is a patient suffering from rheumatoid arthritis; b is a healthy control individual.

Example 5: identification of plasma protein markers

After informed consent was obtained, 40 peripheral blood samples from patients diagnosed with rheumatoid arthritis and 40 healthy persons as normal controls were collected in sodium citrate anticoagulation tubes. The plasma RPN2 protein detection steps were as follows:

1. blood sample collection and plasma separation

(1) Approximately 10ml of venous blood was centrifuged at 500g for 10min at high speed and the upper plasma carefully aspirated for use.

2. Plasma RPN2 protein concentrations were quantified using an ELISA kit for RPN2 protein (MyBioSource, Cat. No.: MBS 9312546). The experiment was performed according to the manual.

Fig. 6 shows the difference in plasma concentrations of RPN2 protein in 40 rheumatoid arthritis patients and 40 control individuals (fig. 6A, N-80; FC-2.12; P < 0.05). The area under the ROC curve (AUC) was 0.672 (FIG. 6B, p < 0.05; 95% confidence interval: 0.55-0.79). The sensitivity and specificity were 65.0% and 67.5% respectively, with 14.29ng/ml as the cut-off point.

Example 6: effect of specific RPN2 genes on cell growth and activation in Jurkat immune cells

The above examples show that: the RPN2mRNA has higher diagnostic value (high sensitivity and specificity) for detecting the peripheral blood mononuclear cells of the rheumatoid arthritis. This example carries out a mechanism of association study between RPN2 gene and rheumatoid arthritis. Jurkat cells are a common CD4+ T lymphocyte cell line. First, the effect of inflammatory factor stimulation on the expression level of RPN2mRNA in Jurkat cells was investigated, and as shown in fig. 7A, Jurkat cells were significantly upregulated in the expression level of RPN2mRNA under PHA stimulation. RPN2 over-expression stably transfected cell lines (OE) and control cells (NC) were constructed in Jurkat cells using PGMLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro vector, where over-expression of RPN2 could affect Jurkat cell cycle (FIG. 7B), promote cell proliferation (FIG. 7C) and PHA-induced cell activation (FIG. 7D).

In summary, according to the above technical scheme, the RPN2 gene biomarker, the corresponding primer set and the probe thereof provided by the invention can be used for preparing a diagnostic kit, and have excellent sensitivity and specificity when being applied to rheumatoid arthritis diagnosis of a peripheral blood mononuclear cell sample. Further, the inventor of the present invention confirms through research that AUC value of RPN2mRNA can reach 0.911, and when the cut-off value is 3.3, sensitivity and specificity are 82.1% and 83.3%, respectively. The AUC value of the RPN2 protein can reach 0.672, the cut-off point is 14.29ng/ml, and the sensitivity and the specificity are 65.0 percent and 67.5 percent respectively. The gene can be used as a biomarker for diagnosing human rheumatoid arthritis peripheral blood mononuclear cell samples, and is favorable for promoting the development of early diagnosis, prediction treatment and relapse monitoring of rheumatoid arthritis in China.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

<110> Suzhou university

<120> biomarker for rheumatoid arthritis diagnosis and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2227

<212> DNA

<213> (Artificial Synthesis)

<400> 1

ggactccggc gggacctgct cggaggaatg gcgccgccgg gttcaagcac tgtcttcctg 60

ttggccctga caatcatagc cagcacctgg gctctgacgc ccactcacta cctcaccaag 120

catgacgtgg agagactaaa agcctcgctg gatcgccctt tcacaaattt ggaatctgcc 180

ttctactcca tcgtgggact cagcagcctt ggtgctcagg tgccagatgc aaagaaagca 240

tgtacctaca tcagatctaa ccttgatccc agcaatgtgg attccctctt ctacgctgcc 300

caggccagcc aggccctctc aggatgtgag atctctattt caaatgagac caaagatctg 360

cttctggcag ctgtcagtga ggactcatct gttacccaga tctaccatgc agttgcagct 420

ctaagtggct ttggccttcc cttggcatcc caagaagcac tcagtgccct tactgctcgt 480

ctcagcaagg aggagactgt gctggcaaca gtccaggctc tgcagacagc atcccacctg 540

tcccagcagg ctgacctgag gagcatcgtg gaggagattg aggaccttgt tgctcgcctg 600

gatgaactcg ggggcgtgta tctccagttt gaagaaggac tggaaacaac agcgttattt 660

gtggctgcca cctacaagct catggatcat gtggggactg agccatccat taaggaggat 720

caggtcatcc agctgatgaa cgcgatcttc agcaagaaga actttgagtc cctctccgaa 780

gccttcagcg tggcctctgc agctgctgtg ctctcgcata atcgctacca cgtgccagtt 840

gtggttgtgc ctgagggctc tgcttccgac actcatgaac aggctatctt gcggttgcaa 900

gtcaccaatg ttctgtctca gcctctgact caggccactg ttaaactaga acatgctaaa 960

tctgttgctt ccagagccac tgtcctccag aagacatcct tcacccctgt aggggatgtt 1020

tttgaactaa atttcatgaa cgtcaaattt tccagtggtt attatgactt ccttgtcgaa 1080

gttgaaggtg acaaccggta tattgcaaat accgtagagc tcagagtcaa gatctccact 1140

gaagttggca tcacaaatgt tgatctttcc accgtggata aggatcagag cattgcaccc 1200

aaaactaccc gggtgacata cccagccaaa gccaagggca cattcatcgc agacagccac 1260

cagaacttcg ccttgttctt ccagctggta gatgtgaaca ctggtgctga actcactcct 1320

caccagacat ttgtccgact ccataaccag aagactggcc aggaagtggt gtttgttgcc 1380

gagccagaca acaagaacgt gtacaagttt gaactggata cctctgaaag aaagattgaa 1440

tttgactctg cctctggcac ctacactctc tacttaatca ttggagatgc cactttgaag 1500

aacccaatcc tctggaatgt ggctgatgtg gtcatcaagt tccctgagga agaagctccc 1560

tcgactgtct tgtcccagaa ccttttcact ccaaaacagg aaattcagca cctgttccgc 1620

gagcctgaga agaggccccc caccgtggtg tccaatacat tcactgccct gatcctctcg 1680

ccgttgcttc tgctcttcgc tctgtggatc cggattggtg ccaatgtctc caacttcact 1740

tttgctccta gcacgattat atttcacctg ggacatgctg ctatgctggg actcatgtat 1800

gtctactgga ctcagctcaa catgttccag accttgaagt acctggccat cctgggcagt 1860

gtgacgtttc tggctggcaa tcggatgctg gcccagcagg cagtcaagag aacagcacat 1920

tagttccaga agaaagatgg aaattctgaa aactgaatgt caagaaaagg agtcaagaac 1980

aattcacagt atgagaagaa aaatggaaaa aaaaaacttt atttaaaaaa gaaaaaagtc 2040

cagattgtag ttatactttt gcttgttttt cagtttcccc aacacacagc agatacctgg 2100

tgagctcaga tagtctcttt ctctgacact gtgtaagaag ctgtgaatat tcctaactta 2160

cccagatgtt gcttttgaaa agttgaaatg tgtaattgtt ttggaataaa gagggtaaca 2220

ataggaa 2227

<210> 2

<211> 631

<212> PRT

<213> (Artificial Synthesis)

<400> 2

Met Ala Pro Pro Gly Ser Ser Thr Val Phe Leu Leu Ala Leu Thr Ile

1 5 10 15

Ile Ala Ser Thr Trp Ala Leu Thr Pro Thr His Tyr Leu Thr Lys His

20 25 30

Asp Val Glu Arg Leu Lys Ala Ser Leu Asp Arg Pro Phe Thr Asn Leu

35 40 45

Glu Ser Ala Phe Tyr Ser Ile Val Gly Leu Ser Ser Leu Gly Ala Gln

50 55 60

Val Pro Asp Ala Lys Lys Ala Cys Thr Tyr Ile Arg Ser Asn Leu Asp

65 70 75 80

Pro Ser Asn Val Asp Ser Leu Phe Tyr Ala Ala Gln Ala Ser Gln Ala

85 90 95

Leu Ser Gly Cys Glu Ile Ser Ile Ser Asn Glu Thr Lys Asp Leu Leu

100 105 110

Leu Ala Ala Val Ser Glu Asp Ser Ser Val Thr Gln Ile Tyr His Ala

115 120 125

Val Ala Ala Leu Ser Gly Phe Gly Leu Pro Leu Ala Ser Gln Glu Ala

130 135 140

Leu Ser Ala Leu Thr Ala Arg Leu Ser Lys Glu Glu Thr Val Leu Ala

145 150 155 160

Thr Val Gln Ala Leu Gln Thr Ala Ser His Leu Ser Gln Gln Ala Asp

165 170 175

Leu Arg Ser Ile Val Glu Glu Ile Glu Asp Leu Val Ala Arg Leu Asp

180 185 190

Glu Leu Gly Gly Val Tyr Leu Gln Phe Glu Glu Gly Leu Glu Thr Thr

195 200 205

Ala Leu Phe Val Ala Ala Thr Tyr Lys Leu Met Asp His Val Gly Thr

210 215 220

Glu Pro Ser Ile Lys Glu Asp Gln Val Ile Gln Leu Met Asn Ala Ile

225 230 235 240

Phe Ser Lys Lys Asn Phe Glu Ser Leu Ser Glu Ala Phe Ser Val Ala

245 250 255

Ser Ala Ala Ala Val Leu Ser His Asn Arg Tyr His Val Pro Val Val

260 265 270

Val Val Pro Glu Gly Ser Ala Ser Asp Thr His Glu Gln Ala Ile Leu

275 280 285

Arg Leu Gln Val Thr Asn Val Leu Ser Gln Pro Leu Thr Gln Ala Thr

290 295 300

Val Lys Leu Glu His Ala Lys Ser Val Ala Ser Arg Ala Thr Val Leu

305 310 315 320

Gln Lys Thr Ser Phe Thr Pro Val Gly Asp Val Phe Glu Leu Asn Phe

325 330 335

Met Asn Val Lys Phe Ser Ser Gly Tyr Tyr Asp Phe Leu Val Glu Val

340 345 350

Glu Gly Asp Asn Arg Tyr Ile Ala Asn Thr Val Glu Leu Arg Val Lys

355 360 365

Ile Ser Thr Glu Val Gly Ile Thr Asn Val Asp Leu Ser Thr Val Asp

370 375 380

Lys Asp Gln Ser Ile Ala Pro Lys Thr Thr Arg Val Thr Tyr Pro Ala

385 390 395 400

Lys Ala Lys Gly Thr Phe Ile Ala Asp Ser His Gln Asn Phe Ala Leu

405 410 415

Phe Phe Gln Leu Val Asp Val Asn Thr Gly Ala Glu Leu Thr Pro His

420 425 430

Gln Thr Phe Val Arg Leu His Asn Gln Lys Thr Gly Gln Glu Val Val

435 440 445

Phe Val Ala Glu Pro Asp Asn Lys Asn Val Tyr Lys Phe Glu Leu Asp

450 455 460

Thr Ser Glu Arg Lys Ile Glu Phe Asp Ser Ala Ser Gly Thr Tyr Thr

465 470 475 480

Leu Tyr Leu Ile Ile Gly Asp Ala Thr Leu Lys Asn Pro Ile Leu Trp

485 490 495

Asn Val Ala Asp Val Val Ile Lys Phe Pro Glu Glu Glu Ala Pro Ser

500 505 510

Thr Val Leu Ser Gln Asn Leu Phe Thr Pro Lys Gln Glu Ile Gln His

515 520 525

Leu Phe Arg Glu Pro Glu Lys Arg Pro Pro Thr Val Val Ser Asn Thr

530 535 540

Phe Thr Ala Leu Ile Leu Ser Pro Leu Leu Leu Leu Phe Ala Leu Trp

545 550 555 560

Ile Arg Ile Gly Ala Asn Val Ser Asn Phe Thr Phe Ala Pro Ser Thr

565 570 575

Ile Ile Phe His Leu Gly His Ala Ala Met Leu Gly Leu Met Tyr Val

580 585 590

Tyr Trp Thr Gln Leu Asn Met Phe Gln Thr Leu Lys Tyr Leu Ala Ile

595 600 605

Leu Gly Ser Val Thr Phe Leu Ala Gly Asn Arg Met Leu Ala Gln Gln

610 615 620

Ala Val Lys Arg Thr Ala His

625 630

<210> 3

<211> 22

<212> DNA

<213> (Artificial Synthesis)

<400> 3

tgacttcctt gtcgaagttg aa 22

<210> 4

<211> 20

<212> DNA

<213> (Artificial Synthesis)

<400> 4

gtgatgccaa cttcagtgga 20

<210> 5

<211> 28

<212> DNA

<213> (Artificial Synthesis)

<400> 5

ccggaattca cctgctcgga ggaatggc 28

<210> 6

<211> 36

<212> DNA

<213> (Artificial Synthesis)

<400> 6

ccgctcgagc taatgtgctg ttctcttgac tgcctg 36

Claims

1. The application of the biomarker in the preparation of products for detecting rheumatoid arthritis is characterized in that the biomarker is RPN2mRNA or RPN2 protein coded by gene RPN 2.

2. The use according to claim 1, wherein the nucleotide sequence of the RPN2mRNA is shown in SEQ ID No. 1.

3. The use according to claim 1, wherein the amino acid sequence of the RPN2 protein is as shown in SEQ ID No. 2.