Application of MID1 as a marker and target for rheumatoid arthritis

Abstract

The present application discloses the application of MID1 as a marker and target for rheumatoid arthritis. The present application discloses that MID1 can be used as a diagnostic marker and therapeutic target for rheumatoid arthritis. By detecting the expression level of MID1, it can be used to diagnose rheumatoid arthritis. By knocking down the expression of MID1, the proliferation, invasion and metastasis of synovial fibroblasts can be inhibited, the secretion of proinflammatory cytokines can be inhibited, and the incidence of collagen-induced arthritis can be reduced.

Description

Technical Field

[0001] The present application relates to the technical field of rheumatoid arthritis, and specifically to the application of MID1 as a marker and target for rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease with erosive, symmetrical polyarthritis as the main clinical manifestation, showing significant clinical heterogeneity. RA causes many changes in the synovium, and the expansion of fibroblast-like synoviocytes (FLS) and changes in their behavior are one of the important differences caused by RA. FLS maintain the homeostasis of synovial fluid and extracellular matrix in healthy joints by producing synovial fluid components. FLS plays a key role in the development of RA pannus. Pannus is composed of new blood vessels, inflammatory cells and hyperplastic synovial tissue, which is the pathological basis for the destruction of articular cartilage and bone in late RA.

[0003] For the diagnosis of RA, the mainstream is still to use a set of criteria formulated by the American College of Rheumatology in 1987, which mainly relies on clinical manifestations and the exclusion of other arthritis to determine whether it is RA. In addition, X-ray changes and rheumatoid factors are also commonly used criteria for RA. However, the above methods are cumbersome and low in specificity, which can easily delay the diagnosis time.

[0004] Some progress has been made at the molecular level in the diagnosis of RA, such as the use of phosphoglycerol kinase 1 (China Patent Publication No. CN104698194A), tenascin C (China Patent Publication No. CN104105967A), microRNA (miRNA) molecular markers (China Patent Publication No. CN103805696A), and autoantibody binding antigens (China Patent Publication No. CN104987366A, CN102796173A), etc. Summary of the invention

[0005] This application analyzed the key signaling pathways involved in the occurrence of RA synovitis from the GEO database, and found that ubiquitin-mediated proteolysis is one of the most enriched pathways in RA. In the GSE89408 and GSE55235 data sets, the common differentially expressed gene MID1 involved in regulating the ubiquitin-mediated proteolysis pathway was highly expressed in the synovial tissue of collagen-induced arthritis model mice compared with the control group, and the difference was more significant. A cell line with knockdown of MID1 was established by lentiviral infection on the RA-FLS cell line MH7A cells to evaluate the biological effects of MID1 on the RA synovial fibroblast cell line MH7A. The results of this application show that knockdown of MID1 can inhibit the proliferation, invasion and metastasis of synovial fibroblasts, inhibit their secretion of proinflammatory cytokines, and reduce the incidence of collagen-induced arthritis.

[0006] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0007] In a first aspect, the embodiment discloses a marker for rheumatoid arthritis, wherein the marker is MID1. MID1 (midline-1, Gene ID: 4281), also known as TRIM18 (tripartite-motif 18), belongs to the TRIM family and has E3 ubiquitin ligase activity.

[0008] In a second aspect, the embodiments disclose the use of MID1 as a diagnostic marker for rheumatoid arthritis in the preparation of a diagnostic reagent or kit for rheumatoid arthritis.

[0009] In an embodiment of the second aspect, the diagnostic reagent or diagnostic kit detects the expression level of MID1 gene or protein in an in vitro sample, and the increased expression indicates that the individual in the in vitro sample suffers from rheumatoid arthritis.

[0010] In an embodiment of the second aspect, the in vitro sample is a sample containing synovial tissue from a subject.

[0011] In an embodiment of the second aspect, the diagnostic reagent or kit comprises primers for specifically amplifying MID1, and the primers comprise DNA molecules as shown in SEQ ID NOs. 1-2.

[0012] In an embodiment of the second aspect, the diagnostic reagent or kit comprises a monoclonal antibody against MID1.

[0013] In a third aspect, the embodiments disclose the use of MID1 as a therapeutic target for rheumatoid arthritis in the preparation of a drug for treating rheumatoid arthritis.

[0014] In an embodiment of the third aspect, the drug is an agent for inhibiting or reducing the expression level of the MID1 gene or protein, or an agent for knocking out the MID1.

[0015] In an embodiment of the third aspect, the reagent includes shRNA as shown in SEQ ID NOs. 3-4 or a plasmid carrying shRNA as shown in SEQ ID NOs. 3-4.

[0016] In an embodiment of the third aspect, the reagent includes a lentivirus carrying the shRNA shown in SEQ ID NOs. 3 to 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The results related to the increased expression of MID1 in the synovial tissue of rheumatoid arthritis provided in the examples. (A) KEGG analysis of differentially expressed genes of the GSE89408 dataset containing RNA sequencing data of synovial tissues of 152 patients with rheumatoid arthritis and 28 healthy controls was performed. (B) Differentially expressed genes enriched in the ubiquitin-mediated proteolytic pathway of the GSE89408 and GSE55235 datasets. (C) mRNA expression levels of MID1, DDB2, FBXW7 and SMURF2 in the synovial tissues of collagen-induced C57BL / 6 arthritis mice. (D) Immunoblotting was used to detect the protein expression level of MID1 in the synovial tissues of normal control mice and collagen-induced arthritis model mice. (EF) Immunohistochemical analysis of the expression level of MID1 in the synovial tissues of collagen-induced and DBA (P=0.0030) and C57BL / 6 (P=0.0018) arthritis model mice compared with the synovial tissues of the control mice (n=3).

[0018] Figure 2 The results of constructing the MH7A cell line with MID1 knockdown provided in the examples. (A, B) After the shRNA (shMID1) targeting MID1 lentiviral vector infected MH7A cells, qRT-PCR and immunoblotting were used to detect and verify whether MID1 was knocked down in MH7A.

[0019] Figure 3 The results of knocking down MID1 to inhibit the in vitro growth of MH7A cells provided in the examples. (A) After 48 hours of CFSE staining, the proliferation of MH7A cells with knockdown of MID1 (shMID1-1 and shMID1-2) was detected by flow cytometry. (B) The viability of MH7A cells with knockdown of MID1 (shMID1-1 and shMID1-2) was determined by CCK8 method. (C) The cell cycle of MH7A cells with stable knockdown of MID1 (shMID1-1 and shMID1-2) was detected by flow cytometry.

[0020] Figure 4The results of knocking down MID1 to inhibit the in vivo growth of MH7A cells provided in the examples. (A) The successfully constructed stable knockdown MID1 (shMID1-1 and shMID1-2) or MH7A cells were injected into the right upper axilla of severely immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice to establish a subcutaneous xenograft model. The MH7A xenograft mass was measured and collected after 14 weeks. n = 5 / group. (B) Statistical analysis of the mass mass of the shscramble control group, the knockdown MID1 group (shMID1-1 and shMID1-2) and the control group, n = 5 / group. (C) After the successfully constructed MH7A cells with stable knockdown of MID1 (shMID1-1 and shMID1-2) were injected into the right upper axilla of severely immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice, the volume of the tumor was measured every other week and recorded and plotted into a growth curve. (D) After 14 weeks, MH7A xenograft tumors were obtained from NSG mice inoculated with MH7A cells with knockdown of MID1 (shMID1-1 and shMID1-2), and the obtained tissues were stained with H&E, Ki67 and DPP4, n=5 / group. (EF) Statistical analysis of the positive rates of Ki67 and DPP4 in the tumors of the shscramble control group and the knockdown of MID1 group (shMID1-1 and shMID1-2), n=5 / group.

[0021] Figure 5 The results of knocking down MID1 to inhibit MH7A cell migration and invasion provided in the examples. (A) In vitro cell scratch wound healing assay detected the changes in cell migration in the shscramble control group, the stable knockdown MID1 groups (shMID1-1 and shMID1-2) and the control group, and the percentage of scratch healing in each group was counted. (B) Invasion and migration assays were used to evaluate the migration and invasion abilities of MH7A cells with stable knockdown of MID1 (shMID1-1 and shMID1-2). Invasion and migration assays The cells on the lower surface of the upper chamber were counted and statistically analyzed. (D) qRT-PCR was used to detect the mRNA expression levels of MMPs in MH7A cells with stable knockdown of MID1 (shMID1-1 and shMID1-2). (E) Western Blot was used to detect the protein expression levels of MMPs in MH7A cells with stable knockdown of MID1 (shMID1-1 and shMID1-2).

[0022] Figure 6The results of MID1 deficiency inhibiting the secretion of proinflammatory cytokines and alleviating collagen-induced arthritis in mice provided in the examples. (A) Protein-protein interaction network analysis showed the interaction between MID1 and RA-related cytokines. (B) qRT-PCR was used to detect the mRNA levels of RA-related cytokines in MID1-knockdown MH7A cells. (C) Wild-type C57BL / 6 (WT) and Mid1- / - mice on the C57BL / 6 background were immunized with type II collagen and Freund's complete adjuvant to establish a collagen-induced arthritis model. (D) Collagen-induced WT and Mid1 - / - Body weight curves of mice. ns, not statistically significant. (E) Collagen-induced WT and Mid1 - / - Images of mouse hind paws and H&E staining of joints. (F) Clinical scores of arthritis in WT and Mid1- / - mice were assessed weekly after collagen induction and recorded and plotted into clinical score curves (n=8 / group). DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0024] qRT-PCR

[0025] Some embodiments provide methods for detecting the mRNA expression level of each gene including MID1 in synovial tissue, including:

[0026] (1) Total RNA extraction

[0027] Trizol reagent was used to extract total RNA from synovial tissue.

[0028] (2) Reverse transcription

[0029] 1 μg of total RNA, 3 μL of 5×gDNAdigester Mix and the remaining DEPC water (15 μL) were used as the genome removal reaction system. Mix well, centrifuge briefly, and heat at 42°C for 2 min to remove the DNA in the genome. Mix well with III SuperMix plus, place in PCR reaction instrument, and incubate at 25℃ for 5 min, 55℃ for 15 min, 85℃ for 5 min, and hold at 4℃ to reverse transcribe RNA into cDNA.

[0030] (3) qRT-PCR

[0031] Prepare qRT-PCR reaction system. The system contains 1 μL cDNA, 5 μL qPCR SYBRGreen Master Mix (No Rox), 0.25 μL 10 μM upstream primer, 0.25 μL 10 μM downstream primer and the rest sterile ultrapure water. The PCR amplification program is: pre-denaturation 95℃ 5min, denaturation 95℃ 10s\annealing 60℃ 20s\extension 72℃ 20s (40 cycles in total), the instrument automatically sets the melting curve. 2 -ΔΔCt Methods The relative expression of target genes was analyzed.

[0032] Among them, the primers used to amplify the human reference gene GAPDH are shown in SEQ ID NOs.1 to 2. The primers used to amplify the mouse reference gene GAPDH are shown in SEQ ID NOs.3 to 4. The primers used to amplify the human MID1 are shown in SEQ ID NOs.5 to 6. The primers used to amplify the mouse MID1 are shown in SEQ ID NOs.7 to 8. The primers used to amplify the human IL-1β are shown in SEQ ID NOs.9 to 10. The primers used to amplify the human IL-6 are shown in SEQ ID NOs.11 to 12. The primers used to amplify the human IL-8 are shown in SEQ ID NOs.13 to 14. The primers used to amplify the human IL-13 are shown in SEQ ID NOs.15 to 16. The primers used to amplify the human MMP2 are shown in SEQ ID NOs.17 to 18. The primers used to amplify the human MMP3 are shown in SEQ ID NOs.19 to 20. The primers used to amplify the human MMP7 are shown in SEQ ID NOs.21 to 22. The primers used to amplify human MMP9 are shown in SEQ ID NOs. 23-24.

[0033] Western Blot

[0034] Some embodiments provide a method for detecting the expression level of DPP4 or MID1 by Western Blot in synovial tissue, including:

[0035] The homogenate of synovial tissue was subjected to 10% PAGE gel electrophoresis, and the gel with a molecular weight greater than 180KD or less than 25KD was removed, and then transferred to a PVDF membrane. After blocking with 5% skim milk for 1 hour, the membrane was washed 4 times with TBST, cut and incubated in the primary antibody solution of the monoclonal antibody of DPP4 (dilution multiple 1:1000) at 4°C for 12-14 hours. Wash with TBST 4 times, add the secondary antibody solution and incubate at room temperature for 1-2 hours, wash the membrane with TBST 4 times, and expose and develop. Among them, the sources of proteins involved: GAPDH, 60004-1-Ig, Proteintech. MID1, Ab70770, Abcam. Mouse anti-HA-Tag antibody, AE008, ABclonal. Rabbit anti-HA-Tag antibody, AE105, ABclonal. Cy3-labeled goat anti-mouse IgG, GB21301, Servicebio. FITC-labeled goat anti-rabbit IgG, GB22303, Servicebio. Construction of lentiviral plasmid for MID1 knockdown

[0036] Some embodiments provide the shRNA sequence as shown in SEQ ID NO. 25 or 28.

[0037] In some embodiments, primer pairs SEQ ID NO. 26-27 (h-Mid1-sh1-F and h-Mid1-sh1-R) for shRNA1 as shown in SEQ ID NO. 25, and primer pairs SEQ ID NO. 29-30 (h-Mid1-sh2-F and h-Mid1-sh2-R) for shRNA2 as shown in SEQ ID NO. 28 are designed. A shRNA primer reaction system is prepared, which contains 1 μL NEB Buffer, 4.5 μL 100 μM shRNA upstream primer, and 4.5 μL 100 μM shRNA downstream primer in 10 μL.

[0038] As shown in Table 3, the annealing product obtained by annealing the shRNA primer was connected to the restriction product of pLKO.1-U6-hPGK-Puro plasmid (NovoPro, product number V010450). The restriction reaction system of pLKO.1-U6-hPGK-Puro plasmid contained 1 μL Fast Digest AgeI, 1 μL Fast Digest EcoRI, 5 μL 10× Fast Digest Buffer, 2.5 μg pLKO.1-U6-hPGK-Puro and the remaining ddH2O in 50 μL, and the restriction was carried out in a thermostatic metal bath preheated at 37°C for 2 hours, and the restriction plasmid product was purified using Fast Pure Gel DNA Extraction Mini Kit. The ligation reaction system of the annealed product and the digested product of the pLKO.1-U6-hPGK-Puro plasmid contains 1 μL of plasmid digested product, 1.5 μL of shRNA primer annealed product and 2.5 μL of Ligation high (TOYOBO Research Reagents, product number LGK-201) in 5 μL. The shRNA primer annealing program includes: 95°C for 5 min for 1 cycle, -0.5°C / cycles for 30 s for 140 cycles, and 25°C for 1 cycle.

[0039] The ligation product was transformed into Escherichia coli, and after positive clones were screened on ampicillin resistance plates, they were transferred to LB liquid medium containing Amp and cultured at 37°C and 200 rpm / min for 2 h to expand the bacteria. The bacterial solution was sequenced (U6 was used as the sequencing primer, and the sequence was SEQ ID NO.31) to verify that a positive clone was obtained, and the MID1 knockdown lentiviral plasmid was extracted from it using the Endo-Free Plasmid Maxi Kit.

[0040] Construction of MID1 knockdown stable MH7A cell line

[0041] 1. Lentiviral plasmid transfection and packaging

[0042] HEK293T cells (Shanghai Cell Bank, Chinese Academy of Sciences) in exponential growth phase with a confluence of about 95% were placed in a 10 cm culture dish and cultured in a cell culture incubator for 6 to 8 hours.

[0043] Prepare transfection mixture 1 and mixture 2. Mixture 1 contains 1mL serum-free DMEM, 12μg MID1 knockdown lentiviral plasmid, 6μg psPAX2 plasmid, and 6μg pMD2.G plasmid. Mixture 2 contains 1mL serum-free DMEM and 48μL PEI. Mix mixture 1 and mixture 2 separately, let stand at room temperature in the dark for 5min, then mix mixture 2 into mixture 1 and incubate at room temperature in the dark for 20min. Gently add the transfection mixture to a 10cm culture dish, mix well and place in a 37℃ cell culture incubator for transfection for 4h. Change the serum-free transfection system in the 10cm culture dish, add 15mL cell growth medium and continue to culture for 48h. Observe the transfection efficiency under a fluorescence microscope. When the transfection efficiency reaches about 80%, collect the supernatant culture solution, filter the virus solution with a 0.22μm filter membrane, centrifuge at low temperature and ultra-high speed, discard the supernatant, and dissolve the virus precipitate with pre-cooled DPBS.

[0044] 2. Construction of Stable MH7A Cell Line

[0045] After digestion, human fibroblast-like synoviocyte cell line MH7A (purchased from RIKEN Cell Bank, Japan) in exponential growth phase with a confluence of about 95% was plated in a 6-well plate and placed in a cell culture incubator for 6-8h. When the cells adhered to the wall and the cell confluence was 60%-70%, the culture medium was discarded and washed once with PBS. 2mL serum-free DMEM was added and placed in a cell culture incubator for starvation culture for 24h. The original serum-free DMEM was discarded and 2mL serum-free DMEM containing 80μL HitransGA (25×) virus infection enhancement solution and 50μL virus concentrate was added. Placed in a cell culture incubator for 12-16h, the supernatant was discarded and washed once with PBS. 2mL DMEM complete culture medium was added and placed in a cell culture incubator for further culture for 48h, the supernatant was discarded and washed once with PBS. 500μL trypsin was added and the digested cells were transferred to a 25T cell culture flask containing 5mL DMEM complete culture medium, and puromycin (8μg / mL) was added for drug screening. The DMEM complete culture medium containing puromycin (8 μg / mL) was replaced every 3 days for 2 weeks to obtain the MH7A cell line stably transfected with MID1.

[0046] 3. Results

[0047] RNA and protein of the constructed stable MH7A cell line were extracted for qRT-PCR and Western Blot to verify the knockdown efficiency of MID1. Example MH7A cells were infected with shRNA lentiviral plasmid targeting MID1, and MID1 knockdown stable MH7A cell lines (shMID1-1, shMID1-2) were successfully established.

[0048] Carboxyfluorescein diacetate succinyl ester (CFSE) labeling test

[0049] The cell CFSE labeling test provided in the embodiment includes:

[0050] Digest the cells that are in the exponential growth phase and have a confluence of about 90% and count the cells. 7 Transfer the cells to a 15 mL sterile centrifuge tube, centrifuge at 800 rpm for 5 min at room temperature, and discard the supernatant. Add 5 mL PBS to wash twice. Resuspend the cells in 1 mL 37°C pre-warmed PBS, add 4 μM CFSE staining solution (CFSE fluorescent dye, HY-D0938, MedChemExpress), and mix well. Incubate at 37°C in the dark for 10 min. Add 5 mL complete culture medium and place on ice for 5 min to terminate staining, centrifuge at room temperature, 800 rpm for 5 min, discard the supernatant, and repeat this step. Add 5 mL PBS to resuspend the cells, centrifuge at room temperature, 800 rpm for 5 min, and discard the supernatant. Add 5 mL PBS to resuspend the cells, take 200 μL of the cell suspension and transfer it to a flow tube, detect with 488 nm excitation light of the flow cytometer, and record it as the mother generation. The remaining cells were plated at a density of 100,000 cells / mL according to the test design, and the cells were collected at the corresponding time points in the test design. After digestion, they were detected using a flow cytometer with 488nm excitation light.

[0051] Cell Counting Kit-8 (CCK-8) Test

[0052] The cell CCK-8 test provided in the embodiment includes:

[0053] Digest the cells that are in the exponential growth phase and have a fusion degree of about 90%, and count the cells. According to the replicate wells and time points of the test design, calculate the number of cells required for the test at a density of 100,000 cells / mL. According to the calculated number of cells and cell density required for the test, calculate the amount of cell suspension that needs to be taken, transfer the cell suspension to a 15mL sterile centrifuge tube, centrifuge at room temperature, 800rpm, for 5 minutes, and discard the supernatant. Resuspend the cells in DMEM complete medium, plate them in 96-well plates at a density of 10,000 cells / well, shake the culture plate in a cross-cross method to spread the cells evenly, and place them in a cell culture incubator. After 6 to 8 hours, wait for the cells to adhere to the wall, discard the supernatant, and add 100μL of CCK-8 working solution configured at a ratio of 1:9 (CCK-8 solution: complete culture medium, CCK-8 proliferation kit, 40203ES76, Yisheng) to each well. Place the culture plate in a 37°C constant temperature incubator away from light and incubate for 1 to 4 hours. For the first test, take out the cells every 30 minutes and use a microplate reader to detect the absorbance at a wavelength of 450nm to select the best incubation time. Subsequent absorbance tests can be performed at the time points designed in the test plan, and the best incubation time selected in the previous step can be used.

[0054] Cell survival rate % = (OD test group - OD blank control group) / (OD negative control group - OD blank control group).

[0055] Cell cycle assay

[0056] The cell cycle test provided in the embodiment includes:

[0057] Digest the cells in the exponential growth phase and count the cells. Plate the cells in a 6-well plate at a cell density of 300,000 cells / mL, 2 mL / well, and divide the cells into groups according to the test design. Take the same amount of cells in each group. When the cell confluence reaches 100%, discard the supernatant. Wash once with 2 mL PBS and add 2 mL serum-free DMEM. After starvation for 60 hours, add 500 μL of trypsin for digestion, transfer the cell suspension to a 15 mL sterile centrifuge tube containing 3 mL serum-free DMEM, centrifuge at room temperature, 800 rpm, and discard the supernatant. Resuspend in 5 mL PBS, take 10 μL, add to a flow tube containing 90 μL PBS, and count with a flow cytometer. Plate the cells in a 12-well plate at a cell density of 100,000 cells / mL, 1 mL / well, and set up 3 replicates for each test group. Take another 100,000 cells and transfer them to a new 1.5 mL EP tube, centrifuge at room temperature, 800 rpm, and centrifuge for 5 minutes. Add 1mL of 75% alcohol precooled on ice and blow thoroughly, fix overnight at -20℃, and record as 0h. In the first test, set a time point every 2h after 8h of plating, collect cells into flow tubes, wash once with 1mL PBS, add 1mL of 75% alcohol precooled on ice and blow thoroughly, and fix overnight at -20℃. Take out the fixed cells, centrifuge at room temperature, 1500rpm, and discard the supernatant. Add 1mL PBS to resuspend the cells, centrifuge at room temperature, 1500rpm, and discard the supernatant. Add 200μL of periodic PI staining working solution (1mg / mL PI 200~500μL+DNase-free RNaseA2mg+Triton X-10010μL+1×PBS to 10mL) to resuspend the cells. Incubate in the dark for 30min, flow detection, and use Modfit LT5 to fit the flow cycle results.

[0058] Cell scratch test

[0059] The cell scratch test provided in the embodiment includes:

[0060] Digest the cells in the exponential growth phase and count the cells. Plate a 6-well plate at a cell density of 300,000 cells / mL, 2mL / well, and divide into groups according to the test design, with the same amount of cells in each group. When the cell confluence reaches 100%, discard the supernatant. Wash once with 2mL PBS and add 2mL serum-free DMEM. After starvation for 60h, use a 200μL pipette tip to gently draw a straight line in the well plate. Wash twice with 2mL PBS, add 2mL serum-free DMEM, capture the image immediately, and capture the image again after 48h, and calculate the percentage of closure of the drawn straight line.

[0061] Transwell cell migration assay

[0062] The cell Transwell cell migration test provided in the embodiment includes:

[0063] Digest the cells in the exponential growth phase and count the cells. Plate a 6-well plate at a cell density of 300,000 cells / mL, 2mL / well, and divide into groups according to the test design, taking the same amount of cells for each group. When the cell confluence reaches 100%, discard the supernatant. Wash once with 2mL PBS and add 2mL serum-free DMEM. After starvation for 48 hours, add 500μL of trypsin for digestion, transfer the cell suspension to a 15mL sterile centrifuge tube containing 3mL serum-free DMEM, centrifuge at room temperature, 800rpm, for 5min, and discard the supernatant. Resuspend in 5mL PBS, take 10μL, add to a flow tube containing 90μL PBS, and count with a flow cytometer. Take out the required amount of cells (based on a density of 50,000 cells / well, and set 3 replicates for each test group), and transfer to a new 15mL sterile centrifuge tube, centrifuge at room temperature, 800rpm, for 5min, and discard the supernatant. Resuspend the cells in serum-free DMEM at 200μL / well, and add the cell suspension to the 24-well The upper chamber (Cat. No. 3422, Costar) was added with 700 μL / well of DMEM complete medium in the lower chamber. Place the culture plate in a CO2 constant temperature cell culture incubator. After 36 hours, take out The upper chamber was washed 3 times with PBS.

[0064] 10) Immerse the cells in 4% paraformaldehyde to fix them for 30 minutes and wash them three times with PBS. Stain them with 1% crystal violet for 30 minutes and wash them three times with PBS. Use a cotton swab to gently wipe the upper surface of the upper chamber to remove the remaining cells. The upper chamber was placed in a clean 24-well plate, dried in a drying oven, and photographed under a microscope.

[0065] Transwell cell invasion assay

[0066] The Transwell cell invasion assay provided in the embodiment includes:

[0067] Digest the cells in the exponential growth phase and count the cells. Plate a 6-well plate at a cell density of 300,000 cells / mL, 2mL / well, and divide into groups according to the test design, taking the same amount of cells for each group. When the cell confluence reaches 100%, discard the supernatant. Wash once with 2mL PBS and add 2mL serum-free DMEM. After starvation for 48 hours, add 500μL of trypsin for digestion, transfer the cell suspension to a 15mL sterile centrifuge tube containing 3mL serum-free DMEM, centrifuge at room temperature, 800rpm, for 5min, and discard the supernatant. Resuspend in 5mL PBS, take 10μL, add to a flow tube containing 90μL PBS, and count with a flow cytometer. Take out the required amount of cells (based on a density of 50,000 cells / well, and set 3 replicates for each test group), and transfer to a new 15mL sterile centrifuge tube, centrifuge at room temperature, 800rpm, for 5min, and discard the supernatant. Resuspend the cells in serum-free DMEM at 200μL / well, and add the cell suspension to the 24-well Upper chamber (Matrigel gel should be prepared one day in advance) DMEM complete medium 700 μL / well was added to the upper chamber of the culture dish, and DMEM complete medium 700 μL / well was added to the lower chamber. Place the culture plate in a CO2 constant temperature cell culture incubator. After 36 hours, take out The upper chamber was washed 3 times with PBS. The upper chamber was soaked in 4% paraformaldehyde to fix the cells for 30 minutes, and then washed 3 times with PBS. The upper chamber was stained with 1% crystal violet for 30 minutes, and then washed 3 times with PBS. The upper surface of the upper chamber was gently wiped with a cotton swab to remove the residual cells on the upper surface of the upper chamber. The upper chamber was placed in a clean 24-well plate, dried in a drying oven, and photographed under a microscope.

[0068] In vivo subcutaneous transplantation test

[0069] In this test, all animal tests were conducted in accordance with the ethical standards of the Animal Care and Use Committee of Huazhong University of Science and Technology (approval number: TJH-202206052). Female NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) severely immunodeficient mice were purchased from Shanghai Nanmo Organisms.

[0070] For subcutaneous transplantation models: 5 × 10 6 DPP4 overexpressing stable cells, MID1 overexpressing stable cells, or DPP4 and MID1 stable cells were injected into the subcutaneous part of the right upper limb axilla of mice. The weight and volume of mice were monitored regularly. When the subcutaneous graft volume reached 100-200mm 3 The mice were euthanized at 14 weeks. After 14 weeks, the subcutaneous masses were weighed, photographed, fixed with 4% paraformaldehyde, dehydrated and embedded, and subsequently subjected to histological staining.

[0071] In the collagen-induced arthritis mouse model, each mouse (including wild-type C57BL / 6 and Mid1- / -) was inoculated with 100 μL of a mixture containing 2 mg / mL chicken type II collagen and 2 mg / mL complete Freund's adjuvant. The mixture was injected subcutaneously at 1-2 cm from the tail root on days 0 and 21, and subcutaneously on the back on day 40 to establish the arthritis model mice. The body weight and redness and swelling of the paws of the mice were observed weekly. After the mice were euthanized, the limbs were fixed in 4% paraformaldehyde, decalcified in EDTA solution decalcification solution, and the decalcification solution was changed every 3 days for a total of 21 days, dehydrated and embedded, and subsequently histologically analyzed. The clinical score of arthritis in mice was up to 16 points, with a maximum of 4 points for each paw. Normal joints were 0 points, only one finger was slightly swollen 1 point, arthritis involved 2 joints 2 points, involved more than two joints 3 points, and severe arthritis of the entire paw 4 points.

[0072] MID1 expression is elevated in synovial tissues of RA patients and mice

[0073] In order to identify the key signaling pathways of RA synovitis, the embodiment analyzed a dataset containing synovial tissue RNA sequencing data of 152 RA patients and 28 healthy controls (i.e., GSE89408 dataset), and performed KEGG analysis on the differentially expressed genes between RA patients and healthy controls. Figure 1 As shown in A, KEGG analysis results showed that ubiquitin-mediated proteolysis pathway was one of the most enriched pathways in RA.

[0074] KEGG enrichment analysis of another RA synovial tissue RNA sequencing dataset GSE55235 also disclosed the ubiquitin-mediated proteolysis pathway. By analyzing the differentially enriched genes in the ubiquitin-mediated proteolysis pathway between the GSE89408 and GSE55235 datasets. Figure 1 As shown in B, a total of 10 common differentially expressed genes were identified: MID1, DDB2, FBXW7, SMURF2, UBE2D1, UBE2J1, UBE2L3, UBE2L6, UBE2A, and UBE2N. Among these 10 common differentially expressed genes, 4 belong to the E3 ubiquitin ligase family: MID1, DDB2, FBXW7, and SMURF2.

[0075] like Figure 1 As shown in C, the mRNA expression level of MID1 was significantly increased in the synovial tissue of collagen-induced C57BL / 6 arthritis mice compared with that of normal C57BL / 6 mice.

[0076] In collagen-induced C57BL / 6 and DBA arthritis models, Western Blot ( Figure 1 D) and immunohistochemistry ( Figure 1 E and 1F) detected the protein expression level of MID1 in the synovial tissue of mice. The results showed that compared with the synovial tissue of control mice, the expression of MID1 in the synovial tissue of RA mice was increased.

[0077] Knockdown of MID1 inhibits proliferation, migration and invasion of synovial fibroblasts

[0078] The above results show that MID1 is significantly increased in RA synovial tissue. In order to understand the effect of abnormal expression of MID1 on RA, especially on RA synovial tissue, the biological effect of MID1 on RA-FLS cell line MH7A will be evaluated next.

[0079] like Figure 2 The RT-qPCR results shown in B show that the mRNA expression of MID1 in the MID1 knockdown cell lines (shMID1-1, shMID1-2) was reduced by more than half compared with the control cells. Figure 2 The Western Blot results shown in A show that the expression level of MID1 protein was reduced in the MID1 knockdown cell lines (shMID1-1, shMID1-2) compared with the control cells. The above results confirm that the MH7A cell line with MID1 knockdown was successfully constructed.

[0080] Cell proliferation was detected using a cell counting kit-8 (CCK-8, 40203ES76, Yisheng) and carboxyfluorescein diacetate succinyl ester (CFSE, HY-D0938, MedChemExpress). Figure 3 As shown, the cell proliferation index of MID1 knockdown cell lines (shMID1-1, shMID1-2) was lower than that of the control cells (shscramble), which inhibited the proliferation of MH7A cells. In addition, compared with the control cells, the MID1 knockdown cell lines increased the proportion of G0 / G1 cells and reduced the number of cells in the S phase.

[0081] like Figure 4 As shown in A to C, compared with the control group (sh scramble), the mass and volume of the tumor in the MID1 knockdown group were significantly reduced. Figure 4 As shown in D-F, compared with the control group, the proportion of Ki67-positive cells in the tumor mass of the MID1 knockdown group was lower and the proportion of DPP4-positive cells was higher. The results confirmed that MID1 inhibited the proliferation of synovial fibroblasts in vivo.

[0082] Knockdown of MID1 reduces the invasion and metastasis of synovial fibroblasts

[0083] To evaluate whether MID1 affects the invasion and migration ability of MH7A cells, we also performed cell scratch wound healing and Invasion and migration assays.

[0084] like Figure 5 As shown, compared with the control group cells (shscramble), the percentage of scratch closure was reduced on MH7A cells with MID1 knockdown (shMID1-1, shMID1-2), and the number of cells on the lower surface of the transwell chamber was reduced. These results indicate that knockdown of MID1 can reduce the migration and invasion ability of synovial fibroblasts.

[0085] Knockdown of MID1 reduces the expression of pro-inflammatory cytokines and alleviates collagen-induced arthritis

[0086] Synovial cells are a key source of inflammatory cytokines, which in turn play an important role in RA.

[0087] Figure 6 As shown in A, a protein-protein interaction network was used to analyze whether there is an association between MID1 and cytokines previously reported to be associated with the occurrence and development of RA. The analysis results showed that there is a close association between MID1 and cytokines previously reported to be associated with the occurrence and development of RA.

[0088] like Figure 6 As shown in B, the mRNA expression levels of cytokines in MH7A cells with knockdown of MID1 were detected by RT-qPCR. The results showed that knockdown of MID1 in MH7A cells led to decreased mRNA levels of pro-inflammatory cytokines (IL-1β, IL-13, IL-6, IL-8) and increased mRNA levels of anti-inflammatory cytokines (IL-10).

[0089] This study used Mid1- / - mice generated by CRISPR-Cas9 technology under contract with Jackson Laboratory to further verify the role of MID1 in the development of rheumatoid arthritis in vivo. Wild-type C57BL / 6 (WT) and C57BL / 6 background Mid1- / - mice were immunized with type II collagen and Freund's complete adjuvant to establish a collagen-induced arthritis model ( Figure 6 C) Figure 6 As shown in Figures 6D and 6E, there was no significant difference in body weight between collagen-induced wild-type (WT) mice and Mid1- / - mice, and no signs of arthritis were observed in Mid1- / - mice. Figure 6 (F) Mid1- / - mice had lower arthritis scores compared with wild-type (WT) mice. This result emphasizes the protective role of MID1 deficiency against collagen-induced arthritis.

[0090] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. Use of MID1 as a diagnostic marker for rheumatoid arthritis in the preparation of a diagnostic reagent or kit for rheumatoid arthritis.

2. The use according to claim 1, wherein the diagnostic reagent or diagnostic kit detects the expression level of MID1 gene or protein in an in vitro sample, and the increased expression indicates that the individual in the in vitro sample suffers from rheumatoid arthritis.

3. The use according to claim 2, wherein the in vitro sample is a sample containing individual synovial tissue or synovial fluid.

4. The use according to claim 2, wherein the diagnostic reagent or kit comprises primers for specific amplification of MID1, and the sequences of the primers are shown in SEQ ID NOs. 5-6.

5. The use according to claim 2, wherein the diagnostic reagent or kit comprises a monoclonal antibody against MID1.

6. Use of an agent for knocking out MID1 in the preparation of a drug for treating rheumatoid arthritis, wherein the agent is a shRNA as shown in SEQ ID NO. 25 or 28, or a plasmid carrying the shRNA as shown in SEQ ID NO. 25 or 28, or a lentivirus carrying the shRNA as shown in SEQ ID NO. 25 or 28.

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