Application of circ-CBLB and targeted TLR3 combination in preparation of medicine for diagnosing and treating rheumatoid arthritis

Abstract

The invention discloses application of a combination of circ-CBLB and targeted TLR3 in preparation of a medicine for diagnosing and treating rheumatoid arthritis. The nucleotide sequence of the circ-CBLB is 5 '-TCAGCTTCCTCATGTT CAGGT-3', and the nucleotide sequence of the circ-CBLB and the nucleotide sequence of the targeted TLR3 are shown in the description. The nucleotide sequence of the targeted TLR3 is 5 '-GCCATGAAGTTGCTGACTG C-3', and the nucleotide sequence of the targeted TLR3 is 5 '- The nucleotide sequence of the circ-CBLB forward primer is 5 '-TCAGCTTCCTCATGTTCA GGT-3', and the nucleotide sequence of the circ-CBLB forward primer is 5 '-TCAGCTTCCTCATGTTCA GGT-3'; the nucleotide sequence of the reverse primer of the circ-CBLB is 5 '-TGCTAACGGACCA GTACACCT-3', and the nucleotide sequence of the reverse primer of the circ-CBLB is 5 '- The nucleotide sequence of the forward primer of the TLR3 is 5 '-GCCATGAAGTTG CTGACTGC-3', and the nucleotide sequence of the forward primer of the TLR3 is 5 '-GCCATGAAGTTG The nucleotide sequence of the reverse primer of the TLR3 is 5 '-TGGCGGCTGGT AATCTTCTG-3', and the nucleotide sequence of the reverse primer of the TLR3 is shown in the description. The circ-CBLB and the targeted TLR3 have an obvious enrichment trend, the circ-CBLB and the targeted TLR3 can be combined, the targeted TLR3 is a target of the circ-CBLB, and expression of M1 macrophage markers CD80 and CD86 can be affected by interfering the circ-CBLB and the targeted TLR3, so that evidence is provided for using the circ-CBLB and targeted TLR3 combination as a rheumatoid arthritis immune inflammation marker.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of the combination of circ-CBLB and a TLR3 target in the preparation of drugs for the diagnosis and treatment of rheumatoid arthritis. Background Art

[0002] Rheumatoid Arthritis (RA) is an autoimmune disease, which is clinically often manifested as joint swelling, pain, deformity, and even involving internal organs. The pathological features of RA include abnormal inflammatory hyperplasia of synovial tissue, angiogenesis, pannus formation, and irreversible damage to articular cartilage and bone. Currently, the etiology and pathogenesis of RA are considered to be related to abnormal immune responses, genetic factors, and environmental factors, but they are still not clear. Given the complexity of the etiology and pathogenesis of RA, it is difficult to make an accurate diagnosis in the early stage of the disease, which also affects the treatment effect. Therefore, finding biomarkers for early diagnosis, targeted treatment, and prognosis evaluation of RA is of great significance for the treatment and prognosis of RA.

[0003] circ-CBLB, that is, circular RNA Cbl proto-oncogene B, plays an important role in rheumatoid arthritis. In the pathogenesis of RA, the abnormal proliferation and insufficient apoptosis of synovial fibroblasts play a key role. Studies have shown that circ-CBLB can inhibit the proliferation of RA-FLS, promote its apoptosis, and increase the levels of anti-inflammatory cytokines such as IL-4 and IL-10, while reducing the levels of pro-inflammatory cytokines such as IL-6 and TNF-α.

[0004] Toll-like receptor 3 (TLR3) is a key member of the Toll-like receptor (TLRs) family, which is mainly expressed in immune cells and various non-immune cells and plays a crucial role in nucleic acid pattern recognition. TLR3 is a nucleotide-sensing TLR that can recognize double-stranded RNA (dsRNA). When TLR3 binds to the ligand dsRNA, it undergoes dimerization and conformational changes, and then recruits the TIR domain-containing adaptor protein (TRIF), initiating a series of signal cascades, activating the transcription factors NF-κB and interferon regulatory factor 3 (IRF3), thereby inducing inflammatory cells to release inflammatory factors and mediating the inflammatory response, and at the same time promoting the release of type I interferon and playing an immune role. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of the combination of circ-CBLB and a TLR3 target in the preparation of drugs for the diagnosis and treatment of rheumatoid arthritis, so as to solve the problems raised in the above background art.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] Use of circ-CBLB in combination with a TLR3 target in the preparation of a medicament for the diagnosis and treatment of rheumatoid arthritis, characterized in that the nucleotide sequence of the circ-CBLB is 5'-TCAGCTTCCTCATGTT CAGGT-3'; the nucleotide sequence of the TLR3 target is 5'-GCCATGAAGTTGCTGACTG C-3'.

[0008] Preferably, the nucleotide sequence of the forward primer of the circ-CBLB is 5'-TCAGCTTCCTCAT GTTCAGGT-3'; the nucleotide sequence of the reverse primer of the circ-CBLB is 5'-TGCTAACGG ACCAGTACACTT-3'.

[0009] Preferably, the nucleotide sequence of the forward primer of the TLR3 is 5'-GCCATGAAGTTGCT GACTGC-3'; the nucleotide sequence of the reverse primer of the TLR3 is 5'-TGGCGGCTGGTAAT CTTCTG-3.

[0010] Preferably, the method for detecting the combination of the circ-CBLB and the TLR3 target in the medicament for the diagnosis and treatment of rheumatoid arthritis includes real-time fluorescence quantitative PCR, RNA pull down experiment and / or immunoblotting method.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: In the pathogenesis of rheumatoid arthritis (RA), the TLR3 signaling pathway plays an important role. The TLR3 target is up-regulated in RFLS, and its activation leads to the production of interferon-β (IFN-β), inducing and participating in the innate immune response; there is an obvious enrichment trend between the circ-CBLB and the TLR3 target in the present invention, indicating that the two can bind, and the TLR3 target is the target of the circ-CBLB. By interfering with the circ-CBLB and the TLR3 target, the expression of M1 macrophage markers CD80 and CD86 can be affected, which provides evidence for the circ-CBLB and TLR3 target combination as an immune inflammation marker for rheumatoid arthritis. Brief Description of the Drawings

[0012] Figure 1 It is a graph showing the results of the expression of circCBLB in healthy people and RA patients;

[0013] Figure 2 It is a graph showing the correlation results of circ-CBLB with immune inflammation indexes ESR, CRP, CCP, and RF in RA;

[0014] Figure 3 It is a correlation result diagram of circ-CBLB with the M1 macrophage marker CD86 and the M2 macrophage marker CD163;

[0015] Figure 4 It is a correlation result diagram of the contents of TNF-a, IL-6, IL-13, and IL-10 in the co-culture system of macrophages and RA-FLS;

[0016] Figure 5 It is a correlation result diagram of the contents of TLR3 and TRAF3 in the co-culture system;

[0017] Figure 6 It is a correlation result diagram of the enrichment of circ-CBLB and TLR3;

[0018] Figure 7 It is an expression result diagram of the M1 macrophage markers CD80 and CD86 after intervening in circ-CBLB. Specific implementation manners

[0019] 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.

[0020] The present invention proposes the application of circ-CBLB and the combination targeting TLR3 in the preparation of drugs for the diagnosis and treatment of rheumatoid arthritis. The nucleotide sequence of the circ-CBLB is 5'-TCAGCTTCCTCATGTT CAGGT-3'; the nucleotide sequence of the TLR3-targeting is 5'-GCCATGAAGTTGCTGACTG C-3'.

[0021] The present invention proposes a pair of primer pairs for detecting circ-CBLB. The nucleotide sequence of the forward primer of the circ-CBLB is 5'-TCAGCTTCCTCATGTTCAGGT-3'; the nucleotide sequence of the reverse primer of the circ-CBLB is 5'-TGCTAACGGACCAGTACACTT-3'.

[0022] The present invention proposes a pair of primer pairs for detecting the TLR3-targeting. The nucleotide sequence of the forward primer of the TLR3 is 5'-GCCATGAAGTTGCTGACTGC-3'; the nucleotide sequence of the reverse primer of the TLR3 is 5'-TGGCGGCTGGTAATCTTCTG-3.

[0023] In the present invention, the detection method of the rheumatoid arthritis diagnosis and treatment drug for the combination of circ-CBLB and the targeted TLR3 includes real-time fluorescence quantitative PCR, RNA pull down experiment and / or immunoblotting method.

[0024] 1. RNA pull down experiment

[0025] Perform an RNA pull-down experiment using an RNA pull-down kit to adsorb the proteins that interact with RNA. The method is as follows:

[0026] 1.1 Cell total protein extraction

[0027] 1) Collect 2×10 7 cell samples and wash them once with PBS;

[0028] 2) Add 1.7 mL of <06>RIP buffer and 17 μL of <21>protease inhibitor, vortex and mix well, incubate on ice for 10 min, vortex and mix again, place at -80 °C for 10 min, and then thaw on ice;

[0029] 3) Centrifuge at 15000 g for 15 min at 4 °C, and transfer the supernatant to a new RNase-free tube.

[0030] 1.2 Removal of nucleic acids and pre-washing of protein samples

[0031] 1) Add 20 μL of <22>DNase and 8.5 μL of <23>DNase salt stock to the above-prepared protein sample, and gently incubate at room temperature (25 °C) for 1 h;

[0032] 2) Add 40 μL of <07>Agarose beads, 8.5 μL of <08>EDTA, 3.4 μL of <09>EGTA, and 17 μL of <24>DTT to the protein sample, and gently rotate at 4 °C for 30 min;

[0033] 3) Centrifuge at 3000 - 5000 g for 1 min at room temperature, and transfer the supernatant to a new centrifuge tube;

[0034] 4) Take 100 μL of the supernatant as the Input group, and divide the remaining supernatant into two tubes at 0.8 mL / tube as RPD and NC samples (labeled RPD and NC respectively), and store them at -80 °C for standby.

[0035] 1.3 RNA secondary structure formation

[0036] 1) Take the corresponding mass of biotin-labeled target RNA probe and NC probe according to the mass ratio of 1 μg / 1000 nt length, add <02>RNase-free water to make up to 30 μL, and denature in a 90 °C water bath for 2 min;

[0037] 2) Quickly transfer and let stand in an ice bath for 2 min;

[0038] 3) Add 50 μL of <01>RNA structure buffer and 20 μL of <02>RNase-free water respectively;

[0039] 4) Incubate at room temperature for 20 min to form RNA secondary structure.

[0040] 1.4. Probe-magnetic bead preparation

[0041] 1) Take 40 μL of <03>Streptavidin magnetic beads respectively and place them in two RNase-free centrifuge tubes. Add 1 mL of <04>1×TES to wash the magnetic beads, and let stand on a magnetic stand for 1 min to remove the TES washing solution;

[0042] 2) Add the RNA probe with secondary structure (about 100 μL), 100 μL of <02>RNase-free water and 200 μL of <05>2×TES to the magnetic beads respectively, and incubate with rotation at 25 °C for 30 min. Let stand on a magnetic stand for 1 min to remove the supernatant;

[0043] 3) Add 0.5 mL of <04>1×TES respectively, gently rotate to wash the magnetic beads, let stand on a magnetic stand for 1 min to remove the supernatant washing solution, and repeat once.

[0044] 1.5. RNA pull down

[0045] 1) Mix the probe-magnetic bead complex with the cell extract and add 5 μL of <25>RNase inhibitor and 5 μL of <26>Yeast tRNA respectively, and gently rotate and bind at room temperature (25 °C) for 2 h;

[0046] 2) Let stand on a magnetic stand for 1 min to collect the magnetic beads and remove the supernatant;

[0047] 3) Add 1 mL of ice-cold <10>NT2 buffer respectively, mix well and wash at 4 °C for 5 min. Let stand on a magnetic stand for 1 min to collect the magnetic beads and remove the supernatant;

[0048] 4) Repeat washing the magnetic beads four times.

[0049] 1.6. Elution of protein products

[0050] 1) Add 60 μL of <11>Protein elution buffer and 0.6 μL of <24>DTT to the magnetic beads in the RPD and NC groups, incubate at 37 °C for 2 h for elution, mix well at intervals, let stand on a magnetic stand for 1 min, collect the magnetic beads, and transfer the supernatant to a new centrifuge tube;

[0051] 2) The protein samples can be stored at -80 °C for later use.

[0052] 2. WB Verification

[0053] 2.1. Preparation of SDS-PAGE Gel

[0054]

[0055] 2.2. Loading and Electrophoresis

[0056] The protein samples can be directly loaded into the sample wells of the SDS-PAGE gel; the voltage used for the stacking gel is 80 v and the time is 30 min; the voltage used for the separating gel is 120 v and the time is 1 h; the electrophoresis time can be adjusted appropriately.

[0057] 2.3. Blotting

[0058] Cut the filter paper and PVDF membrane (pre-soaked in methanol for 3 minutes) to the same size as the gel strip in advance, immerse them in the transfer buffer for 5 minutes; place the transfer device in order from top to bottom as anode plate, 3 layers of filter paper, PVDF membrane, gel, 3 layers of filter paper, cathode plate, align the filter paper, gel, and PVDF membrane precisely, and remove air bubbles at each step; connect the power supply and transfer at a constant current of 300 mA.

[0059] 2.4. Blocking

[0060] After blotting, immediately place the protein membrane into the pre-prepared Western washing solution, rinse for 5 minutes to wash off the transfer solution on the membrane; add Western blocking solution (5% non-fat milk powder), gently shake on a shaker, and block at room temperature for 2 hours.

[0061] 2.5. Incubation with Primary Antibody

[0062] Refer to the instruction manual of the primary antibody, dilute it with the primary antibody diluent at a ratio of 1:1000, incubate with gentle shaking at 4 °C overnight; add the washing solution (PBST), wash for 10 min each time, and wash 3 times in total.

[0063] 2.6. Incubation with Secondary Antibody

[0064] Refer to the instruction manual of the secondary antibody, dilute the horseradish peroxidase (HRP)-labeled secondary antibody with the secondary antibody diluent at a ratio of 1:5000, and incubate at room temperature for 2 h; add the washing solution (PBST), wash for 10 min each time, and wash 3 times in total.

[0065] 2.7. Protein detection

[0066] In the darkroom, mix the two reagents of ECLA solution and ECLB solution in a 1:1 ratio in a centrifuge tube. Place the PVDF membrane with the protein side up in the center of the exposure board, add the mixed ECL solution and react fully. After 1 - 2 min, remove all the residual liquid and adjust the exposure conditions according to different luminescence intensities.

[0067] 3. Real-time fluorescence quantitative PCR

[0068] 3.1. RNA extraction

[0069] 1) Collect the cell pellet and add 1 mL of TRIzol for lysis;

[0070] 2) Add 0.2 mL of chloroform, shake vigorously for 15 s, and let stand at room temperature for 5 min;

[0071] 3) Centrifuge at 12000 rpm for 10 min at 4 °C, and transfer the supernatant (about 500 μL) to another EP tube;

[0072] 4) Add 0.5 mL of pre-cooled isopropanol, mix gently, and incubate on ice for 30 min;

[0073] 5) Centrifuge at 12000 rpm for 15 min at 4 °C and discard the supernatant;

[0074] 6) Add 1 mL of pre-cooled 75% ethanol, centrifuge at 12000 rpm for 5 min at 4 °C, and discard the supernatant;

[0075] 7) Repeat step 6);

[0076] 8) Dry the RNA pellet at room temperature, add 20 - 50 μL of DEPC water, and store at -80 °C for later use.

[0077] 3.2. RT reaction

[0078] 1) In a 0.2 mL EP tube, add total RNA (with a mass of 1 μg), 4.0 μL of 5×RT MasterMix, 1.0 μL of 20×OligodT, and make up to 20 μL with DEPC water. Mix gently and centrifuge briefly;

[0079] 2) Heat on a PCR instrument at 37 °C for 30 min, at 85 °C for 5 min, and immediately place on ice for 1 min;

[0080] 3) Take out the above reaction solution, which is the cDNA, and store it at -20 °C for later use.

[0081] 4. Fluorescent quantitative PCR reaction

[0082] 1) Take out the cDNA as the template for fluorescent quantitative analysis. The reaction system is as follows:

[0083]

[0084]

[0085] 2) The reaction conditions are as follows:

[0086]

[0087] 3) Primers for each detection index:

[0088]

[0089]

[0090] 5. Result description

[0091] Figure 1 It reflects the expression levels of circ-CBLB in the sera of RA patients and healthy controls (HC). The results of real-time quantitative PCR show that: compared with the HC group, the expression level of circ-CBLB in the RA group is significantly down-regulated (P < 0.05). This result indicates that in RA patients, circ-CBLB is in a low-expression state, providing a new perspective for studying the role of circ-CBLB in the pathogenesis of RA.

[0092] Figure 2 It reflects the correlations between circ-CBLB and the immune-inflammatory indexes ESR, CRP, RF, and CCP of RA. The results show that circ-CBLB is negatively correlated with ESR, CRP, RF, and CCP.

[0093] Figure 3 It reflects the correlations between circ-CBLB and the M1 macrophage marker CD86 and the M2 macrophage marker CD163. The results show that circ-CBLB is negatively correlated with CD86 and positively correlated with CD163. This result indicates that circ-CBLB is closely related to macrophage polarization.

[0094] Figure 4Reflects the correlation results of the contents of TNF-a, IL-6, IL-13, and IL-10 in the co-culture system of macrophages and RA-FLS. Real-time quantitative PCR results showed that after knocking down circ-CBLB, the expressions of TNF-α and IL-6 were significantly increased (P<0.01), while the expressions of IL-13 and IL-10 were significantly decreased (P<0.01). After overexpressing circ-CBLB, the expressions of TNF-α and IL-6 were significantly decreased (P<0.01), while the expressions of IL-13 and IL-10 were significantly increased (P<0.01). These results suggest that overexpression of circ-CBLB can inhibit the expression of pro-inflammatory factors.

[0095] Figure 5 Reflects the correlation results of the contents of TLR3 and TRAF3; Real-time quantitative PCR results showed that when circ-CBLB was overexpressed, the expressions of TLR3 and TRAF3 were decreased (P<0.01). It shows that overexpression of circ-CBLB can down-regulate the expression levels of TLR3 / TRAF3.

[0096] Figure 6 Reflects the correlation results of the enrichment of circ-CBLB and TLR3; The results of the co-immunoprecipitation experiment showed an obvious enrichment trend between circ-CBLB and TLR3 (P<0.05), indicating that TLR3 and TRAF3 can specifically bind.

[0097] Figure 7 Reflects the expression results of the M1 macrophage markers CD80 and CD86 after intervening in circ-CBLB. Real-time quantitative PCR results showed that when circ-CBLB was overexpressed, the expressions of CD80 and CD86 were decreased (P<0.01). It shows that overexpression of circ-CBLB can inhibit the polarization of macrophages towards the M1 direction. These results demonstrate the potential biological functions of circ-CBLB in RA and provide important clues for further research on the diagnosis and treatment methods of RA.

[0098] Examples of the present invention can show that: compared with the healthy control group (HC), the expression of circ-CBLB in the synovial tissue of RA patients is significantly reduced, and it is negatively correlated with the disease activity indicators ESR, CRP, RF, CCP, and the M1 macrophage marker CD80, and positively correlated with the M2 macrophage marker CD163. The RIP experiment was used to prove that there is an obvious enrichment trend between circ-CBLB and TLR3, indicating that the two can bind, and TLR3 is the target of circ-CBLB. Further, by interfering with circ-CBLB and TLR3, the expression of the M1 macrophage markers CD80 and CD86 can be affected. This provides evidence for the combination of circ-CBLB targeting TLR3 as an immune-inflammatory marker for rheumatoid arthritis. The examples of the present invention verified the expression and function of circ-CBLB and its target TLR3 in RA, providing ideas and methods for the research on evaluating the disease activity, targeted treatment, and prognosis evaluation of RA.

[0099] 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 the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, in any aspect, 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.

Claims

1. Use of circ-CBLB in combination with targeting TLR3 in the preparation of a drug for the diagnosis and treatment of rheumatoid arthritis, characterized in that, The nucleotide sequence of the circ-CBLB is 5'-TCAGCTTCCTCATG TTCAGGT-3'; the nucleotide sequence targeting TLR3 is 5'-GCCATGAAGTTGCTGAC TGC-3'.

2. Use of the combination of circ-CBLB and targeting TLR3 according to claim 1 in the preparation of a drug for the diagnosis and treatment of rheumatoid arthritis, characterized in that, The nucleotide sequence of the forward primer of the circ-CBLB is 5'-TCAGCTTCCTCATGTTCAGGT-3'; the nucleotide sequence of the reverse primer of the circ-CBLB is 5'-TGCTAACGGACCAGTACACTT-3'.

3. Use of the combination of circ-CBLB and targeting TLR3 according to claim 1 in the preparation of a medicament for the diagnosis and treatment of rheumatoid arthritis, characterized in that, The nucleotide sequence of the forward primer of the TLR3 is 5'-GCCATGAAGTTGCTGACTGC-3'; the nucleotide sequence of the reverse primer of the TLR3 is 5'-TGGCGGCTGGTAATCTTCTG-3'.

4. Use of the combination of circ-CBLB and targeting TLR3 according to claim 1 in the preparation of a medicament for the diagnosis and treatment of rheumatoid arthritis, characterized in that, The detection method of the rheumatoid arthritis diagnosis and treatment drug for the combination of circ-CBLB and targeting TLR3 includes real-time fluorescence quantitative PCR, RN A pull down experiment, and / or immunoblotting method.

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