Application of combination of circular RNA and targeting ETS-1 as rheumatoid arthritis marker

Abstract

The invention discloses an application of a combination of circular RNA and targeted ETS-1 as a rheumatoid arthritis marker. The nucleotide sequence of the circular RNAcirc-CBLB is SEQ ID NO. 1, and the nucleotide sequence of the circular RNAcirc-CBLB is SEQ ID NO. The nucleotide sequence of the target ETS-1 is shown as SEQ ID NO. 2. The combination of the circular RNAcirc-CBLB and the targeted ETS-1 can be used as a marker for diagnosing RA, can evaluate RA inflammation and disease activity degree, and proves that the circular RNAcirc-CBLB and the targeted ETS-1 can be used for treating inflammation-related rheumatism.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and specifically to the application of a combination of circular RNA and a target ETS-1 as a biomarker for rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is an autoimmune disease mainly manifested by chronic and invasive arthritis. Clinical manifestations often include swelling, pain, deformity, and even loss of function in small joints such as the symmetrical hands and wrists, with a high disability rate. If not treated regularly, it can affect the lungs and kidneys in the later stage, causing irreversible serious damage. At the same time, the pathogenesis of RA is still unclear. The pathophysiological changes of RA occur throughout the body without treatment. The lack of effective early diagnosis and treatment can lead to severe disability and even death. Currently, the clinical diagnosis of RA is mainly based on blood tests and clinical symptoms, and it is often difficult to diagnose in the early stage, thus delaying the condition. Early diagnosis and control of the disease activity of RA can significantly improve the prognosis of patients. Exploring more laboratory test items that reflect the disease activity is of great significance for the evaluation of the condition. Circular RNA (circRNA) and transcription factors (TFs) are involved in regulating the physiological and pathological processes of various diseases, such as inflammatory arthritis diseases like osteoarticular diseases. The "inflammatory polarization" of RA is of great significance, among which macrophages are involved in the occurrence and development of RA. After polarization, M1 macrophages secrete a large amount of pro-inflammatory cytokines (such as IL-1β, IL-6, IL-23, TNF-α, etc.), chemokines, etc., which activate fibroblasts and osteoclasts, recruit inflammatory cells, and trigger a series of inflammatory reactions, resulting in joint cartilage damage. M2 macrophages highly express anti-inflammatory cytokines (such as IL-4, IL-10, etc.) and play an anti-inflammatory role. Effectively intervening in M1 macrophages and converting them into M2 macrophages to restore the dynamic balance of M1 / M2 is beneficial to promoting the regression of RA inflammation and the repair of synovial joint tissues.

[0003] The imbalance mechanism of RA "inflammatory polarization" is related to the abnormal activation of circular RNA (circRNA), transcription factors (TFs), and signaling pathways. CircRNA can regulate the inflammatory polarization state. Therefore, it is of great significance to study RA "inflammatory polarization" from the perspective of circRNA / TF. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of a combination of circular RNA and a target ETS-1 as a biomarker for rheumatoid arthritis to solve the problems raised in the above background art.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] Use of a reagent for quantitatively detecting the combination of circular RNA and targeted ETS-1 in the preparation of a reagent for diagnosing rheumatoid arthritis, wherein the nucleotide sequence of the circular RNA circ-CBLB is SEQ ID NO.1;

[0007] The nucleotide sequence of the targeted ETS-1 is SEQ ID NO.2.

[0008] As a further aspect of the present invention, the reagent comprises:

[0009] A forward primer for circular RNA circ-CBLB, with the sequence 5'-TCCTGCTAGAAGGTTA CCAG-3';

[0010] A reverse primer for circular RNA circ-CBLB, with the sequence 5'-TTGCTAACGGACCAGT ACAC-3'.

[0011] As a further aspect of the present invention, it is characterized in that the reagent comprises:

[0012] A forward primer for targeted ETS-1, with the sequence 5'-CCATTCTGGAGAGGGACTTC-3';

[0013] A reverse primer for targeted ETS-1, with the sequence 5'-TGCTGTAAAACCCAGAGTGT-3'.

[0014] As a further aspect of the present invention, the reagent comprises a reagent applicable to at least one of the following methods: fluorescence dye method, digital PCR, resonance light scattering method, real-time fluorescence quantitative PCR, sequencing or biomass spectrometry.

[0015] In the early stage, the inventors of the present application screened differentially expressed circRNAs and mRNAs based on peripheral blood macrophages of RA patients using high-throughput gene sequencing. The results showed that through circRNAs gene sequencing analysis, a total of 165 differentially expressed circRNAs (fold change ≥ 2 and P ≤ 0.05) were found, with 109 up-regulated and 56 down-regulated. Real-time quantitative fluorescence PCR was used for verification, and the results were consistent with the sequencing results. Among them, the circular RNA Cbl proto-oncogene B (circ-CBLB) had the most obvious expression difference, and subsequent in-depth research was carried out. Bioinformatics GO and KEGG pathway analysis were used to analyze the mRNAs targeted by hsa_circ_0066715 (circ-CBLB), and it was found that the direct target gene of hsa_circ_0066715 (circ-CBLB) was E26 transformation-specific sequence-1 (ETS-1). The two are expected to become new intervention targets for the diagnosis and prevention of RA disease.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The combination of circular RNA circ-CBLB and the targeted ETS-1 in the present invention can be used as a biomarker for diagnosing RA, and can evaluate the degree of RA inflammation and disease activity, and it is confirmed that the two can be used to treat rheumatism related to inflammation.

[0017] The functional axis formed by circular RNA circ-CBLB and the targeted ETS-1 forms a targeted regulatory relationship. Overexpression and interference are carried out on it, and WB method and RT-qPCR method are used for detection and verification, as well as correlation analysis to verify the functional axis; In RA patients, the expression levels of circular RNA circ-CBLB and the targeted ETS-1 decrease, and the two are positively correlated. The inflammatory indicators ESR and CRP increase, the specific diagnostic antibodies RF and CCP increase, and the M1-type inflammatory pro-inflammatory cytokines IL-23 and TNF-α in macrophages increase, while the M2-type inflammatory anti-inflammatory cytokine IL-4 decreases.

[0018] Circular RNA circ-CBLB is positively correlated with the targeted ETS-1 and the M2-type inflammatory anti-inflammatory cytokine IL-4. Circular RNA circ-CBLB, the targeted ETS-1 are negatively correlated with the inflammatory indicators ESR, CRP, specific diagnostic antibodies RF, CCP, disease activity DAS28, and M1-type inflammatory pro-inflammatory cytokines IL-23 and TNF-α. Brief Description of the Drawings

[0019] Figure 1 It is a graph showing the expression levels of circ-CBLB and ETS-1 in peripheral blood PBMCs of RA patients and healthy controls (HC);

[0020] Figure 2 Results diagram of the targeting relationship between circ-CBLB and ETS-1;

[0021] Figure 3 Results diagram of the expression of M1 and M2 cytokines in RA patients;

[0022] Figure 4 Results diagram of the correlation between the expression level of circ-CBLB and RA inflammation-related indicators in RA patients;

[0023] Figure 5 Results diagram of the correlation between the expression level of circ-CBLB and RA diagnosis-related antibodies in RA patients;

[0024] Figure 6 Results diagram of the correlation between the expression level of ETS-1 and RA inflammation-related indicators in RA patients;

[0025] Figure 7 Results diagram of the correlation between the expression level of ETS-1 and RA diagnosis-related antibodies in RA patients;

[0026] Figure 8 Results diagram of the relationship between the expression levels of circ-CBLB and ETS-1 and the RA disease activity DAS28 in RA patients. Specific implementation manners

[0027] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0028] I. Clinical research

[0029] Fifteen RA patients included were from the outpatient and inpatient departments of the Rheumatology Department of Anhui Provincial Hospital of Traditional Chinese Medicine, and fifteen healthy individuals (HC group) were from the physical examination center of Anhui Provincial Hospital of Traditional Chinese Medicine. All the included subjects signed the informed consent form. The RA diagnosis criteria refer to the RA classification criteria and scoring system revised by ACR and EULAR in 2010 and ACR in 1987.

[0030] In the HC group, there were 8 males and 22 females, with an average age of (43.69 ± 5.23) years. In the RA group, there were 8 males and 22 females, with an average age of (46.98 ± 4.00) years. There were no statistically significant differences in the age (t = -1.940, P = 0.063) and gender (χ 2 = 0.000, P = 1.000) between the two groups of subjects (P > 0.05), and they were comparable.

[0031] Detection index: 4 mL of fasting forearm venous blood was collected from the enrolled subjects using an erythrocyte sedimentation rate tube (black tube), and PBMCs were extracted using Ficoll and stored in a -80 °C refrigerator for later use.

[0032] The Erythrocyte Sedimentation Rate (ESR) was detected using an automatic erythrocyte sedimentation analyzer (Vital Monitor-20); for biochemical indices such as c-reactive protein (CRP), Rheumatoid Factor (RF), and Anti-Cyclic Citrullinated Peptide Antibody (CCP): 5 mL of forearm venous blood was collected from the enrolled subjects using a biochemical tube (yellow tube). After low-temperature coagulation at 3 °C - 5 °C, it was centrifuged at 4000 r / min for 15 min in a centrifuge. After separating the serum, it was stored in a -80 °C refrigerator. After all the specimens were collected, they were detected using an automatic biochemical analyzer (HITACHI 7600-020), and the 28-joint Disease Activity Score (DAS28) of RA patients was calculated.

[0033] II. In vitro study

[0034] 1. Cell culture: FLS cells and RA-FLS cells (iCell, Shanghai, China) were taken out of the incubator, and the culture medium (iCell, Shanghai, China) was discarded; after washing twice with PBS (Hyclone, Shanghai, China), 1 mL of trypsin (Beyotime, Shanghai, China) was added and digested at 37 °C for 2 minutes; when the cells were significantly shrunk and rounded, the digestion was terminated and fresh culture medium was added; the cells were pipetted to make them detached and collected into a 15 mL centrifuge tube, and centrifuged at 1000 r / min for 5 minutes; subculture was performed at a ratio of 1:1 or 1:2 according to the cell number, and subculture was carried out once every 2 days. After subculturing to the 7th generation, the next relevant experimental operations were carried out.

[0035] 2. Cell transfection: After digesting RA-FLS cells, centrifuge to discard the culture medium, wash twice with PBS, resuspend with the medium, and seed 5×10^5 cells per well into a 6-well plate. Incubate overnight in a 37°C, 5% CO2 cell culture incubator; after observing that the cell confluence reaches 70% - 80%, perform transfection on each group of cells; Add 125 μL of DEPC water (Generay Biotech, Shanghai, China) to the freeze-dried powders of pcDNA3.1 / siRNA-circCBLB, ETS-1, and negative control (pcDNA3.1-NC / si-NC), and the diluted concentration is 20 μmol / L; Add 5 μL of 20 μmol / L pcDNA 3.1 / siRNA / NC to 250 μL of serum-free medium and mix well; Take 5 μL of Lipofectamine TM 2000 (Invitrogen, Carlsbad, CA, USA) and dilute it in 250 μL of serum-free medium, and incubate at room temperature for 5 minutes; Mix the two solutions and Lipofectamine TM 2000, and place at room temperature for 20 minutes; Add 500 μL of the transfection solution to each well of cells, and detect the transfection efficiency by real-time quantitative PCR; 24 hours after transfection, digest the cells with 2.5 g / L trypsin, wash twice with PBS, collect the cell pellet, and store it in an -80°C refrigerator for subsequent experiments; The circ-CBLB overexpression plasmid, circ-CBLB, ETS-1 small interfering RNAs, and related negative controls were all constructed by Shanghai GenePharma.

[0036] 3. Detection of related protein expression by Western Blotting: Cell samples of each group were collected, and total proteins were extracted using RIPA lysis buffer containing PMSF (Beyotime, Shanghai, China). Subsequently, proteins were separated by SDS-PAGE gel. SDS (Solarbio, Beijing, China) and PAGE gel accelerator (Solarbio, Beijing, China) from Solarbio were used for electrophoresis. Then, the proteins were transferred onto a PVDF membrane (Millipore, Bedford, MA, USA), and the transfer time for ETS-1 (49 kDa) was 25 - 50 minutes. After transfer, the membrane was blocked with 5% non-fat milk powder (Beyotime, Shanghai, China) at room temperature for 2 hours to block non-specific binding. Subsequently, specific primary antibodies were used for overnight incubation at 4°C: ETS-1 (rabbit anti, Bioss, Beijing, China). After incubation, the membrane was washed three times with PBST buffer (Zs-Bio, Beijing, China) for 10 minutes each time. Then, a goat anti-rabbit secondary antibody labeled with horseradish peroxidase (HRP) diluted 1:20,000 (Zsbio, Beijing, China) was used for incubation at room temperature for 1.2 hours, and washed three times again. Finally, protein bands were detected using an ECL hypersensitive luminescence kit (Thermo Corp, Waltham, MA, USA), and quantitative analysis was performed using ImageJ software to ensure the reliability and accuracy of the experimental results.

[0037] 4. Detection of related gene expression by real-time quantitative PCR:

[0038] 4.1 RNA extraction

[0039] 1) Cell pellets were collected and lysed by adding 1 ml of TRIzol.

[0040] 2) After complete lysis, 0.2 mL of chloroform was added, and the mixture was vigorously shaken for 15 seconds and left at room temperature for 5 minutes.

[0041] 3) Centrifuge at 12,000 rpm at 4°C for 10 minutes, and take the supernatant (about 500 μl) and transfer it to another EP tube.

[0042] 4) Add 0.5 mL of pre-cooled isopropanol, gently mix, and incubate at -20°C for 30 minutes.

[0043] 5) Centrifuge at 12,000 rpm at 4°C for 15 minutes and discard the supernatant.

[0044] 6) Add 1 mL of pre-cooled 75% ethanol (anhydrous ethanol diluted with DEPC water). Centrifuge at 12,000 rpm at 4°C for 5 minutes and discard the supernatant.

[0045] 7) Repeat step 6).

[0046] 8) Dry the RNA pellet at room temperature, add 20 - 50 μL of DEPC water, and store it at -80 °C for later use.

[0047] 4.2 RT Reaction

[0048] 1) Genomic DNA removal reaction: In a 0.2 mL EP tube

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[0050]

[0051] Gently mix and briefly centrifuge.

[0052] 2) Heat at 42 °C for 2 min on a PCR instrument and immediately ice-bath for 1 min.

[0053] 3) Add to the above EP tube

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[0055] 4) 37 °C, 15 min; 85 °C, 5 s.

[0056] 5) Take out the above reaction solution, which is the cDNA, and store it at -80 °C for later use.

[0057] 4.3 Fluorescent Quantitative PCR Reaction

[0058] 1) Take out the cDNA as the template for fluorescent quantitative PCR, and the reaction system is as follows:

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[0060] 2) The reaction conditions are as follows:

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[0063] Quantitative analysis is performed according to the 2 -ΔΔC t method. The primers used in this study are shown in Table 1.

[0064] Table 1: List of primer sequences used in each PCR reaction system.

[0065]

[0066] Among them, the sequence of β-actin is SEQ ID NO.3, which is used as an internal reference;

[0067] The sequence of circ-CBLB is: SEQ ID NO.1

[0068] The sequence of ETS-1 is: SEQ ID NO.2

[0069] 5. Detection of related macrophage inflammatory factor levels by ELISA

[0070] The levels of cytokines M1 type IL-4, IL-23, and M2 type TNF-α in peripheral blood cells of the subjects were detected by ELISA, and the operation was completed according to the instructions of the kit (Gene Mei, Wuhan, China).

[0071] 6. Statistical methods

[0072] SPSS 23.0 was used to analyze the data, Graphpad 9.0 was used for chart drawing. Measurement data were expressed as mean ± standard deviation. Independent sample t-test was used for comparison between two groups, one-way ANOVA was used for comparison among multiple groups, and Pearson test or Spearman test was used for correlation analysis. All statistical tests were two-sided tests. P < 0.05 was considered as having statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001.

[0073] 6. Result description

[0074] Figure 1 It reflects the expression levels of circ-CBLB and ETS-1 in peripheral blood PBMC of RA patients and healthy controls (HC). The results of real-time quantitative PCR showed that: compared with the HC group, the expression levels of circ-CBLB mRNA and ETS-1 mRNA in the RA group decreased (P < 0.01), where A is circ-CBLB and B is ETS-1.

[0075] Figure 2 It reflects the targeting relationship between circ-CBLB and ETS-1. The expression level of circ-CBLB in RA patients was positively correlated with the expression level of ETS-1 (P < 0.01). The results of real-time quantitative PCR showed that: compared with FLS, the expression level of ETS-1 mRNA in RA-FLS decreased (P < 0.01); compared with RA-FLS, the expression level of ETS-1 mRNA in the overexpression group increased (P < 0.01); the expression level of ETS-1 mRNA in the interference group decreased (P < 0.05); the expression level of ETS-1 mRNA in the overexpression interference group decreased (P < 0.01); compared with the overexpression interference group, the expression level of ETS-1 mRNA in the interference group decreased (P < 0.01), and the expression level of ETS-1 mRNA in the overexpression group increased (P < 0.01).

[0076] WB results showed that the expression level of ETS-1 protein in RA-FLS was decreased compared with that in FLS (P<0.01). By constructing the overexpression plasmid pcDNA3.1-circ-CBLB and its negative control, and small interfering siRNA-circ-CBLB and its negative control, it was found that the expression level of ETS-1 protein in the overexpression group was increased compared with that in RA-FLS (P<0.01), and the expression level of ETS-1 protein in the interference group was decreased compared with that in RA-FLS (P<0.01). Among them, A was the correlation analysis between the two, B was ETS-1 protein, C was the semi-quantitative analysis of ETS-1 protein, and D was the semi-quantitative analysis of ETS-1 mRNA.

[0077] Figure 3 Reflected the expression of M1 and M2 type cytokines in RA patients. ELISA results showed that: compared with the HC group, the expression level of IL-4 in the RA group was decreased, while the expression levels of IL-23 and TNF-α were increased (P<0.01). Among them, A was IL-4, B was IL-23, and C was TNF-α.

[0078] Figure 4 Reflected the correlation between the expression level of circ-CBLB in RA patients and RA inflammation-related indicators. The correlation analysis results showed that circ-CBLB was positively correlated with IL-4 (P<0.05), and circ-CBLB was negatively correlated with ESR, CRP, IL-23, and TNF-α (P<0.01). Among them, A was ESR, B was CRP, C was IL-4, D was IL-23, and E was TNF-α.

[0079] Figure 5 Reflected the correlation between the expression level of circ-CBLB in RA patients and RA diagnosis-related antibodies. The correlation analysis results showed that circ-CBLB was negatively correlated with RF and CCP (P<0.01). Among them, A was CCP and B was RF.

[0080] Figure 6 Reflected the correlation between the expression level of ETS-1 in RA patients and RA inflammation-related indicators. The correlation analysis results showed that ETS-1 was negatively correlated with ESR, CRP, IL-23, and TNF-α (P<0.05, P<0.01). Among them, A was ESR, B was CRP, C was IL-4, D was IL-23, and E was TNF-α.

[0081] Figure 7 Reflected the correlation between the expression level of ETS-1 in RA patients and RA diagnosis-related antibodies. The correlation analysis results showed that ETS-1 was negatively correlated with CCP and RF (P<0.05, P<0.01). Among them, A was CCP and B was RF.

[0082] Figure 8 It reflects the relationship between the expression levels of circ-CBLB and ETS-1 in RA patients and the disease activity DAS28 of RA. The results of the correlation analysis show that: circ-CBLB is negatively correlated with DAS28 (P<0.01), and ETS-1 is negatively correlated with DAS28 (P<0.01); for circ-CBLB and ETS-1, values below the average of DAS28 are 1, and values above the average are 2. Drawing the ROC curve shows that the areas under the curves (AUC) of circ-CBLB and ETS-1 are 0.747 (P = 0.021) and 0.809 (P = 0.004) respectively, and the 95% CIs are 0.5705 - 0.9229 and 0.6486 - 0.9692 respectively. circ-CBLB and ETS-1 can be used as markers for predicting high disease activity in RA patients. A and B are for the correlation analysis, and C and D are for the ROC prediction curves.

[0083] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claimed rights.

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Claims

1. Use of a reagent for quantitatively detecting a combination of circular RNA and targeting ETS-1 in the preparation of a diagnostic reagent for rheumatoid arthritis, characterized in that: The nucleotide sequence of the circular RNAcirc-CBLB is SEQ ID NO.1; The nucleotide sequence targeting ETS-1 is SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The reagents include: The forward primer of circular RNAcirc-CBLB had the sequence of 5′-TCCTGCTAGAAGGTTA CCAG-3′; The reverse primer of circular RNA circ-CBLB had the sequence 5′-TTGCTAACGGACCAGT ACAC-3′.

3. The use according to claim 1, characterized in that: The reagents include: The forward primer targeting ETS-1 had the sequence 5′-CCATTCTGGAGAGGGACTTC-3′; The reverse primer targeting ETS-1 had the sequence 5'-TGCTGTAAAACCCAGAGTGT-3'.

4. The use according to claim 1, characterized in that: The reagents include reagents suitable for at least one of the following methods: fluorescent dye method, digital PCR, resonance light scattering method, real-time fluorescence quantitative PCR, sequencing or biological mass spectrometry.

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