CN109022568B - MicroRNA biomarker for rheumatoid arthritis diagnosis and application thereof - Google Patents

Abstract

The invention discloses a microRNA biomarker for rheumatoid arthritis diagnosis and application thereof, and belongs to the technical field of biological detection. The 3 microRNA biomarkers, the corresponding primer sets and the probes 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 a peripheral blood mononuclear cell sample. The AUC value of the combination of the 3 kinds of microRNAs can reach 0.745%, and the sensitivity and specificity are 73.5% and 76.7% respectively. The 3 kinds of microRNAs can be used as biomarkers for diagnosing human rheumatoid arthritis peripheral blood mononuclear cell samples, and are favorable for promoting the development of early diagnosis, prediction treatment and relapse monitoring of rheumatoid arthritis in China.

Description

MicroRNA biomarker for rheumatoid arthritis diagnosis and application thereof

Technical Field

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

Background

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease with erosive arthritis as the major manifestation. At present, about 1% of people all over the world suffer from the disease, wherein the prevalence rate of developed countries is 0.5-1%, the prevalence rate of new cases is 5-50/10 ten thousand every year, the prevalence rate of continental areas of China is 0.2-0.36%, and the total number of cases exceeds 500 thousand. The clinical manifestations are symmetry, persistent joint swelling and pain, often accompanied by bone loss, bone erosion, loss of articular cartilage, synovial inflammation and joint capsule swelling. If the patient does not control the disease, the disease will gradually cause the incompetence of muscles and bones, the hypofunction of the body and the induction of symbiotic diseases, the disability rate in the later period is high, the life quality of the patient is seriously affected, and the heavy social and economic burden is accompanied. 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.

At present, the pathogenesis and mechanism of RA are not clear, but RA is generally considered to be a complex disease which is jointly regulated by environmental factors and genetic factors. The genetic factors are important influencing factors of the onset of RA, and the genetic factors account for about 50-60% of the onset of RA. Previous genome-wide association studies have found more than 100 susceptibility genes associated with RA, and many of them are immunologically relevant. However, the molecular mechanism of RA pathogenesis is not researched and matured, the discovered genetic susceptibility sites are combined to explain that the RA inheritance rate is very small (15%), and a large number of genetic factors are still to be discovered. Epigenetic modifications are important contributors to the development and progression of many diseases, and are involved in affecting late onset, sex differences, fluctuations in symptoms, etc., of complex diseases. microRNA (miRNA) plays a role in multiple levels of genome as an important component of epigenetic modification, and is expressed in regulating and controlling other epigenetic factors such as DNA methylation and modifying the expression of genes encoded by proteins by recruiting specific protein complex binding promoters.

Researches show that 1/3 gene in human body is regulated by miRNA, which is closely related to not only proliferation, differentiation, apoptosis, stress, fat metabolism, growth and development of normal cells, and function regulation of heart, but also generation and development of complex diseases such as malignant tumor and cardiovascular diseases. Mirnas have been reported to play an important role in the pathogenesis of RA as an important loop in epigenetic modification. It can participate in influencing multiple RA-related cytokine signal pathways and immune inflammatory response through targeted regulation and control of genes, or directly act on CD4⁺Immune cells such as T cells, synovial fibroblasts, synovial tissues, joint bones and the like, and further cause the occurrence and development of RA diseases.

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 miRNA abnormally expressed in RA is screened from peripheral blood mononuclear cells 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 problem, the peripheral blood mononuclear cell microRNA biomarker for diagnosing the rheumatoid arthritis is 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 purpose of the invention is to provide a microRNA biomarker for rheumatoid arthritis diagnosis, wherein the microRNA biomarker is any one or a combination of more than one of has-miR-99b-5p, has-miR-26b-5p or has-miR-7641.

In one embodiment of the invention, the nucleotide sequence of hsa-miR-99b-5p is shown in SEQ ID NO: 1, and the nucleotide sequence of the has-miR-26b-5p is shown as SEQ ID NO: 2, the nucleotide sequence of the has-miR-7641 is shown as SEQ ID NO: 3, respectively.

In one embodiment of the invention, the has-miR-99b-5p, has-miR-26b-5p and has-miR-7641 are derived from peripheral blood mononuclear cells.

The second purpose of the invention is to provide a combination of a microRNA primer group and a probe for rheumatoid arthritis diagnosis, which comprises one or more than one of a has-miR-99b-5p primer and a probe, a has-miR-26b-5p primer and a probe or a has-miR-7641 primer and a probe.

The third purpose of the invention is to provide a kit for diagnosing rheumatoid arthritis, which comprises a microRNA primer group and a microRNA probe combination for detecting the expression quantity of any one or more than one combination of has-miR-99b-5p, has-miR-26b-5p or has-miR-7641 in peripheral blood mononuclear cells.

In one embodiment of the invention, the microRNA primer set comprises a reverse transcription primer, a pre-PCR primer and a post-PCR primer of microRNA.

In one embodiment of the invention, the reverse transcription primer sequence of hsa-miR-99b-5p is shown in SEQ ID NO: 4, the sequence of the primer before PCR is shown as SEQ ID NO: 7, the primer sequence after PCR is shown as SEQ ID NO: 10 is shown in the figure; the reverse transcription primer sequence of has-miR-26b-5p is shown in SEQ ID NO: 5, the sequence of the primer before PCR is shown as SEQ ID NO: 8, the primer sequence after PCR is shown as SEQ ID NO: 10 is shown in the figure; the reverse transcription primer sequence of has-miR-7641 is shown as SEQ ID NO: 6, the sequence of the primer before PCR is shown as SEQ ID NO: 9, the primer sequence after PCR is shown as SEQ ID NO: shown at 10.

In one embodiment of the invention, the kit further comprises conventional components for qPCR amplification detection, wherein the conventional components for qPCR amplification detection comprise reverse transcriptase, buffer, dNTPs, MgCl and dNTPs₂、dd H₂O, fluorescent dye, Taq enzyme, standard and control.

The fourth purpose of the invention is to provide a chip for diagnosing rheumatoid arthritis, which comprises a microRNA probe for detecting any one or more than one of has-miR-99b-5p, has-miR-26b-5p or has-miR-7641.

In one embodiment of the invention, the nucleotide sequence of the microRNA probe is completely complementary to the full-length mature microRNA of the detection object of the microRNA probe.

The fifth purpose of the invention is to provide the application of the microRNA biomarker in the preparation of products for detecting rheumatoid arthritis.

The application of the microRNA biomarker in the diagnosis of rheumatoid arthritis comprises the following steps:

determining the expression level of a biomarker of peripheral blood mononuclear cells from the subject, wherein the biomarker is selected from any one or more than one combination of has-miR-99b-5p, has-miR-26b-5p and has-miR-7641, so as to obtain a determination value; and comparing the measured value with a reference value, and determining that the subject has rheumatoid arthritis if the measured value of has-miR-99b-5p is higher than the reference value and the measured values of has-miR-26b-5p and has-miR-7641 are lower 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 reverse transcription primer sequence of the hsa-miR-99b-5p is shown in SEQ ID NO: 4, the sequence of the primer before PCR is shown as SEQ ID NO: 7, the primer sequence after PCR is shown as SEQ ID NO: 10 is shown in the figure; the reverse transcription primer sequence of has-miR-26b-5p is shown in SEQ ID NO: 5, the sequence of the primer before PCR is shown as SEQ ID NO: 8, the primer sequence after PCR is shown as SEQ ID NO: 10 is shown in the figure; the reverse transcription primer sequence of has-miR-7641 is shown as SEQ ID NO: 6, the sequence of the primer before PCR is shown as SEQ ID NO: 9, the primer sequence after PCR is shown as SEQ ID NO: shown at 10.

In an embodiment of the present invention, the application specifically includes:

extracting biomarkers in peripheral blood mononuclear cells of the subject;

providing a detection chip, wherein the detection chip is loaded with a miRNA probe with a complete complementary sequence with the full-length mature miRNA of the biomarker;

and measuring the detection value by using the detection chip.

The invention has the beneficial effects that: the 3 microRNA biomarkers, the corresponding primer sets and the probes 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 a peripheral blood mononuclear cell sample. The AUC value of the combination of the 3 kinds of microRNAs can reach 0.745%, and the sensitivity and specificity are 73.5% and 76.7% respectively. The 3 kinds of microRNAs can be used as biomarkers for diagnosing human rheumatoid arthritis peripheral blood mononuclear cell samples, and are favorable for promoting the development of early diagnosis, prediction treatment and relapse monitoring of rheumatoid arthritis in China.

Drawings

FIG. 1 is a graph showing the results of analysis of significantly differentially expressed microRNAs (p <0.05, difference not less than 2-fold) generated by cluster analysis;

FIG. 2 is a ROC curve chart of the microRNA of the present invention for distinguishing rheumatoid arthritis patients from normal controls; wherein, A to C are respectively used independently for 3 microRNAs, and D is used jointly for 3 microRNAs;

FIG. 3 is a graph showing the expression level changes of 3 miRNAs of the present invention in 35 cases of rheumatoid arthritis and 35 cases of healthy human peripheral blood mononuclear cell samples (P < 0.05);

FIG. 4 shows miRNA expression levels after successful construction of miR-99b-5p overexpression cell lines;

FIG. 5 is a graph of the effect of miR-99b-5p overexpression on cell proliferation of Jurkat cells;

FIG. 6 is a graph of the effect of miR-99b-5p overexpression on apoptosis in Jurkat cells;

FIG. 7 is a graph of the effect of miR-99b-5p overexpression on the cell cycle of Jurkat cells;

FIG. 8 is a graph of the effect of miR-99b-5p overexpression on cell activation of Jurkat cells;

FIG. 9 is a graph of the effect of miR-99b-5p overexpression on proinflammatory cytokines in T cells.

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 provide some remarks: 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 for differential expression of microRNA (miRNA) in peripheral blood mononuclear cells

1. After informed consent was obtained, 25 peripheral blood samples from the first civilian hospital affiliated to Suzhou university were collected from sodium citrate anticoagulation tubes as confirmed rheumatoid arthritis patients and 18 healthy persons as normal controls.

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) peripheral blood samples were centrifuged and allowed to separate, 2000rpm × 2 min.

(2) 15ml of the separated liquid was added to a leucosep separation tube, and centrifuged at 5100g for 1 minute.

(3) The supernatant of the layered peripheral blood sample was collected in a 15ml centrifuge tube, and centrifuged at 2500g for 15 min. Then, the mixture was dispensed into 2ml centrifuge tubes and stored in a freezer at-80 ℃.

(4) 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.

(5) The mixture was transferred to the leucosep tube in (2), and centrifuged at 5300g for 20 minutes.

(6) The supernatant in the separator 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.

(7) The supernatant in the 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.

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

3. In the discovery experiment, mirVana was used from peripheral blood mononuclear cells of TRIzol-added rheumatoid arthritis patients and healthy controls^TMTotal RNA was extracted by miRNA Isolation Kit (ambion), and miRNA expression was detected by whole genome expression profiling chip. The chip (Affymetrix miRNA 4.0) covers all miRNA mature bodies of Sanger miRBase 20.0 version 203 species, including 2578 human mature miRNAs. The chip experiment procedure has been standardized and can be referred to as www.thermofisher.com.

4. Data extraction and 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. And finally, performing exponential conversion on the preprocessed data to obtain an miRNA expression value.

And carrying out miRNA differential expression analysis on the preprocessed miRNA expression profile data. The analysis mainly comprises the following steps: comparing the miRNA expression difference of 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 are that the expression difference is not less than 2 times (FC ≥ 2 or ≤ 0.5) or p < 0.05. And then performing cluster analysis on the miRNA with differential expression obtained by screening by using Cluster 3.0.

5. Differential expression analysis is carried out on peripheral blood mononuclear cells of 25 cases of rheumatoid arthritis patients and 18 cases of normal controls by using a miRNA chip, and 18 miRNAs with differential expression are screened out in total, namely hsa-miR-101-3p, hsa-miR-1184, hsa-miR-1246, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-195-5p, hsa-miR-26b-5p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-3201, hsa-miR-3607-5p, hsa-miR-3613-3p, hsa-miR-3651, hsa-miR-4448, hsa-miR-4668-5p, hsa-miR-7641, hsa-miR-8084 and hsa-miR-99b-5 p. In addition to the upregulation of has-miR-99b-5p, the remaining miRNAs were downregulated in rheumatoid arthritis peripheral blood mononuclear cells compared to normal controls (see FIG. 1).

Example 2: qRT-PCR experiment of miRNA in peripheral blood mononuclear cells

1. According to the result of the miRNA chip, 9 miRNAs are selected from 18 miRNAs with differential expression according to the following conditions for carrying out qRT-PCR verification. The concrete conditions are as follows: (1) the miRNA has not been previously reported to have been validated by RT-PCR in the RA population; (2) the miRNA has a high FC value; (3) the miRNA has higher expression level in the chip. The sequences of selected hsa-miR-99b-5p, hsa-miR-26b-5p, hsa-miR-7641, hsa-miR-142-5p, hsa-miR-29c-3p, hsa-miR-3607-5p, hsa-miR-3651, hsa-miR-4448 and hsa-miR-1184 are shown in Table 1. And (3) carrying out qRT-PCR detection on miRNA on peripheral blood mononuclear cells of rheumatoid arthritis patients and normal controls, implementing strict quality control in the whole research, and continuously detecting each sample at least three times.

TABLE 1 primer sequence Listing

2. After informed consent was obtained, 35 peripheral blood samples from the first civilian hospital affiliated to Suzhou university were collected from sodium citrate anticoagulation tubes as 35 patients diagnosed with rheumatoid arthritis and 35 healthy persons as normal controls.

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) extracting total RNA of the sample, reversely transcribing miRNA and quantitatively PCR (polymerase chain reaction) of cDNA (complementary deoxyribonucleic acid) on the peripheral blood mononuclear cells added with TRIzol. The routine experimental procedures are as follows: extraction of sample RNA, digestion of residual genome DNA in RNA, reverse transcription reaction and qPCR reaction.

5. And (3) 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 the miRNAs with differential expression, adopting RNU48 as an internal reference for standardization, and applying relative quantification

The method calculates the expression difference of miRNA between the case group and the control group. Comparison between groups Using t-test to compare differences, P<0.05 was considered statistically different. ROC curve analysis was performed with MedCalc 15.8 software and sensitivity and specificity were calculated.

6. Data analysis results show that the differential expression of hsa-miR-99b-5p, hsa-miR-26b-5p and hsa-miR-7641 has significance (figure 2, p is less than 0.05), and the areas under ROC curves (AUC) of 3 miRNAs are respectively as follows: hsa-miR-99b-5p, 0.632 (95% confidence interval, 0.503-0.748); hsa-miR-26b-5p, 0.660 (95% confidence interval, 0.532-0.773); hsa-miR-7641, 0.701 (95% confidence interval, 0.575-0.808) (FIG. 2A-FIG. 2C). At the optimal cutoff value, the sensitivity and specificity of mirnas are as follows: hsa-miR-99b-5p, 50.0% and 80.6% respectively, hsa-miR-26b-5p, 55.9% and 96.8% respectively, and hsa-miR-7641, 52.9% and 83.9% respectively. The 3 microRNAs combined AUC value can reach 0.745, (95% confidence interval, 0.621-0.846), and the sensitivity and specificity are 73.5% and 76.7% respectively (FIG. 2D), which are superior to single miRNA. These results show that hsa-miR-99b-5p, hsa-miR-26b-5p and hsa-miR-7641 are combined to detect the peripheral blood mononuclear cells of the rheumatoid arthritis, and the diagnosis of the rheumatoid arthritis has very high sensitivity and specificity.

Example 3: effect of specific miRNAs (miR-99b-5p) on cell growth and inflammatory cytokines in Jurkat immune cells

The above examples show that hsa-miR-99b-5p, hsa-miR-26b-5p and hsa-miR-7641 have high diagnostic value (high sensitivity and specificity) for detecting the peripheral blood mononuclear cells of the rheumatoid arthritis, and the example carries out the correlation research between the peripheral blood mononuclear cells and the rheumatoid arthritis around miR-99b-5 p.

1. Construction of miR-99b-5p overexpression cell line in Jurkat cells by using pCDH-CMV-MCS-EF1-copGFP vector

1.1 construction of miR-99b-5p Lentiviral vectors

And designing primers according to the complete sequence of the hsa-miR-99b-5p and the sequences of 1000bp at the upstream and downstream of the complete sequence, and preparing a PCR product of a target sequence. And carrying out electrophoretic verification on the obtained PCR product. After verification, the PCR product was purified (Qiaquick PCR purification kit), digested (EcoRI, XbaI), repurified (Qiaquick gel recovery kit), and enzymatically ligated to lentiviral expression vector pCDH with T4DNA ligase. After connecting overnight at 16 ℃, the recombinant lentivirus expression vector is transformed, and the monoclonal colony is picked for amplification culture and simultaneously subjected to sequencing verification.

1.2 construction of miR-99b-5p Lentiviral cell line

And carrying out plasmid extraction of the miR-99b-5p lentivirus expression vector on the bacterial liquid subjected to amplification culture after verification. The lentivirus was then packaged with a lentivirus-associated packaging plasmid (psPAX/pMD2G, SBI). The packaged lentiviruses were virus concentrated and the concentrated lentiviruses were titered using a qPCR titer detection kit (abm). And then constructing a lentivirus overexpression cell line of miR-99b-5p in Jurkat cells by using the prepared lentivirus, wherein the steps are as follows:

(1) taking Jurkat cells with good growth state, inoculating the Jurkat cells into a 96-well plate, and culturing the Jurkat cells at 1X 10⁵Cells/dish, culture volume 100 ul/dish, and set 3 multiple wells. The plates were placed in a 37 ℃ cell incubator overnight.

(2) The number of Jurkat cells in the culture dish was as long as about 30% the following day before infection with virus. 1ul of 7.5mg/ul polybrene was diluted with 1ml of medium and 100ul of polybrene-containing medium was added to each well. Jurkat cells were infected by virus instillation in MOI 20, 200, 2000 gradients. Then placing the mixture in an incubator for culture.

(3) After culturing for 48h, the growth of the cells was observed by an inverted fluorescence microscope. The lentivirus infected Jurkat cells are transferred into a 12-well plate for continuous expansion culture.

(4) Preparing a miR-99b-5p negative control strain (NC) according to the method: and infecting Jurkat cells with good growth state by using pCDH no-load slow viruses, and performing cell infection by using the virus concentration consistent with miR-99b-5p overexpression cells to obtain a negative control strain for continuous amplification culture.

After 4 days, cells of the miR-99b-5p Overexpression (OE) group and the negative control NC group after amplification culture are centrifugally collected, Jurkat healthy cells are set as a control group, and the transfection efficiency of the miR-99b-5pOE group and the NC group is detected by a flow cytometry analyzer.

1.3 identification of miR-99b-5p overexpressing cell lines

Taking miR-99b-5p over-expression OE group cells with good growth state and high positive rate and negative control NC group cells, inoculating the cells in a 6-well plate, wherein the cell amount of each well is about 5 multiplied by 10⁵And (4) respectively. After 2 days of culture, centrifuging and collecting, adding 1ml of Trizol, blowing, beating and mixing evenly, and then carrying out total RNA extraction, cDNA reverse transcription and miR-99b-5p fluorescence quantitative PCR experiment.

1.3.1 extraction of Total RNA from cells

(1) Taking out the cells at the early stage, adding 1ml of Trizol to blow and beat the uniformly mixed cells: cells of miR-99b-5p OE group, NC group and normal Jurkat group.

(2) To the above lysate was added 1/5 volumes of chloroform (200 ul). And (5) tightly covering the centrifugal tube, then violently shaking for 15s, and standing for 2min at room temperature.

(3) Centrifuge at 12000rpm, 4 ℃ for 15 min. After centrifugation, the mixture was divided into 3 layers: the upper colorless water sample layer (containing RNA), the middle layer and the lower red organic phenol chloroform layer. The middle layer and the organic phase are protein and DNA.

(4) Carefully pipette 500ul of the upper aqueous phase into a new centrifuge tube and add an equal volume of isopropanol (500ul) to avoid contaminating the genome by pipetting into the middle layer. The mixture was inverted and left at room temperature for 10 min.

(5) Centrifuge at 12000rpm, 4 ℃ for 10 min. A gelatinous precipitate formed on the tube side or bottom after centrifugation.

(6) The supernatant was carefully discarded and 1ml of 75% ethanol in DEPC water was added. The tube cover and the tube wall are fully washed, and the tube bottom is flicked to suspend the sediment.

(7) Centrifuge at 12000rpm, 4 ℃ for 3 min. The supernatant was carefully discarded to prevent loss of the RNA pellet.

(8) Standing at room temperature for 2min, and air drying. Adding 30ul DEPC water, taking a small amount of the dissolved RNA after the dissolved RNA is completely dissolved, and detecting the light absorption values and the concentrations of the RNA samples at the positions with the wavelengths of 260nm and 280nm by using an ultraviolet spectrophotometer. When the ratio of OD260/OD280 is between 1.8 and 2.0, the RNA extraction is qualified. If the ratio is less than 1.8, protein or other organic contamination is possible, and if the ratio is greater than 2.2, RNA may have been hydrolyzed.

1.3.2 RNA tailing reaction and first Strand cDNA Synthesis

The TransScript miRNA First-Strand cDNA Synthesis SuperMix reverse transcription kit is adopted in the experiment, and the experimental steps are as follows:

(1) preparation of a cDNA reverse transcription System (20 ul): x ul RNA; 1ul TransScript miRNA RT Enzyme Mix; 10ul 2xTS miRNA Reaction Mix; 9-x ul RNase-Free Water.

The RNA in the system is 2ug, and the corresponding RNA volume and the corresponding water volume are obtained by calculation according to the RNA concentration.

(2) Mix gently, incubate the template RNA and primer mixture for 1h at 37 ℃ and heat to 85 ℃ for 5s to inactivate RT Enzyme Mix.

1.3.3 Real-Time qPCR

Configuring a qPCR reaction system (20 ul): 1ul cDNA; 0.5ul Forward Primer (10 uM); 0.5ul Universal miRNA qPCR Primer (10 uM); 10ul GoTaq qPCR Master Mix, 2 ×; 8ul ddH₂O。

The cDNA obtained by reverse transcription was diluted 5-fold and used. Each set of experiments was set up in 3 replicates.

The hsa-miR-99b-5p upstream primer is a specific primer, the downstream primer is a universal primer, U6 is selected as an internal reference Real-Time PCR reaction, and ABI QuantStudio is utilized^TMAnd 6, an amplification standard program of a Flex full-functional real-time fluorescence quantitative PCR system. After the reaction is finished, an amplification curve and a dissolution curve of Real Time PCR are prepared, and the expression value of the gene is used

And (4) showing. The result shows that the expression quantity of miR-99b-5P in miR-99b-5P overexpression cells is 5.23 times of that of a negative control group (P)<0.01, fig. 4). miR-99b-5p recombinant lentiviral vector constructed by researchCan effectively infect Jurkat cells and obviously improve the expression level of miR-99b-5p in the cells.

Effect of miR-99b-5p overexpression on Jurkat T cell growth

2.1 CCK-8 cell proliferation assay

Taking miR-99b-5p overexpression cells with good growth state and high positive rate and NC control cells, inoculating the cells and the NC control cells into a 96-well plate (100 ul/well), wherein the cell amount of each well is about 5 multiplied by 10⁴For each cell, 5 parallel rows were established. Four time gradient groups of 0h/12h/24h/48h are sequentially set in the experiment. To prevent errors due to media evaporation, PBS was added to each well at the four sides of the experimental wells (not used as an indicator detection well). A CCK-8 blank (containing medium and CCK-8 only and no cells) was also set up.

The cells are cultured in an incubator for 0h/12h/24h/48h and then taken out, 10ul of CCK-8 solution is added into each hole, and the absorbance during detection is prevented from being influenced by bubbles during sample adding. And then, the culture plate is put back to the incubator to be incubated for 1.5h, and the absorbance at the wavelength of 450nm is detected by an enzyme-linked immunosorbent assay instrument after the incubation is finished.

2.2 Annexin V/PI double-staining apoptosis assay

Inoculating miR-99b-5p over-expression OE cells, NC control cells and normal Jurkat cells with good growth state and high positive rate into 6-well plate (cell amount is not less than 1 × 10)⁶) Each cell was set up in 3 parallel rows. After 24h incubation, centrifugation was carried out at 1000rpm × 5min, and the supernatant was discarded and washed 2 times with PBS. Finally, the cells were resuspended with 1ml of 1 × Annexin V binding buffer. Preparation of dead cells: mixing 100ul Jurkat cells and 100ul NC cells to obtain 200ul Jurkat and NC mixed solution, taking 50ul mixed solution, placing the mixed solution and another 100ul Jurkat cells in a water bath kettle with a constant temperature of 99 ℃ for 5min, and cooling to room temperature to obtain dead cells. The apoptosis experiment was designed as follows:

TABLE 2 Experimental design for apoptosis

The compensation group was set with normal compensation and GFP compensation, which was set with Jurkat and NC (containing GFP) mixed liquor group and its death half group for modulating the effect of GFP fluorescence on apoptosis.

Three parallel rows are set for each cell in the detection group, 5ul PE and 5ul 7-AAD are added according to the design respectively, and after being mixed evenly and mildly, the cells are protected from light and incubated for 15min at room temperature. After the incubation is finished, 400ul of 1 × Annexin V binding buffer is added, and after the mixture is gently mixed, a flow cytometer is used for analyzing the apoptosis.

2.3 PI staining cell cycle test

Taking miR-99b-5p overexpression cells with good growth state and high positive rate and NC control cells, inoculating the cells in a 12-well plate (the cell amount is about 1 × 10)⁵) Each cell was arranged in 3 parallel rows. After preculture for 24h, the cells were pelleted by centrifugation at 1000rpm × 5 min. The supernatant was then carefully aspirated, the cells were washed with 1ml of pre-cooled PBS and resuspended in a 1.5ml centrifuge tube. After centrifugation again, the supernatant was carefully aspirated and gently flicked to disperse the cells properly at the bottom of the tube, preventing cell clumping. 50ul of supernatant was properly retained each time the supernatant was aspirated away to avoid cell loss by aspiration.

1ml of precooled 70% ethanol is added into the washed cell sediment, and the cell sediment is gently blown, beaten, mixed evenly and then fixed in a refrigerator at 4 ℃ overnight. After 24h, the mixture is taken out and centrifuged at 1000rpm × 5 min. After carefully aspirating the supernatant, the cells were washed with 1ml of precooled PBS, centrifuged again and the supernatant was aspirated, gently flicked against the tube wall to prevent cell clumping.

Preparing an propidium iodide staining solution: 535ul of final system contained 500ul of staining buffer, 25ul of propidium iodide staining solution (20X), and 10ul of RNase A (50X). 500ul of the mixed system was added to each tube of the cell sample, and after the cells were slowly and sufficiently resuspended, the mixture was incubated at 37 ℃ for 30min in the dark. Then, the sample was stored on ice for flow detection.

2.4 detection of CD69/CD25 cell Activity

Taking miR-99b-5p overexpression cells with good growth state and high positive rate and NC control cells, inoculating the cells in a 12-well plate (the cell amount is about 1 × 10)⁵) 5ug/ml phytohemagglutinin (PHA; beyotime Biotechnology) or not. Each cell was arranged in 3 parallel rows. After 48h of incubation, cells were collected and washed. Followed by the addition of 3 uLAPC-human CD69 or human CD2Antibody 5 (BioLegend, USA). After incubation for 30 minutes in the absence of light, the positive cell rate of CD69 or CD25 was determined by flow cytometry.

2.5 data analysis

The experimental results show that compared with negative control cells, the cell proliferation activity of miR-99b-5P overexpression cells is remarkably higher than that of a control group (P <0.05, figure 5), and the apoptosis rate is remarkably lower than that of the negative control group (P <0.001, figure 6). Cells were multi-retained in the G2/M phase in the miR-99b-5p overexpression state (FIG. 7), suggesting that miR-99b-5p stimulates cell differentiation and proliferation. CD69 and CD25 are activation markers for T lymphocyte prophase and metaphase, respectively. After PHA stimulation, the positive rates of two antibodies of the miR-99b-5P overexpression cell group and the negative control group are obviously increased (P <0.01, figure 8), and the positive rate of the overexpression group is obviously higher than that of the negative control group (P <0.05, figure 8). In conclusion, miR-99b-5p overexpression can effectively promote cell proliferation of Jurkat T cells, inhibit apoptosis and promote cell activation.

Effect of miR-99b-5p overexpression on inflammatory cytokines

Taking miR-99b-5p over-expression OE group cells with good growth state and high positive rate and negative control NC group cells, inoculating the cells in a 6-well plate, wherein the cell amount of each well is about 5 multiplied by 10⁵And (4) respectively. After 2 days of culture, the cells were collected by centrifugation and cellular RNA was extracted by TRIzol. The RNA was subsequently reverse transcribed to cDNA (Promega GoScript reverse transcription System kit) using the following experimental procedure:

(1) taking a certain amount of template RNA, adding a primer, and reacting in a reaction system (10 ul): x ul RNA; 1ul Random Primers; 1ul Oligo (dT)₁₅Primer；8-x ul Nuclease-Free Water。

The RNA in the system is 2ug, and the corresponding RNA volume and the corresponding water volume are obtained by calculation according to the RNA concentration.

(2) The mixture of template RNA and primers was pre-denatured at 70 ℃ for 5min, and after completion, taken out and placed on ice.

(3) Preparing RT-Mix (10 ul): 1.6ul Nuclear-Free Water; 4ul GoScript^TM5×Reaction Buffer；2ul MgCl₂(25mM)；1ul PCR Nucleotide Mix；0.4ul Recombinant Rnasin Ribonuclease Inhibitor；1ul GoScript^TM Reverse Transcriptase。

(4) Reverse transcription program was set, annealing (25 deg.C, 5min), extension (42 deg.C, 60min), reverse transcriptase inactivation (70 deg.C, 15 min). After completion of the procedure, cDNA was obtained.

The synthesized cDNA was subjected to qPCR using Promega GoTaq qPCR Master Mix in the following reaction scheme (15 ul): 4.5ul Nuclear-Free Water; 1ul of upstream primer (10 uM); 1ul of downstream primer (10 uM); 7.5ul GoTaq qPCR Master Mix, 2 ×; 1ul cDNA.

3 parallels are set in each group of experiment, GAPDH is selected as an internal reference gene, and the expression of cytokines such as IL-1 beta, IL-2, IL-4, IL-6, IL-8, TNF-alpha, IFN-gamma and the like is detected by PCR reaction. Real-Time PCR reaction Using ABI QuantStudio^TMAnd 6, an amplification standard program of a Flex full-functional real-time fluorescence quantitative PCR system. After the reaction, the amplification curve and the dissolution curve of Real Time PCR were obtained, and the expression value of the gene was expressed by 2- Δ CT. The results show that the proinflammatory cytokines IL-2, IL-6, TNF-alpha and IFN gamma are remarkably and highly expressed (P) in miR-99b-5P overexpression cells<0.05, fig. 9), suggesting that miR-99b-5p is involved in regulating the immune inflammatory response in T cells.

Example 4: qPCR kit for diagnosing human rheumatoid arthritis

The above examples show that hsa-miR-99b-5p, hsa-miR-26b-5p and hsa-miR-7641 have high diagnostic value (high sensitivity and specificity) for detecting rheumatoid arthritis peripheral blood mononuclear cells, and therefore, a kit for diagnosing human rheumatoid arthritis can be prepared based on hsa-miR-99b-5p, hsa-miR-26b-5p and hsa-miR-7641. The kit comprises an hsa-miR-99b-5p primer, an hsa-miR-26p-5p primer, an hsa-miR-7641 primer and a universal probe. Each primer specifically comprises a reverse transcription primer, a quantitative PCR pre-primer and a universal quantitative PCR post-primer, and the universal quantitative PCR post-primer and the universal probe are premixed together for convenient use. Of course, the kit should also include conventional enzymes and reagents required for the corresponding PCR reaction, such as reverse transcriptase, buffer, dNTPs, MgCl₂、dd H₂O, fluorescent dye, Taq enzyme, standard substance, control substance and the like. The design of primers and probes is a matter of routine skill in the art and can be designed in other sequencesAnd (4) columns. The kit has the value that only a peripheral blood mononuclear cell sample is needed, and a tissue and peripheral blood mononuclear cell sample is not needed, so that the kit can be used for diagnosing the rheumatoid arthritis.

Example 5: chip kit for diagnosing human rheumatic arthritis

Similarly, chip-based miRNA detection can also be used for diagnosis of rheumatoid arthritis peripheral blood mononuclear cells. The expression of hsa-miR-99b-5p, hsa-miR-26b-5p and hsa-miR-7641 can be detected by manufacturing a chip with a small amount of probes, so that the rheumatoid arthritis diagnosis is performed on the peripheral blood mononuclear cell sample.

1. All designed miRNA probe sequences in the miRNA chip are completely complementary with full-length mature miRNAs detected correspondingly. The miRNA to be detected comprises hsa-miR-99b-5p, hsa-miR-26b-5p and hsa-miR-7641.

2. Total RNA from peripheral blood mononuclear cells was extracted using TRIzol reagent, and small RNA was extracted using the mirNAISATION Kit from Ambion. Small RNAs were labeled using T4DNA ligase labeling technique, i.e., 1. mu.g of small RNA was mixed with 4uL of Flashtag Ligation Mix Biotin (Genisphere) and labeled with T4DNA ligase (New England Biolabs). The labeling reaction was carried out at 25 ℃ for 30 minutes. Taking out after the incubation is finished, performing instantaneous centrifugation to collect the solution at the bottom of the tube, adding the stop solution, fully and uniformly mixing, and placing on ice.

3. The chip hybridization solution was first placed on a constant temperature metal bath, incubated at 99 ℃ for 5 minutes, then at 45 ℃ for 5 minutes, removed, centrifuged at 13200rpm for 5 minutes, and injected into the chip. The chip was then placed in a hybridization oven (hybridization oven 640) at 60rpm and 48 ℃ overnight for 16 hours.

4. And after the hybridization of the chip is finished, washing the chip by using a washing solution, washing and dyeing the chip by using AGCC software, and scanning the washed chip. After scanning with a GeneChip Scanner 3000 Scanner (Affymetrix), AGCC software (see

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 provided human peripheral blood mononuclear cell samples were diagnosed according to Logistic regression analysis equation. Chip detection is carried out on hsa-miR-99b-5P, hsa-miR-26b-5P and hsa-miR-7641 in 35 rheumatoid arthritis and 35 healthy human peripheral blood mononuclear cells, and results show that miRNAs in two groups of human peripheral blood mononuclear cells have significant difference (P-miR-7641)<0.05) as in fig. 3.

In conclusion, according to the technical scheme, the 3 microRNA biomarkers, the corresponding primer sets and the probes 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 a peripheral blood mononuclear cell sample. Further, the inventor of the present invention confirms through research that the AUC value of the combination of the 3 kinds of micrornas can reach 0.745, and the sensitivity and specificity are 73.5% and 76.7%, respectively. The 3 kinds of microRNA can be used as biomarkers for diagnosing human rheumatoid arthritis peripheral blood mononuclear cell samples, and the sensitivity and specificity of combined diagnosis are higher than those of single microRNA diagnosis, so that the development of early diagnosis, prediction treatment and relapse monitoring of rheumatoid arthritis in China can be promoted.

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.

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Claims (10)

1. A microRNA biomarker for diagnosing rheumatoid arthritis is characterized in that the microRNA biomarker is a combination of has-miR-99b-5p, has-miR-26b-5p and has-miR-7641.

2. The microRNA biomarker of claim 1, wherein the nucleotide sequence of the hsa-miR-99b-5p is represented by SEQ ID NO: 1, and the nucleotide sequence of the has-miR-26b-5p is shown as SEQ ID NO: 2, the nucleotide sequence of the has-miR-7641 is shown as SEQ ID NO: 3, respectively.

3. The microRNA biomarker of claim 1, wherein the has-miR-99b-5p, has-miR-26b-5p and has-miR-7641 are derived from peripheral blood mononuclear cells.

4. A kit for diagnosing rheumatoid arthritis is characterized by comprising a microRNA primer group and a microRNA probe which are used for detecting the expression quantity of a combination of has-miR-99b-5p, has-miR-26b-5p and has-miR-7641 in peripheral blood mononuclear cells.

5. The kit according to claim 4, wherein the microRNA primer set comprises reverse transcription primers, pre-PCR primers and post-PCR primers of microRNA.

6. The kit of claim 5, wherein the reverse transcription primer sequence of hsa-miR-99b-5p is shown in SEQ ID NO: 4, the sequence of the primer before PCR is shown as SEQ ID NO: 7, the primer sequence after PCR is shown as SEQ ID NO: 10 is shown in the figure; the reverse transcription primer sequence of has-miR-26b-5p is shown in SEQ ID NO: 5, the sequence of the primer before PCR is shown as SEQ ID NO: 8, the primer sequence after PCR is shown as SEQ ID NO: 10 is shown in the figure; the reverse transcription primer sequence of has-miR-7641 is shown as SEQ ID NO: 6, the sequence of the primer before PCR is shown as SEQ ID NO: 9, the primer sequence after PCR is shown as SEQ ID NO: shown at 10.

7. The kit of claim 5, further comprising a conventional component of a qPCR amplification assayConventional modules include reverse transcriptase, buffer, dNTPs, MgCl₂、dd H₂O, fluorescent dye, Taq enzyme, standard and control.

8. A chip for diagnosing rheumatoid arthritis is characterized by comprising a microRNA probe for detecting the combination of has-miR-99b-5p, has-miR-26b-5p and has-miR-7641.

9. The chip of claim 8, wherein the nucleotide sequence of the microRNA probe is completely complementary to the full-length mature microRNA of the detection object of the microRNA probe.

10. Use of the microRNA biomarker of any one of claims 1 to 3 in the preparation of a product for detecting rheumatoid arthritis.

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