CN113970641A

CN113970641A - Application of Cdc37 in preventing or treating rheumatoid arthritis

Info

Publication number: CN113970641A
Application number: CN202111256998.8A
Authority: CN
Inventors: 徐华; 陈民浩; 孙魏巍; 王友华
Original assignee: Affiliated Hospital of Nantong University
Current assignee: Affiliated Hospital of Nantong University
Priority date: 2021-10-27
Filing date: 2021-10-27
Publication date: 2022-01-25

Abstract

The invention discloses application of Cdc37 (cell division cyclin 37) as a target for preventing and treating rheumatoid arthritis. The invention also discloses application of the Cdc37 gene inhibitor in preparation of a medicament for preventing or treating 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 reaction and migration capability of FLS, thereby achieving the effect of preventing/treating RA. The invention provides application of Cdc37 as a target for preventing and treating rheumatoid arthritis, and finds that Cdc37 is highly expressed in RA synovial tissue for the first time, and the inhibition of Cdc37 can obviously inhibit FLS proliferation and weaken the inflammatory reaction and migration capacity of FLS. Cdc37 has great potential as a specific diagnosis and treatment target of RA patients and provides a theoretical basis for developing new anti-RA drugs.

Description

Application of Cdc37 in preventing or treating rheumatoid arthritis

Technical Field

The invention belongs to the technical field of biological medicines, and relates to application of Cdc37 (cell division cyclin 37) in prevention or treatment of rheumatoid arthritis, in particular to application of Cdc37 serving as a target point for prevention and treatment of rheumatoid arthritis, application of a Cdc37 gene inhibitor in preparation of a medicament for prevention or treatment of rheumatoid arthritis, application of a Cdc37 gene inhibitor in preparation of a medicament for prevention or treatment of rheumatoid arthritis, and application of Cdc37 in screening of medicaments for prevention/treatment of rheumatoid arthritis.

Background

Rheumatoid Arthritis (RA) is a multi-system inflammatory autoimmune disease that mainly affects peripheral joints. RA is stubborn in illness and extremely high in disability rate, and the progress to the later stage seriously affects daily life and working capacity, so that RA is one of main diseases causing labor loss and disability in China. 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 has the same proliferative capacity as tumor cells, which is one of the pathogenesis of abnormal RA cell proliferation. Cytokines and other factors secreted by FLS, such as Interleukin (IL) -6, IL-1 β, Matrix Metalloproteinase (MMP) -3, and MMP-9, are thought to play a major role in the development of sustained inflammation, synovial hyperplasia, and joint destruction. At present, the exact cause of the disease is unknown. The inflammatory process of RA is very complex, and is a complex immune inflammatory process involving various cytokines, adhesion molecules, chemokines, and the like, and biological treatment of RA has been developed sufficiently in recent years, but its therapeutic effect is still very limited. Therefore, the search for RA-specific preventive/therapeutic targets is imperative.

Cyclin 37(Cdc37) is involved as a chaperone in many physiological processes, such as cell cycle progression, cell signaling, cell proliferation. Cdc37 recruits labile kinases to Hsp90 and regulates cellular signaling pathways such as the ERK and PI3K/AKT signaling pathways that have been shown to be closely related to the development of RA, however, the biological function of Cdc37 in RA has not been reported. If Cdc37 can be used as a specific target for treating RA and corresponding drugs are developed, the Cdc37 has important significance for the diagnosis and treatment of RA.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems or the defects in the prior art, the invention provides the application of Cdc37 (cell division cyclin 37) in the prevention or treatment of rheumatoid arthritis, in particular relates to the application of Cdc37 as the prevention and treatment target of rheumatoid arthritis, the application of Cdc37 gene inhibitor in the preparation of drugs for preventing or treating rheumatoid arthritis, the application of Cdc37 gene inhibitor in the preparation of drugs for preventing or treating rheumatoid arthritis, and the application of Cdc37 in the screening of drugs for preventing or treating rheumatoid arthritis.

In order to achieve the above object, the present invention provides the application of Cdc37 as a target for preventing and treating rheumatoid arthritis.

The embodiment of the invention also provides application of the Cdc37 gene inhibitor in preparing a medicament for preventing or treating rheumatoid arthritis.

Furthermore, the inhibitor of the Cdc37 gene reduces the expression of Cdc37 in FLS, thereby inhibiting the proliferation of FLS and weakening the inflammatory reaction and migration capacity of FLS, thereby achieving the effect of preventing/treating RA.

Further, the inhibitor of the Cdc37 gene is selected from Cdc37-siRNA specific to Cdc37 gene.

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

cdc37-siRNA sense strand sequence is 5'-CGAAUAGAGAAGGCCAUGATT-3';

cdc37-siRNA antisense strand sequence is 5'-UCAUGGCCUUCUCUAUUCGTT-3'.

The embodiment of the invention also provides a reagent or a kit for diagnosing rheumatoid arthritis, which is characterized by comprising an antibody with detection specificity on Cdc37 and an immunohistochemical kit.

The embodiment of the invention also provides application of the Cdc37 in screening of drugs for preventing/treating rheumatoid arthritis, which is characterized in that a reagent or a kit for detecting Cdc37 expression quantity is adopted in screening; the biological sample used was synovial tissue.

The embodiment of the invention also provides a Cdc37 protein detection method, which is characterized by comprising the following steps: (1) the method comprises the steps of (1) extracting synovial membrane tissues and FLS, (2) preparing paraffin sections, (3) obtaining total protein by a protein cracking method, measuring protein concentration, and detecting expression of Cdc37 by an immune protein blotting method and immunohistochemistry.

The embodiment of the invention also provides application of the Cdc37 gene in preparation of a rheumatoid arthritis targeted therapy preparation, which is characterized in that the rheumatoid arthritis targeted therapy preparation is obtained by preparing a Cdc37 specific interference sequence, and the Cdc37 specific interference sequence is used for knocking down Cdc37 to express in cells.

The technical scheme of the invention has the following beneficial effects: the invention provides an application of Cdc37 in RA prevention/treatment medicines. The examples of the present invention demonstrate: the expression of Cdc37 in RA synovial tissue is obviously increased, the Cdc37 specific interference sequence of the invention obviously reduces the expression of Cdc37 in FLS, and simultaneously obviously inhibits the proliferation, inflammatory reaction and migration capability of FLS. The invention firstly verifies the expression and biological functions of Cdc37 in RA, and the discovery further perfects the research of RA pathogenesis. The invention provides a new theoretical basis for developing and treating RA drugs, and screens out the Cdc37 specific inhibitor as a candidate drug for RA prevention/treatment by taking Cdc37 as a drug target, thereby laying a foundation for clinically improving the RA treatment effect.

Drawings

FIG. 1 is a graph showing the Arthritis score on rats on day 2 after the booster immunization, in which a Collagen-Induced Arthritis model (CIA) was established in example 1 of the present invention.

FIG. 2 is a statistical chart of Cdc37 expression of primary CIA-FLS extracted and cultured in example 2 of the present invention.

FIG. 3 is a graph showing the proliferation of CIA-FLS by EdU staining and flow cytometry after Cdc37-siRNA was used to inhibit the expression of Cdc37 in example 2 of the present invention.

FIG. 4 is a graph showing the change in MMP-3 and MMP-9 expression in CIA-FLS after TNF- α treatment using qPCR in example 3 of the present invention.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.

Example 1: detection of Cdc37 expression levels in collagen-induced arthritis model

Firstly, establishing a collagen-induced arthritis model:

1-1, animals were randomized into two groups (control and experimental). The control group (n ═ 10) did not undergo any experimental manipulations, while the experimental group (n ═ 10) was treated with incomplete freund's adjuvant and bovine type II collagen in a 1: 1, and emulsifying. Finally, 200ul of the mixture was injected into the rat tail root by intradermal injection.

After 1-2, 7 days, 100ul of immunopotentiator was injected at the same site in the same manner. After 28 days, 300mg/kg chloral hydrate was intraperitoneally injected into the rat operating room at the laboratory animal center of southeast university for anesthesia. No signs of peritonitis, pain or discomfort were observed following intraperitoneal injection of 10% chloral hydrate.

And II, evaluating by using an Arthritis Index (AI): there is no swelling at 0 point, interphalangeal joints are affected at 1 point, joints and toes are slightly swollen at 2 points, toes and ankles are swollen at 3 points, and severe arthritis of the whole foot is severe at 4 points. Each limb has a maximum of 4 points, 16 points in total. If the total score exceeds 6 points, the modeling is successful.

And thirdly, extracting primary synovial cells by adopting the synovial membrane of the knee joint of the rat after successful modeling, and performing histology, immunohistochemical observation and immunoblot analysis. Taking rat ankle joints for paraffin section. Rats were sacrificed with excess chloral sodium hydrate.

3-1, Western blot analysis: the proteins were loaded on SDS-polyacrylamide gel electrophoresis and then transferred to PVDF membrane. The membrane was incubated with the specific antibody overnight at 4 ℃ and then with the secondary antibody. And detecting the protein band by using an ECL chemiluminescence kit.

3-2, immunohistochemistry: tissues were deparaffinized in xylene, then soaked with endogenous peroxidase blockers for 20 minutes, and then rinsed with PBS. The specific antibody was allowed to stand overnight at 4 ℃. Diaminobenzidine was used as the chromogenic substrate. The sections were then observed for immunostaining.

The results are shown in FIG. 1, and compared to the control group, appearance, H & E staining, safranin O staining of CIA rats showed joint swelling, cartilage erosion, pannus formation, and synovial membrane hyperplasia. The expression of the Cdc37 protein in the synovial tissue of the CIA rat model is detected by an immune protein blotting method, and is obviously increased compared with a control group; it can be seen that Cdc37 expression was significantly increased in synovial tissue in the collagen-induced arthritis model. Immunohistochemical analysis was consistent with the results of immunoblotting.

Example 2: in vitro knockdown of Cdc37 inhibits FLS proliferation

Firstly, FLS separation and culture: centrifuging and washing fresh synovial tissue for 2 times (2500r/min, centrifuging for 6min), sucking supernatant, sucking collagenase II into a centrifuge tube, lightly blowing to resuspend tissue blocks, putting the tissue blocks into a CO2 incubator for digestion, observing the tissue blocks to be flocculent, adding 1mL of FBS with the volume fraction of 20% to stop digestion, centrifuging (2500r/min, centrifuging for 6min), removing supernatant, putting the tissue blocks into a culture bottle, manually attaching a small amount of FBS with the volume fraction of 20% to the wall, adding 3mL of FBS with the volume fraction of 20% to culture in a CO2 incubator, and adopting cells of 3 rd to 8 th generations in the experiment.

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

The sequence of Cdc37-siRNA includes:

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

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

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

NC-siRNA：5'-UUC UCC GAA CGU GUC ACG UTT-3'，5'-ACG UGA CAC GUU CGG AGAATT-3'。

EdU staining: cdc 37-FLS after siRNA transfection at 5X 10⁴The cells were plated in 96-well plates overnight at a density of 100. mu.l. The EDU DNA cell proliferation kit was then used for analysis as per instructions.

Cell cycle analysis: after transfection with Cdc37-siRNA, cells were washed with PBS and fixed with 70% ethanol overnight at-20 ℃. Cells were incubated with RNaseA (1mg/ml) at 37 ℃ for 30 min and then stained with propidium iodide (50. mu.g/ml) for 20 min. Finally, cells were analyzed using facscalibur (bd).

The results are shown in FIG. 2 and FIG. 3, and Cdc37 is highly expressed in CIA-FLS as shown in FIG. 2. After TNF-alpha is used for stimulating CIA-FLS, the expression of Cdc37 is increased in a time-dependent manner, the expression reaches a peak value around 48h, and the expression of CyclinD1 and PCNA is also increased in a time-dependent manner. As shown in FIG. 3, the proliferation rate of FLS was significantly lower in Cdc37-siRNA group than in control group after TNF- α stimulation. And the expression of CyclinD1 and PCNA is reduced. Therefore, after transfection of Cdc37-siRNA, Cdc37 is knocked down, and after Cdc37 is knocked down, the proliferation capacity of FLS is obviously reduced, and meanwhile, the expression of proliferation related markers CyclinD1 and PCNA is reduced.

As can be seen from FIG. 2 and FIG. 3, among the 3 Cdc 37-siRNAs, Cdc37-siRNA #2 can obviously knock down Cdc37, the proliferation capability of FLS is obviously reduced, and the expression of proliferation-related markers CyclinD1 and PCNA is most obviously reduced, so that the Cdc37-siRNA can be preferably used as a Cdc37-siRNA inhibitor.

Example 3: knockdown of Cdc37 expression inhibited the inflammatory response and migratory invasion capacity of FLS.

Firstly, RNA separation and quantitative polymerase chain reaction: total RNA was extracted using a UNlQ-10 column Trizol Total RNA isolation kit, and then the concentration was determined. After reverse transcription into cDNA using HiScriptIIQRTSeupermMorqPCR (+), mRNA expression was analyzed by Q-PCR amplification. The ratio analysis and comparison of the experimental group target gene (MMP) and the control group housekeeping Gene (GAPDH) are carried out by adopting a relative quantitative algorithm 2-delta Ct method (Livak method). The primers used were as follows:

cell scratching: FLS was placed in 6-well plates and 48 hours after transfection, incubated with serum-free DMEM-F12 for 12 hours. The cell monolayer was scratched with a 10 μ l sterile tip. Immediately after scraping, the medium was switched to fresh medium and the cells were cultured for 24 hours before taking pictures under the microscope.

Transwell experiment: after transfection of FLS with Cdc37-siRNA, it was resuspended in serum-free DMEM-F12 medium to a final concentration of 2X 104/mL. 200 microliters of the cell suspension was placed in the upper chamber of a Transwell, and the lower chamber was 600 μ L of DMEM-F12 medium containing 10% fetal bovine serum. After 12 hours the cells adhering to the bottom of the membrane were fixed with 4% paraformaldehyde, stained with crystal violet and finally observed with a microscope.

The results are shown in FIG. 4, Cdc37-siRNA significantly inhibited TNF- α mediated release of MMP-3 and MMP-9 from CIA-FLS. Cell scratch test results show that the knock-down of Cdc37 significantly reduced cell migration. In addition, Transwell experiments show that silencing Cdc37 can obviously inhibit the invasion capacity of CIA-FLS after TNF-alpha treatment. Therefore, Cdc37-siRNA (Cdc37-siRNA #2) is transfected, Cdc37 is knocked down, the expressions of MMP-3 and MMP-9 in FLS are obviously reduced, and the migration and invasion capabilities of the FLS are also inhibited.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

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Claims

The application of Cdc37 (cell division cyclin 37) as a target for preventing and treating rheumatoid arthritis.
Application of Cdc37 gene inhibitor in preparing medicine for preventing and treating rheumatoid arthritis.
3. The use as claimed in claim 2, wherein the inhibitor of Cdc37 gene reduces Cdc37 expression in FLS, thereby inhibiting FLS proliferation and reducing FLS inflammatory response and migration ability, thereby achieving the effect of preventing/treating RA.
4. The use as claimed in claim 2, wherein the inhibitor of the Cdc37 gene is selected from Cdc37-siRNA specific to Cdc37 gene.
5. The use of claim 4, wherein the Cdc37-siRNA has the following sequence:

cdc37-siRNA sense strand sequence is 5'-CGAAUAGAGAAGGCCAUGATT-3'; cdc37-siRNA antisense strand sequence is 5'-UCAUGGCCUUCUCUAUUCGTT-3'.
6. A reagent or a kit for diagnosing rheumatoid arthritis, characterized by comprising an antibody having detection specificity for Cdc37 and an immunohistochemical kit.
The application of the Cdc37 in screening the medicines for preventing/treating the rheumatoid arthritis is characterized in that a reagent or a kit for detecting the expression quantity of Cdc37 is adopted in screening; the biological sample used was synovial tissue.
8. A Cdc37 protein detection method is characterized by comprising the following steps: (1) the method comprises the steps of (1) extracting synovial membrane tissues and FLS, (2) preparing paraffin sections, (3) obtaining total protein by a protein cracking method, measuring protein concentration, and detecting expression of Cdc37 by an immune protein blotting method and immunohistochemistry.
9. An application of a Cdc37 gene in preparation of a rheumatoid arthritis targeted therapy preparation is characterized in that the rheumatoid arthritis targeted therapy preparation is obtained by preparing a Cdc37 specific interference sequence, and the Cdc37 specific interference sequence is used for knocking down Cdc37 to express in cells.