Application of Cdc37 in the prevention or treatment of rheumatoid arthritis

Abstract

The present invention discloses the application of Cdc37 (cell division cycle protein 37) as a target for the prevention and treatment of rheumatoid arthritis. The present invention also discloses the application of an inhibitor of the Cdc37 gene in the preparation of a drug for the prevention or treatment of rheumatoid arthritis. The inhibitor of the Cdc37 gene reduces the expression of Cdc37 in FLS, thereby inhibiting the proliferation of FLS, and weakening the inflammatory response and migration ability of FLS, thereby achieving the effect of preventing / treating RA. The present invention provides the application of Cdc37 as a target for the prevention and treatment of rheumatoid arthritis. It is found for the first time that Cdc37 is highly expressed in RA synovial tissue, and inhibiting Cdc37 can significantly inhibit the proliferation of FLS, and weaken the inflammatory response and migration ability of FLS. Cdc37 has great potential as a specific diagnostic and therapeutic target for RA patients, and provides a theoretical basis for the development of new anti-RA drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to the application of Cdc37 (cell division cycle protein 37) in the prevention or treatment of rheumatoid arthritis, and specifically relates to the application of Cdc37 as a prevention and treatment target for rheumatoid arthritis, the application of Cdc37 gene inhibitors in the preparation of drugs for the prevention or treatment of rheumatoid arthritis, the application of Cdc37 gene inhibitors in the preparation of drugs for the prevention or treatment of rheumatoid arthritis, and the application of Cdc37 in the screening of drugs for the prevention / treatment of rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a multisystem inflammatory autoimmune disease that mainly affects peripheral joints. RA is a stubborn disease with a high disability rate. In the late stage, it seriously affects daily life and work ability. It is one of the main diseases causing labor force loss and disability in my country. Various types of cells, such as fibroblast-like synoviocytes (FLS) and macrophages, are involved in the chronic inflammatory process of RA. Previous studies have shown that FLS activated by proinflammatory cytokines in RA joints have the same proliferation ability as tumor cells, which is one of the pathogenesis of abnormal proliferation of RA cells. Cytokines and other factors secreted by FLS, such as interleukin (IL)-6, IL-1β, matrix metalloproteinase (MMP)-3 and MMP-9, are believed to play a major role in the development of persistent inflammation, synovial hyperplasia and joint destruction. At present, its exact cause is still unclear. The inflammatory process of RA is extremely complex. It is a complex immune inflammatory process involving multiple cytokines, adhesion molecules and chemokines. In recent years, biological treatment of RA has made sufficient progress, but its efficacy is still very limited. Therefore, it is imperative to find specific prevention / treatment targets for RA.

[0003] As a molecular chaperone, cell division cyclin 37 (Cdc37) participates in many physiological processes, such as cell cycle progression, cell signaling, and cell proliferation. Cdc37 recruits unstable kinases to Hsp90 and regulates cell signaling pathways, such as the ERK and PI3K / AKT signaling pathways that have been shown to be closely related to the occurrence of RA. However, the biological function of Cdc37 in RA has not been reported. If Cdc37 can be used as a specific target for the treatment of RA and the corresponding drugs can be developed, it will be of great significance for the diagnosis and treatment of RA. Summary of the invention

[0004] Purpose of the invention: In view of the problems or shortcomings in the prior art, the present invention provides the application of Cdc37 (cell division cycle protein 37) in the prevention or treatment of rheumatoid arthritis, specifically to the application of Cdc37 as a prevention and treatment target for rheumatoid arthritis, the application of Cdc37 gene inhibitors in the preparation of drugs for the prevention or treatment of rheumatoid arthritis, the application of Cdc37 gene inhibitors in the preparation of drugs for the prevention or treatment of rheumatoid arthritis, and the application of Cdc37 in screening drugs for the prevention / treatment of rheumatoid arthritis.

[0005] To achieve the above-mentioned purpose of the invention, the embodiments of the present invention provide the application of Cdc37 as a target for the prevention and treatment of rheumatoid arthritis.

[0006] The embodiments of the present invention also provide the use of Cdc37 gene inhibitors in the preparation of drugs for preventing or treating rheumatoid arthritis.

[0007] Furthermore, inhibitors of the Cdc37 gene reduce the expression of Cdc37 in FLS, thereby inhibiting the proliferation of FLS and weakening the inflammatory response and migration ability of FLS, thereby achieving the effect of preventing / treating RA.

[0008] Furthermore, the inhibitor of the Cdc37 gene is selected from Cdc37-siRNA specific to the Cdc37 gene.

[0009] Preferably, the sequence of the Cdc37-siRNA is as follows:

[0010] The sequence of the Cdc37-siRNA sense strand was 5′-CGAAUAGAGAAGGCCAUGATT-3′;

[0011] The sequence of the Cdc37-siRNA antisense strand was 5′-UCAUGGCCUUCUCUAUUCGTT-3′.

[0012] An embodiment of the present invention further provides a reagent or kit for diagnosing rheumatoid arthritis, characterized in that it comprises an antibody with detection specificity for Cdc37 and an immunohistochemical kit.

[0013] The embodiments of the present invention also provide an application of Cdc37 in screening drugs for preventing / treating rheumatoid arthritis, characterized in that the screening uses a reagent or kit for detecting the expression level of Cdc37; and the biological sample used is synovial tissue.

[0014] An embodiment of the present invention also provides a method for detecting Cdc37 protein, which is characterized by comprising the following steps: (1) extracting synovial tissue and FLS, (2) preparing paraffin sections, (3) obtaining whole protein by protein lysis method, determining protein concentration, and detecting the expression of Cdc37 by immunoblotting and immunohistochemistry.

[0015] An embodiment of the present invention further provides an application of a Cdc37 gene in the preparation of a targeted therapeutic preparation for rheumatoid arthritis, characterized in that the targeted therapeutic preparation for rheumatoid arthritis is obtained by preparing a Cdc37-specific interfering sequence, and the Cdc37-specific interfering sequence is used to knock down the expression of Cdc37 in cells.

[0016] The beneficial effects of the above technical scheme of the present invention are as follows: The present invention provides the application of Cdc37 in RA prevention / treatment drugs. The embodiment study of the present invention confirms that the expression of Cdc37 in RA synovial tissue is significantly increased, and the Cdc37-specific interference sequence of the present invention significantly reduces the expression of Cdc37 in FLS, and at the same time significantly inhibits the proliferation, inflammatory response, and migration ability of FLS. The present invention verifies the expression and biological function of Cdc37 in RA for the first time. This discovery will further improve the research on the pathogenesis of RA. The present invention provides a new theoretical basis for the development of drugs for the treatment of RA. Using Cdc37 as a drug target, Cdc37-specific inhibitors are screened as candidate drugs for the prevention / treatment of RA, laying the foundation for improving the clinical treatment effect of RA. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The collagen-induced arthritis (CIA) model was established in Example 1 of the present invention. The arthritis scores of rats were evaluated on the second day after booster immunization.

[0018] Figure 2 This is a statistical diagram of Cdc37 expression when primary CIA-FLS were extracted and cultured in Example 2 of the present invention.

[0019] Figure 3 This is a diagram showing the proliferation of CIA-FLS detected by EdU staining and flow cytometry after Cdc37-siRNA was used to inhibit the expression of Cdc37 in Example 2 of the present invention.

[0020] Figure 4 This is a graph showing changes in the expression of MMP-3 and MMP-9 in CIA-FLS after TNF-α treatment detected by qPCR method in Example 3 of the present invention. DETAILED DESCRIPTION

[0021] In order to make the technical problems to be solved, technical solutions and advantages of the present invention more clear, they are described in detail below in conjunction with specific embodiments.

[0022] Example 1: Detection of Cdc37 expression level in collagen-induced arthritis model

[0023] 1. Establishment of collagen-induced arthritis model:

[0024] 1-1. The animals were randomly divided into two groups (control group and experimental group). The control group (n=10) did not undergo any experimental operation, while the experimental group (n=10) was emulsified with incomplete Freund's adjuvant and bovine type II collagen at a ratio of 1:1. Finally, 200ul of the mixture was injected intradermally into the base of the rat's tail.

[0025] After 1-2 and 7 days, 100ul of immune enhancer was injected in the same way at the same site. After 28 days, 300mg / kg chloral hydrate was injected intraperitoneally in the rat operating room of the Experimental Animal Center of Nantong University for anesthesia. No signs of peritonitis, pain or discomfort were observed after intraperitoneal injection of 10% chloral hydrate.

[0026] Second, the arthritis index (AI) was used for evaluation: no swelling 0 points, interphalangeal joint involvement 1 point, mild swelling of joints and toes 2 points, swelling of toes and ankles 3 points, and severe arthritis of the entire foot 4 points. Each limb has a maximum of 4 points, for a total of 16 points. If the total score exceeds 6 points, the modeling is successful.

[0027] 3. Extract primary synovial cells from rat knee synovium after successful modeling, observe histology, immunohistochemistry and immunoblot analysis. Paraffin sections were taken from the rat ankle joints. Rats were killed with an overdose of sodium chloral hydrate.

[0028] 3-1. Protein immunoblotting analysis: The proteins were loaded on SDS-polyacrylamide gel electrophoresis and then transferred to PVDF membrane. The membrane was incubated with specific antibodies overnight at 4°C and then incubated with secondary antibodies. Protein bands were detected using an ECL chemiluminescence kit.

[0029] 3-2. Immunohistochemistry: The tissue was dewaxed in xylene, then soaked in endogenous peroxidase blocker for 20 minutes, and then rinsed with PBS. The specific antibody was incubated at 4°C overnight. Diaminobenzidine was used as a chromogenic substrate. The immunostaining of the sections was then observed.

[0030] The results are as follows Figure 1As shown in the figure, compared with the control group, the appearance, H&E staining, and Safranin O staining of CIA rats showed joint swelling, cartilage erosion, pannus formation, and synovial hyperplasia. The expression of Cdc37 protein in the synovial tissue of the CIA rat model was detected by immuno-western blotting, which was significantly increased compared with the control group; it can be seen that the expression of Cdc37 in the synovial tissue of the collagen-induced arthritis model was significantly increased. The results of immunohistochemical analysis were consistent with those of immuno-blotting.

[0031] Example 2: In vitro knockdown of Cdc37 inhibits FLS proliferation

[0032] 1. Isolation and culture of FLS: Wash fresh synovial tissue twice by centrifugation (2500r / min, 6min), discard the supernatant, aspirate collagenase II into a centrifuge tube, gently blow to resuspend the tissue blocks, put them into a CO2 incubator for digestion, observe the tissue into flocculence, add 1mL of 20% FBS to terminate digestion, centrifuge (2500r / min, 6min), discard the supernatant, put them into a culture bottle, use a small amount of 20% FBS to artificially adhere to the wall, and then add 3mL of 20% FBS in a CO2 incubator for culture. This experiment used cells of the 3rd to 8th generations.

[0033] 2. Cdc37-siRNA transfection of FLS: The cells were seeded in 6-well plates at a density of 40%, and NC-siRNA and Cdc37-siRNA were transfected into FLS using Lip2000, and the culture medium was replaced with DMEM-F12 containing 20% ​​fetal bovine serum after 6 hours. The cells were further cultured for 48 hours and used for subsequent experiments.

[0034] The sequences of Cdc37-siRNA include:

[0035] Cdc37-siRNA#1: 5'-GGACUACAGCGUUUGGGAUTT-3', 5'-AUCCCAAACGCUGUAGUCCTT-3';

[0036] Cdc37-siRNA#2: 5'-CGAAUAGAGAAGGCCAUGATT-3', 5'-UCAUGGCCUUCUCUAUUCGTT-3',

[0037] Cdc37-siRNA#3: 5'-CUUCGAGAAAGACAUCAGUTT-3', 5'-ACUGAUGUCUUUCUCGAAGTT-3';

[0038] NC-siRNA: 5'-UUC UCC GAA CGU GUC ACG UTT-3', 5'-ACG UGA CAC GUU CGGAGAATT-3'.

[0039] EdU staining: FLS after Cdc37-siRNA transfection were stained with 5×10 4 The cells were seeded at a density of 100 μl / well in a 96-well plate overnight, and then analyzed using the EdU DNA Cell Proliferation Kit according to the instructions.

[0040] Cell cycle analysis: After Cdc37-siRNA transfection, cells were washed with PBS and fixed with 70% ethanol at -20°C overnight. Cells were incubated with RNaseA (1 mg / ml) at 37°C for 30 minutes and then stained with propidium iodide (50 μg / ml) for 20 minutes. Finally, cells were analyzed using FACSCalibur (BD).

[0041] The results are as follows Figure 2 and Figure 3 As shown, Figure 2 As shown in Figure 2, Cdc37 is highly expressed in CIA-FLS. After TNF-α stimulation of CIA-FLS, the expression of Cdc37 increased in a time-dependent manner, reaching a peak at around 48h, and the expression of CyclinD1 and PCNA also increased in a time-dependent manner. Figure 3 As shown in the figure, after TNF-α stimulation, the proliferation rate of FLS in the Cdc37-siRNA group was significantly lower than that in the control group. At the same time, the expression of CyclinD1 and PCNA decreased. It can be seen that after transfection of Cdc37-siRNA, Cdc37 was knocked down. After knocking down Cdc37, the proliferation ability of FLS was significantly reduced, and the expression of proliferation-related markers CyclinD1 and PCNA decreased.

[0042] pass Figure 2 and Figure 3 It can be seen that among the three groups of Cdc37-siRNA, Cdc37-siRNA#2 can significantly knock down Cdc37, the proliferation ability of FLS is significantly reduced, and the expression of proliferation-related markers CyclinD1 and PCNA is most significantly reduced, which can be used as the preferred Cdc37-siRNA inhibitor.

[0043] Example 3: Knockdown of Cdc37 expression inhibits the inflammatory response and migration and invasion ability of FLS.

[0044] 1. RNA isolation and quantitative polymerase chain reaction: Total RNA was extracted using the UNlQ-10 column Trizol total RNA isolation kit, and then the concentration was determined. After reverse transcription into cDNA using HiScriptIIQRTSuperMorqPCR(+), the expression of mRNA was analyzed by Q-PCR amplification. The relative quantitative algorithm 2-ΔΔCt method (Livak method) was used to perform ratio analysis and comparison of the target gene (MMP) in the experimental group and the housekeeping gene (GAPDH) in the control group. The primers used are as follows:

[0045]

[0046] Cell scratching: FLS were placed in a 6-well plate and incubated with serum-free DMEM-F12 for 12 hours after transfection for 48 hours. The cell monolayer was scratched with a 10 μl sterile tip. After scraping, the culture medium was immediately switched to fresh medium and the cells were photographed under a microscope after 24 hours of culture.

[0047] Transwell experiment: After transfecting FLS with Cdc37-siRNA, resuspend them in serum-free DMEM-F12 medium to a final concentration of 2×104 / mL. Place 200 μL of cell suspension in the upper chamber of the Transwell, and 600 μL of DMEM-F12 medium containing 10% fetal bovine serum in the lower chamber. After 12 hours, fix the cells attached to the bottom of the membrane with 4% paraformaldehyde, stain with crystal violet, and finally observe under a microscope.

[0048] The results are as follows Figure 4 As shown in the figure, Cdc37-siRNA significantly inhibited the release of MMP-3 and MMP-9 in CIA-FLS mediated by TNF-α. The results of the cell scratch assay showed that the knockdown of Cdc37 significantly reduced cell migration. In addition, the Transwell experiment showed that silencing Cdc37 could significantly inhibit the invasion ability of CIA-FLS after TNF-α treatment. It can be seen that after Cdc37-siRNA (Cdc37-siRNA#2) transfection, the expression of MMP-3 and MMP-9 in FLS was significantly decreased by knocking down Cdc37, and its migration and invasion ability were also inhibited.

[0049] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> The Affiliated Hospital of Nantong University <120> Application of Cdc37 in the prevention or treatment of rheumatoid arthritis <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty one <212> DNA / RNA <213> Cdc37-siRNA#1 positive strand sequence (Cdc37-siRNA#1-F ) <400> 1 ggacuacagc guuugggaut t 21 <210> 2 <211> twenty one <212> DNA / RNA <213> Cdc37-siRNA#1 antisense sequence (Cdc37-siRNA#1-R) <400> 2 aucccaaacg cuguagucct t 21 <210> 3 <211> twenty one <212> DNA / RNA <213> Cdc37-siRNA#2 positive strand sequence (Cdc37-siRNA#2-F ) <400> 3 cgaauagaga aggccaugat t 21 <210> 4 <211> twenty one <212> DNA / RNA <213> Cdc37-siRNA#2 antisense sequence (Cdc37-siRNA#2-R) <400> 4 ucauggccuu cucuauucgt t 21 <210> 5 <211> twenty one <212> DNA / RNA <213> Cdc37-siRNA#3 positive strand sequence (Cdc37-siRNA#3-F ) <400> 5 cuucgagaaa gacaucagut t 21 <210> 6 <211> twenty one <212> DNA / RNA <213> Cdc37-siRNA#3 antisense sequence (Cdc37-siRNA#3-R ) <400> 6 acugaugucu uucucgaagt t 21 <210> 7 <211> twenty one <212> DNA / RNA <213> NC-siRNA positive strand sequence (NC-siRNA -F) <400> 7 uucuccgaac gugucacgut t 21 <210> 8 <211> twenty one <212> DNA / RNA <213> NC-siRNA antisense sequence (NC-siRNA -R) <400> 8 acgugacacg uucggagaat t 21

Claims

1. Use of an inhibitor of Cdc37 gene in the preparation of a drug for preventing or treating rheumatoid arthritis, characterized in that: The inhibitor of the Cdc37 gene is selected from Cdc37-siRNA specific to the Cdc37 gene, and the sequence of the Cdc37-siRNA is as follows: The sequence of the Cdc37-siRNA sense strand was 5′-CGAAUAGAGAAGGCCAUGATT-3′; The sequence of the Cdc37-siRNA antisense strand was 5′-UCAUGGCCUUCUCUAUUCGTT-3′.

2. The use according to claim 1, characterized in that: Cdc37 gene inhibitors reduce the expression of Cdc37 in FLS, thereby inhibiting the proliferation of FLS and weakening the inflammatory response and migration ability of FLS, thereby achieving the effect of preventing / treating RA.

3. Use of an inhibitor targeting the Cdc37 gene in the preparation of a targeted therapeutic preparation for rheumatoid arthritis, characterized in that: The inhibitor is the Cdc37-siRNA selected from the Cdc37 gene specificity as described in claim 1, and the rheumatoid arthritis targeted therapeutic preparation is obtained by preparing a Cdc37-specific interfering sequence, and the Cdc37-specific interfering sequence is used to knock down the expression of Cdc37 in cells.

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