Application of TAOK3 biomarker in diagnosis, treatment and prognosis monitoring of rheumatoid arthritis

Abstract

The invention relates to the technical field of molecular biology of rheumatism immune diseases, in particular to application of a TAOK3 biomarker in diagnosis, treatment and prognosis monitoring of rheumatoid arthritis. According to the application disclosed by the invention, the difference of the TAOK3 protein in patients with rheumatoid arthritis (RA) in an active stage and a remission stage is found for the first time through combination of high-depth plasma proteome and single cell sequencing data, and the TAOK3 protein is verified to be capable of mediating bone destruction in cell and animal experiments for the first time, which indicates that the TAOK3 protein can be used for monitoring the bone destruction of the recurrent RA; potential molecular markers closely related to RA bone destruction are disclosed through differential protein analysis and trend clustering analysis; random forest analysis shows that the diagnosis accuracy (AUC = 0.915) of the recurrent RA can be remarkably improved by combining the TAOK3 protein with the CRP, and the diagnosis accuracy is superior to that of any marker which is independently used.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology for rheumatic and immunological diseases, and in particular to the application of a TAOK3 biomarker in the diagnosis, treatment and prognostic monitoring of rheumatoid arthritis (RA). Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by systemic inflammation and joint destruction. Without proper treatment, RA can lead to severe joint deformities, functional impairment, and a decline in quality of life. Bone destruction is a typical pathological feature of RA, including bone erosion and periarticular bone loss, which in severe cases can cause irreversible damage to joint function.

[0003] Plasma proteins are core components of blood that perform vital biological functions, playing a crucial role in various physiological and pathological processes. In recent years, blood proteomics technology has been widely applied in disease research, providing new possibilities for clinical translation and precision medicine. The application of proteomics technology has opened up new avenues for disease research, enabling not only the identification of potential biomarkers and therapeutic targets but also a deeper understanding of the intrinsic mechanisms of diseases.

[0004] The etiology and pathogenesis of rheumatoid arthritis (RA) are complex. To further explore the pathogenesis of RA, in recent years, an increasing number of experts and scholars have used proteomics methods to systematically analyze samples from synovial membranes, synovial fluid, and peripheral blood of RA patients, aiming to discover key biomarkers and elucidate its pathological mechanisms. Tabushi et al. studied the synovial fluid of RA patients using MALDI-TOF / MS proteomics analysis and found significantly elevated expression levels of citrullinated fibrinogen, vimentin, and fibronectin, and that these proteins are closely related to the pathogenesis of RA. Segigawa et al. analyzed plasma proteins in RA patients after anti-TNF-α treatment using 2D-LC-MS / MS technology and discovered several differentially expressed proteins associated with RA pathogenesis. Among them, FAM62A and MBC2 are associated with TNF-α-mediated activation of the nuclear factor-kappa B (NF-κB) pathway, while CTGF is closely related to articular cartilage metabolism. Hu, by comparing serum protein abundance in normal individuals and RA patients, found a significant difference in ORM1 expression, which was positively correlated with disease activity. Studies by Liu et al. have shown that FGL1 is a novel biomarker capable of predicting RA disease activity and prognosis. However, current research on blood proteomics targeting RA bone destruction-related proteins is still lacking, and further exploration is urgently needed to reveal the potential mechanisms of bone destruction and discover new monitoring and therapeutic targets, thereby improving clinical management and enhancing long-term patient outcomes.

[0005] Therefore, while some differentially expressed proteins associated with the pathogenesis of rheumatoid arthritis (RA) have been identified, in-depth research on specific biomarkers and therapeutic targets for RA bone destruction remains lacking. Thus, developing new biomarkers for the diagnosis and monitoring of RA bone destruction, exploring new therapeutic targets, and developing corresponding auxiliary diagnostic reagents are of significant clinical value for improving early diagnosis, targeted therapy, and prognostic monitoring of RA patients. Summary of the Invention

[0006] In view of this, the purpose of this invention is to propose the application of TAOK3 biomarker in the diagnosis, treatment and prognosis monitoring of rheumatoid arthritis (RA), so as to solve the problem of the lack of specific biomarkers and therapeutic targets for bone destruction and disease activity in RA in the prior art.

[0007] To achieve the above objectives, this invention provides an application of the TAOK3 biomarker in the diagnosis, treatment, and prognosis monitoring of rheumatoid arthritis; the biomarker is the TAOK3 protein.

[0008] The amino acid sequence of the TAOK3 protein (SEQ ID NO:1):

[0009] MRKGVLKDPEIADLFYKDDPEELFIGLHEIGHGSFGAVYFATNAHTSEVVAIKKMSYSGKQTHEKWQDILKEVKFLRQLKHPNTIEYKGCYLKEHTAWLVMEYCLGSASDLLEVHKKPLQEVEIAAITHGALHGLAYLHSHALIHRDIKAGNILLTEPGQVKLADFGSASMASPANSFVGTPYWMAPEVILAMDEGQYDGKVDIWSLGITCIELAERKPPLFNMNAMSALYHIAQNDSPTLQSNEWTDSFRRFVDYCLQKIPQERPTSAELLRHDFVRRDRPLRVLIDLIQRTKDAVRELDNLQYRKMKKILFQETRNGPLNESQEDEEDSEHGTSLNREMDSLGSNHSIPSMSVSTGSQSSSVNSMQEVMDESSSELVMMHDDESTINSSSSVVHKKVGFLVPSTEDHVFIRDEAGHGDPRPEPRPTQSVQSQALHYRNRERFATIKSASLVTRQIHEHEQENELREQMSGYKRMRRQHQKQLIALENKLKAEMDEHRLKLQKEVETHANNSSIELEKLAKKQVAIIEKEAKVAAADEKKFQQQILAQQKKDLTTFLESQKKQYKICKEKIKEEMNEDHSTPKKEKQERISKHKENLQHTQAEEEAHLLTQQRLYYDKNCRFFKRKIMIKRHEVEQQNIREELNKKRTQKEMEHAMLIRHDESTRELEYRQLHTLQKLRMDLIRLQHQTELENQLEYNKRRERELHRKHVMELRQQPKNLKAMEMQIKKQFQDTCKVQTKQYKALKNHQLEVTPKNEHKTILKTLKDEQTRKLAILAEQYEQSINEMMASQALRLDEAQEAECQALRLQLQQEMELLNAYQSKIKMQTEAQHERELQKLEQRVSLRRAHLEQKIEEELAALQKERSERIKNLLERQEREIETFDMESLRMGFGNLVTLDFPKEDYR;

[0010] The mRNA sequence of the TAOK3 gene (SEQ ID NO:2):

[0011]

[0012] It should be noted that this invention, by analyzing the molecular differences between the relapse and remission phases of RA, further reveals the potential pathological mechanisms of disease relapse and provides important clues for the development of novel therapeutic targets. To explore novel monitoring and therapeutic targets for RA bone destruction in plasma, this invention employed high-resolution mass spectrometry with data-independent acquisition (DIA) to conduct in-depth analysis of 200 plasma samples, systematically depicting the proteomic characteristics of RA remission and relapse phases. Furthermore, by combining single-cell sequencing results, multiplex immunohistochemistry, ELISA, and in vitro and in vivo functional validation, the inventors have for the first time confirmed and revealed the potential of TAO kinase 3 (TAOK3) as a novel monitoring and therapeutic target for RA bone destruction.

[0013] Furthermore, the TAOK3 biomarker is applied to the diagnosis, treatment, and prognostic monitoring of bone destruction in recurrent rheumatoid arthritis.

[0014] This application is the first to discover TAOK3 protein as a novel biomarker for bone destruction in recurrent rheumatoid arthritis (RA): through high-depth plasma proteomics combined with single-cell sequencing analysis, it was found for the first time that TAOK3 protein is significantly highly expressed in the plasma of patients with active RA and is positively correlated with the Sharp score of bone destruction, filling the gap in blood proteomics research on RA bone destruction; this discovery provides a new molecular target for the early monitoring of RA bone destruction.

[0015] This application further confirms that TAOK3 protein can serve as a therapeutic target for recurrent RA bone destruction: cell and animal experiments have demonstrated that knocking down TAOK3 protein can significantly inhibit the proliferation of RA-FLS and promote its apoptosis, and TAOK3 targeting inhibitors can effectively alleviate bone destruction in CIA mice; this discovery provides a new direction for drug development in the treatment of RA bone destruction and expands the range of targets for RA treatment.

[0016] The application of a TAOK3 biomarker in the preparation of reagents or kits for detecting bone destruction in rheumatoid arthritis.

[0017] Furthermore, the reagent or kit determines the bone destruction and disease activity of rheumatoid arthritis by detecting the expression level of TAOK3 protein in the subject's plasma; when the expression level of TAOK3 protein is elevated, it is determined that the subject has bone destruction of rheumatoid arthritis or is in the active phase of the disease.

[0018] Furthermore, the detection method includes qualitative or quantitative analysis of TAOK3 protein in plasma using data-independent acquisition proteomics technology or ELISA method.

[0019] Application of a TAOK3 biomarker in the preparation of a drug for treating bone destruction in rheumatoid arthritis.

[0020] Furthermore, the drug targets the TAOK3 protein and contains components that inhibit the expression or activity of the TAOK3 protein, thereby achieving the therapeutic purpose of treating rheumatoid arthritis by inhibiting the expression or activity of the TAOK3 protein.

[0021] Furthermore, the drug contains a TAOK3 protein-targeting inhibitor, such as SBI-581.

[0022] The application of a TAOK3 and CRP biomarker in the combined preparation of a reagent or kit for detecting bone destruction in recurrent rheumatoid arthritis.

[0023] Furthermore, by simultaneously detecting the expression levels of TAOK3 protein and CRP in the plasma of subjects, the diagnostic accuracy of recurrent RA was improved. Random forest analysis showed that the AUC value for diagnosing recurrent RA when TAOK3 and CRP were used in combination reached 0.915, which was superior to using TAOK3 protein or CRP alone. This combined detection strategy provides a more reliable method for the accurate clinical diagnosis of recurrent RA, and can improve the accuracy and timeliness of disease diagnosis.

[0024] The beneficial effects of this invention are:

[0025] This invention is the first to discover the difference in TAOK3 protein between patients with active and remission RA using high-depth plasma proteomics combined with single-cell sequencing data, and the first to verify in cell and animal experiments that TAOK3 protein can mediate bone destruction, indicating that TAOK3 protein can monitor bone destruction in recurrent RA; and through differential protein analysis and trend clustering analysis, it reveals potential molecular markers closely related to bone destruction in RA.

[0026] Furthermore, random forest analysis showed that the combination of TAOK3 protein and CRP significantly improved the diagnostic accuracy of recurrent RA (AUC = 0.915), which was superior to using either biomarker alone.

[0027] The inventors also discovered that knocking down the TAOK3 encoding gene can significantly inhibit the proliferation of RA-FLS and promote its apoptosis; at the same time, targeted inhibition of TAOK3 protein can effectively alleviate bone destruction in CIA mice, providing a new approach for the treatment of RA bone destruction. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1-A , 1-B : Flowchart of DIA proteomics analysis and differential expression diagram of TAOK3 protein in the plasma of RA patients in active and remission phases; among which, Figure 1-A For data-independent acquisition (DIA) proteomics analysis workflow; Figure 1-B A graph showing the differential expression of TAOK3 in the plasma of RA patients in active and remission phases;

[0030] Figure 2 : Spearman correlation analysis results between plasma differentially expressed proteins and Sharp bone destruction scores during active and remission phases; red dots indicate proteins with significant correlation (P<0.05), and the top 10 proteins with the strongest correlation are marked;

[0031] Figure 3 Multicolor immunohistochemistry and ELISA were used to verify the expression levels of TAOK3 protein in synovial tissue and paired plasma samples from patients with active and remission RA.

[0032] Figure 4 Random forest model, ROC curve and correlation validation plot of important expressed proteins related to bone destruction;

[0033] Figure 5 Figure: Analysis of the effects of TAOK3 gene knockdown on the biological function of synovial fibroblasts (RA-FLS) in rheumatoid arthritis;

[0034] Figure 6 The effect of targeted inhibition of TAOK3 protein on bone destruction in CIA mice. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0036] like Figure 1-A As shown, the following embodiments of the present invention employ data-independent acquisition (DIA) proteomics technology to analyze plasma samples (development set) from 80 patients with sustained remission of RA, 80 patients with relapsed RA, and 40 healthy controls. In addition, plasma samples from 50 RA patients (validation set 1) and 10 RA patients (validation set 2) were recruited independently from the outpatient and inpatient departments, respectively.

[0037] Embodiments 1-9 of this invention utilize Orbitrap TM Astral TM High-depth plasma proteomic analysis was performed on 200 plasma samples using a high-depth plasma proteomic analyzer. Differential protein analysis and trend clustering analysis revealed potential molecular markers closely related to bone destruction in rheumatoid arthritis (RA). Furthermore, key bone destruction proteins were screened by combining bone destruction Sharp scores and single-cell RNA sequencing data, and validated in plasma and synovial tissue samples by multiplex immunohistochemistry and ELISA.

[0038] Meanwhile, the effects of small interfering RNA (siRNA) knockdown on TAOK3 expression on RA-FLS proliferation and apoptosis were evaluated. The effects of TAOK3 on the biological behavior of RA-FLS were assessed using CCK-8 assays and flow cytometry, and the expression levels of MMPs were analyzed by Western blotting. In Example 10, the therapeutic effect of the TAOK3-targeting inhibitor on joint bone destruction was further verified in a mouse CIA model. Micro-CT, H&E staining, and Safranin-Fix-Green staining techniques were used to evaluate the ameliorative effect of TAOK3-targeting inhibition on joint bone erosion and histological damage.

[0039] Example 1: Collection and Processing of Plasma Samples

[0040] 1.1 Plasma samples from all RA patients were collected prior to the initiation of any clinical treatment, and fasting blood samples were collected from all participants;

[0041] 1.2 After the samples were allowed to stand for 1 hour to separate into layers, the blood samples were centrifuged at 3000 r / min for 10 min at room temperature. After separating the plasma, the samples were aliquoted into Eppendorf tubes and immediately placed in liquid nitrogen to freeze for 15 min.

[0042] 1.3 Subsequently, all samples were transferred to an environment of -80°C for long-term storage, and any serum samples with slight hemolysis were excluded.

[0043] Example 2: Bone Destruction Sharp Score

[0044] In the development cohort, which included 160 RA patients and 40 healthy controls, all 160 RA patients underwent hand X-ray examination, and the Sharp / van der Heijde scoring method was used to assess bone erosion and joint space narrowing (JSN) in the metacarpophalangeal joints (MCP), proximal interphalangeal joints (PIP), and wrist joint.

[0045] Example 3: Proteomics Analysis

[0046] Using Orbitrap TM Astral TM High-depth plasma proteomic analysis was performed on 200 plasma samples (80 patients with sustained remission of RA, 80 patients with relapsed RA, and 40 healthy controls).

[0047] 3.1 Sample preparation: 100 μL of each sample was taken, and low-abundance proteins were enriched using the magnetic bead method; the specific procedures are as follows:

[0048] Mix 100 μL of plasma with 4 μL of magnetic beads (pre-washed twice with washing buffer) thoroughly and incubate at room temperature for 2 h;

[0049] Subsequently, the sample was placed on a magnetic rack, the supernatant was removed, and the washing reagent was added. The sample was inverted and mixed for 5 minutes each time, and the process was repeated 5 times to obtain the protein solution of the sample. Equal amounts of each sample were mixed to form a quality control sample (QC).

[0050] DTT was added to each sample to reduce disulfide bonds, and the reaction was carried out at 37°C for 1.5 h; then IAA was added to block the reduced cysteine ​​residues, and the reaction was carried out at room temperature in the dark for 30 min. Trypsin was added, and the mixture was incubated at 37°C for 15-18 h at an enzyme-to-protein ratio of 1:50.

[0051] The digested peptides were desalted on an MCX desalting column, concentrated by vacuum centrifugation, and then redissolved in 20 μL of an aqueous solution containing 0.1% formic acid. The peptide content was estimated by 280 nm UV spectral density. In the DIA experiment, iRT calibration peptides were added to the sample.

[0052] 3.2 DIA mass spectrometry analysis:

[0053] Using Orbitrap TM Astral TM The mass spectrometer was connected to the Vanquish Neo system liquid chromatograph, and peptides in each sample were analyzed in DIA mode. The precursor ion scan range was 380-980 m / z, the MS1 resolution was 240,000 at 200 m / z, the normalized AGC target value was 500%, and the maximum ion implantation time was 5 ms. In the MS2 scan, 299 windows were set in DIA mode, the isolation window was 2 m / z, the HCD collision energy was 25 eV, the normalized AGC target value was 500%, and the maximum ion implantation time was 3 ms.

[0054] DIA data were analyzed using DIA-NN 1.8.1 software, with the following main parameter settings: the enzyme digestion was trypsin, the maximum missed cleavage site was 1, the fixed modification was carbamide methylation (C), and the dynamic modifications were oxidation (M) and acetylation (N-terminus of the protein); all reported data were based on a 99% confidence level for protein identification, with a false discovery rate (FDR) ≤ 1%.

[0055] 3.3 Differential protein analysis: Proteins expressed in more than 50% of the samples were selected for differential expression analysis; differentially expressed proteins were compared among the three groups of remission, active phase and healthy population using analysis of variance. A two-sample t-test was used, and proteins with p-value <0.05 and fold change >1.5 or <0.67 were defined as differentially expressed proteins.

[0056] Figure 1-B A graph showing the differential expression of TAOK3 in the plasma of RA patients in active and remission phases: (Image provided by...) Figure 1-B It can be seen that there are significant differences in TAOK3 expression in the plasma of patients in the active and remission phases, and the expression level of TAOK3 in the active phase is higher than that in the remission phase.

[0057] Example 4: Single-cell data acquisition and analysis

[0058] Using the Seurat R package, dimensionality reduction analysis (tSNE and UMAP) and cluster analysis were performed on single-cell data, and cell subpopulation annotations were further refined by combining cell characteristic genes.

[0059] Given the crucial role of FLS in bone destruction in RA, we analyzed the differentially expressed genes of RA-FLS during the relapse and remission phases and drew a violin diagram.

[0060] By analyzing the intersection of differentially expressed genes and plasma proteins, potential targets related to bone destruction can be further screened.

[0061] Figure 2 Screening for proteins related to bone destruction: by Figure 2 It is known that the expression levels of proteins such as TAOK3, ATE1, FKBP3, SYNCRIP, SAA2, PLEKHF2, RAP1GAP2, and STK24 in plasma are positively correlated with the bone destruction score (Sharp score).

[0062] To further explore the relationship between bone destruction-related proteins and disease recurrence, and to evaluate their potential value in differentiating between remission and relapsed RA patients, a random forest model was used to assess feature importance, and the top 20 key features were selected, in order of importance: SAA2, CRP, SAA1, TAOK3, CXCL10, HPSE, PLA2G2A, AKAP1, ZC3HAV1, CFHR3, GSPT2, UBE2H, ORM1, PPBP, PF4, LRG1, IGHV4.34, IGHV1.69.2, ORM2, and PPP2R5A. Analysis Figure 4 As shown in (a, b), the importance scores of SAA2, CRP, SAA1, and TAOK3 are significantly higher than those of other features, indicating that they play a key role in the prediction model, while the importance distribution of the remaining features is relatively uniform.

[0063] At the same time, the inventors unexpectedly discovered that the combined use of TAOK3 and CRP significantly improved predictive performance (AUC = 0.915), outperforming the use of either marker alone. Figure 4 (c, d). This finding suggests that TAOK3 may serve as a novel biomarker, which, when used in conjunction with existing biomarkers, could further improve the accuracy of RA recurrence prediction.

[0064] Example 5 Multiple Immunohistochemistry

[0065] Tissue sections were dewaxed using xylene, followed by rehydration, antigen retrieval, and non-specific binding blocking.

[0066] Follow the manufacturer's instructions (PerkinElmer, Opal kit) to perform multiple staining on the slides. The antibodies used include CD90 (Abcam, #b16449) and TAOK3 (Proteintech, #67451-1-Ig).

[0067] After washing, the sections were incubated with HRP-conjugated secondary antibody at room temperature for 30 min, then counterstained with DAPI, and scanned and analyzed using the PerkinElmerVectra3 platform;

[0068] Finally, the proportion of positive cells was calculated using ImageJ software.

[0069] The results are as follows Figure 3 As shown in (a, b), CD90 in the synovial tissue of patients with recurrent RA + TAOK3 + The proportion of FLS increased significantly, and it was mainly expressed in the underlayer of the lining.

[0070] Example 6: ELISA detection

[0071] In the independent validation cohort, the absolute concentration of selected proteins was quantitatively determined using human enzyme-linked immunosorbent assay (ELISA) kits, all provided by Mlbio.

[0072] In the independent validation set 1, changes in the expression levels of CRP and TAOK3 in plasma were detected using an ELISA kit:

[0073] The results are as follows Figure 4 As shown in (e, f), both CRP and TAOK3 were significantly elevated in the plasma of patients with recurrent RA, and the combined use of the two further improved the diagnostic efficacy of recurrent RA (AUC = 0.894), further verifying that TAOK3 can be used as a new biomarker and can be used in combination with existing biomarkers to synergistically improve the accuracy of RA recurrence prediction.

[0074] In the independent validation set 2, changes in TAOK3 levels in the plasma of all participants were detected using an ELISA kit: results are as follows. Figure 4 As shown in c: ELISA results showed that TAOK3 levels in the plasma of patients with recurrent RA were significantly elevated.

[0075] In summary, by combining bone destruction Sharp score and single-cell RNA sequencing data to screen key bone destruction proteins, and by validating them in plasma and synovial tissue samples using multiplex immunohistochemistry and ELISA, TAOK3 protein was finally identified as a key biomarker closely related to bone destruction in RA.

[0076] Example 7: Extraction and Cultivation of Primary RA-FLS

[0077] Synovial tissue (STs) was obtained from 10 RA patients in independent validation cohort 2 who underwent knee synovectomy at Shanghai Guanghua Integrated Traditional and Western Medicine Hospital;

[0078] Under aseptic conditions, STs were cut into 1mm pieces. 3 Small pieces were washed with PBS and then digested with 100 U / ml type II collagenase solution at 37°C for 1 hour to isolate synovial cells.

[0079] Cells were cultured in DMEM medium (Gibco, #11965092) containing 10% fetal bovine serum (FBS, Gibco, #10091148) and grown at 37°C and 5% CO2. When the cell density reached approximately 80%-90%, the cells were passaged using trypsin (Gibco, #25300062). Examples 8-9 below all used RA-FLS cells from the 3rd to the 6th generation.

[0080] Example 8 CCK8 Detection

[0081] The CCK8 kit (Cell Counting Kit-8, Beyotime, China) was used to analyze the effect of TAOK3 knockdown on cell proliferation.

[0082] Cells transfected with Si-TAOK3 or Si-NC were seeded in 96-well plates at a density of 1 × 10⁶ cells per well. 5 Cells; at sampling points of 0, 24 and 48 h respectively, 10 μl of CCK8 reagent was added to each well and incubated for 2 h at 37 °C and 5% CO2.

[0083] Subsequently, the absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader to assess cell proliferation capacity.

[0084] like Figure 5 As shown in a, the CCK-8 experimental results showed that the OD values ​​of the Si-TAOK3#1 and Si-TAOK3#2 knockdown groups were lower than those of the Si-NC group at 24h and 48h, and the differences were statistically significant (p<0.05; p<0.001), indicating that TAOK3 gene knockdown can effectively reduce the proliferation of RA-FLS.

[0085] Example 9: Flow Cytometry Detection of Apoptosis

[0086] After collecting the cells, wash them with PBS;

[0087] Cells were stained for apoptosis using the Annexin V-FITC apoptosis detection kit (Beyotime);

[0088] Apoptosis was then detected by flow cytometry, and data analysis was performed using CytExpert software.

[0089] Annexin V-FITC / PI staining results further showed that TAOK3 gene knockdown significantly increased apoptosis in RA-FLS cells (5b, c). Compared with the Si-NC group, the proportion of Annexin V+PI+ cells in the Si-TAOK3#2 group was significantly increased (p<0.01), suggesting that TAOK3 gene knockdown significantly increased the level of apoptosis in RA-FLS cells.

[0090] These results indicate that knockdown of the TAOK3 gene significantly inhibits the proliferation of RA-FLS and promotes its apoptosis, suggesting that the TAOK3-encoded gene may play an important role in the biological function of RA-FLS.

[0091] Example 10: Construction of a mouse model of collagen-induced arthritis

[0092] The experimental mice were 8-10 week old male DBA / 1 mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in the animal room of the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. All animal experiments complied with animal ethics guidelines and were reviewed by the Experimental Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine.

[0093] Mice were allowed free access to food and water at a standard temperature of 22℃ and a 12-hour light-dark cycle. After one week of acclimatization, the mice were randomly divided into four groups: control group, CIA model group, CIA+SBI-581 group, and positive treatment group. The method for establishing the CIA mouse model is as follows:

[0094] For the first immunization, bovine type II collagen (2 mg / ml, dissolved in 0.05 M acetic acid) and an equal volume of complete Freund's adjuvant are fully emulsified in an ice box. After emulsification, a drop of emulsion is dropped onto the water surface. If the emulsion does not disperse immediately, it indicates that the emulsification is successful.

[0095] Mice were anesthetized with a 3.5% isoflurane / oxygen mixture and 0.1 ml of the emulsion was injected subcutaneously at the base of the tail. A second immunization was performed 21 days later, using bovine type II collagen emulsified with an equal volume of incomplete Freund's adjuvant and injected, with an injection volume of 0.1 ml.

[0096] Dosing began on day 35. The CIA+SBI-581 group received intraperitoneal injection of SBI-581 (10 mg / kg / day), while the positive treatment group received intraperitoneal injection of MTX (2 mg / kg / 3 days), for a total of 28 days.

[0097] Starting with the second immunization, two independent, double-blind observers scored the severity of arthritis until the end of the experiment (day 63).

[0098] like Figure 6 As shown, the effects of targeted inhibition of TAOK3 protein on improving joint bone erosion and histological damage were evaluated using techniques such as Micro-CT, H&E staining, and Safranin-Fix-Green staining. The results showed that targeted inhibition of TAOK3 protein effectively alleviated bone destruction in CIA mice, indicating that TAOK3 protein targeted inhibitors may be a novel strategy for treating bone destruction in RA.

[0099] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention (including the claims) is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in the details for the sake of brevity.

[0100] This invention is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. The application of a TAOK3 biomarker in the diagnosis, treatment, and prognostic monitoring of rheumatoid arthritis, characterized in that, The biomarker mentioned is the TAOK3 protein.

2. The application according to claim 1, characterized in that, The TAOK3 biomarker is used for the diagnosis, treatment, and prognostic monitoring of bone destruction in recurrent rheumatoid arthritis.

3. The application of a TAOK3 biomarker in the preparation of reagents or kits for detecting bone destruction in rheumatoid arthritis.

4. The application according to claim 3, characterized in that, The reagent or kit is used to determine the bone destruction and disease activity of rheumatoid arthritis by detecting the expression level of TAOK3 protein in the plasma of the subject; when the expression level of TAOK3 protein is elevated, it is determined that the subject has bone destruction of rheumatoid arthritis or is in the active phase of the disease.

5. The application according to claim 4, characterized in that... The detection methods include qualitative or quantitative analysis of TAOK3 protein in plasma using data-independent acquisition proteomics technology or ELISA.

6. The application of a TAOK3 biomarker in the preparation of a drug for treating bone destruction in rheumatoid arthritis.

7. The application according to claim 6, characterized in that, The drug targets the TAOK3 protein and contains components that inhibit the expression or activity of the TAOK3 protein.

8. The application according to claim 7, characterized in that, The drug contains a TAOK3 protein-targeting inhibitor.

9. The application of a TAOK3 and CRP biomarker in the combined preparation of a reagent or kit for detecting bone destruction in recurrent rheumatoid arthritis.

10. The application according to claim 9, characterized in that, By simultaneously detecting the expression levels of TAOK3 protein and CRP in the plasma of subjects, the diagnostic accuracy of recurrent rheumatoid arthritis (RA) can be improved.

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