Use of linc02224 in preventing or treating gout

Abstract

The application relates to the field of biological medicine, in particular to application of LINC02224 in prevention or treatment of gout, and discloses application of a LINC02224 gene in preparation of a product for diagnosing or treating gout. The LINC02224 gene is down-regulated in THP-1 macrophages induced by MSU, overexpression of the LINC02224 gene can effectively inhibit the expression level of inflammatory factors in THP-1 macrophages induced by MSU, which indicates that the LINC02224 plays an anti-inflammatory role and can be used as a target point for diagnosis and treatment of gout.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to the application of LINC02224 in the prevention or treatment of gout. Background Technology

[0002] Gout is a common type of inflammatory arthritis in men. Studies have found that the deposition of monosodium urate (MSU) crystals in joints and periarticular tissues induces monocyte differentiation into macrophages, which is associated with the occurrence, progression, and resolution of acute gouty inflammation. This is attributed to the fact that MSU crystals can increase the secretion of inflammatory factors, including TNF-α, IL-1β, and IL-6, by macrophages and monocytes. Colchicine and nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to treat acute gouty arthritis, but these drugs have limited efficacy and long-term use can cause side effects such as gastrointestinal bleeding, gastrointestinal toxicity, and nephrotoxicity. Therefore, the treatment of gout requires the search for more effective and less toxic drugs.

[0003] Long non-coding RNAs (lncRNAs) have shown crucial roles in inflammation-related diseases, with specific mechanisms including regulating the levels of inflammatory factors and proteins related to inflammatory signaling pathways through epigenetic, transcriptional, and post-transcriptional regulation. LINC02224 is a newly discovered lncRNA predicted to be associated with inflammation and immune responses, mitochondrial processes, and metabolic processes; however, whether LINC02224 can serve as a target for gout treatment requires further validation. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention provides the application of LINC02224 in the prevention or treatment of gout.

[0005] This invention relates to the application of LINC02224 as a molecular marker for aiding diagnosis or predicting treatment efficacy.

[0006] This invention also provides an application of a reagent for detecting the expression level of long non-coding LINC02224 in gout.

[0007] Furthermore, the reagent is used to prepare an auxiliary diagnostic agent for gout.

[0008] Furthermore, the reagent is used to prepare a gout efficacy prediction agent.

[0009] Furthermore, the reagent includes:

[0010] Oligonucleotide probes that specifically recognize LINC02224;

[0011] Alternatively, primers that specifically amplify LINC02224;

[0012] Alternatively, antibodies or ligands that specifically bind to the expression product of primers that bind to LINC02224.

[0013] Furthermore, the gout auxiliary diagnostic or efficacy prediction agent is a real-time fluorescence quantitative detection reagent.

[0014] Furthermore, the real-time fluorescence quantitative detection reagent contains primer sequences for real-time fluorescence quantitative detection of LINC02224 expression:

[0015] Upstream primer: 5'-ACAAGCGGTGCCTGAGCC-3';

[0016] Downstream primer: 5'-CCGACTCTCCTACACATCCACAG-3'.

[0017] The present invention also provides the use of a LINC02224 expression promoter in the preparation of a medicament for the prevention and / or treatment of gout.

[0018] The present invention also provides a medicament for the prevention and / or treatment of gout, characterized in that it comprises a medically acceptable carrier and an effective amount of an expression promoter of LINC02224.

[0019] The main beneficial effects of this invention are:

[0020] This invention discloses the application of the LINC02224 gene in the preparation of products for the diagnosis or treatment of gout. The LINC02224 gene is downregulated in MSU-induced THP-1 macrophages, and overexpression of the LINC02224 gene effectively inhibits the expression levels of inflammatory factors in MSU-induced THP-1 macrophages, indicating that LINC02224 exerts an anti-inflammatory effect and can serve as a target for the diagnosis and treatment of gout. Attached Figure Description

[0021] Figure 1 Expression of LINC02224 in MSU-induced THP-1 macrophages;

[0022] Figure 2 Effects of LINC02224 overexpression or silencing on the secretion of inflammatory factors in MSU-induced THP-1 macrophages;

[0023] Figure 3 Effects of LINC02224 overexpression or silencing on the expression of inflammatory cytokine mRNA in MSU-induced THP-1 macrophages. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described and discussed below with reference to the accompanying drawings. Obviously, what is described here is only a part of the examples of the present invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0025] To facilitate understanding of the embodiments of the present invention, further explanations and descriptions will be provided below with reference to the accompanying drawings and specific embodiments, and these embodiments do not constitute a limitation on the embodiments of the present invention.

[0026] Example 1 of this invention relates to the application of LINC02224 in the prevention or treatment of gout. The specific experimental steps regarding the MSU-induced decrease in LINC02224 expression in THP-1 macrophages are as follows:

[0027] (1) Cell culture:

[0028] THP-1 cells were purchased from the American Type Culture Collection (ATCC) and cultured at 37°C and 5% CO2 in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. THP-1-derived macrophages were obtained by treating THP-1 cells with 100 ng / mL myristate acetate (PMA) for 48 hours. THP-1 macrophages were induced for 24 hours with different doses (0, 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) of MSU.

[0029] (2) ELISA detection of inflammatory factors in cell extracts:

[0030] ELISA was used to detect the expression levels of inflammatory factors in cell extracts from each group. The main experimental steps are as follows: Cells were centrifuged (500×g, 5 minutes), and the supernatant was collected and washed three times with cold PBS. Every 10... 6Resuspend cells in 150-200 μL of PBS and lyse them by sonication. Centrifuge the extract at 1500 × g for 10 minutes at 4°C and collect the supernatant for detection. After removing the kit from the refrigerator, allow it to equilibrate at room temperature (25-28°C) for 20 minutes, then dilute the standards and samples according to the instructions of the human ELISA kit. Add 100 μL of sample or different concentrations of standard to the corresponding well, seal the reaction wells with clear film, and incubate at room temperature for 120 minutes. After washing the plate 4 times, add 100 μL of biotinylated antibody working solution to the 96-well plate and continue incubation for 60 minutes. Wash the plate 5 times again, add 100 μL of horseradish peroxidase-labeled streptavidin to the 96-well plate, and incubate at room temperature in the dark for 20 minutes. Next, wash the plate 4 times, add 100 μL of chromogenic reagent to the well, and incubate at 37°C in the dark for 20 minutes. Finally, add 50 μl of stop solution per well, mix well, and immediately measure the OD450 value using a microplate reader.

[0031] The results are as follows Figure 1 As shown in AB, MSU promoted the secretion of IL-1β and caspase-1 in THP-1 macrophages in a concentration-dependent manner.

[0032] (3) Detection of LINC02224 expression by QRT-PCR:

[0033] TRIzol reagent (12183555, Thermo Fisher Scientific, USA) was used to extract total RNA from cells. The concentration and purity of total RNA were determined using a NanoDrop spectrophotometer. When the A260 / A280 ratio was between 1.8 and 2.0, RNA was reverse transcribed into cDNA using a reverse transcription kit and stored at -20°C for later use. The cDNA was used as a template for qPCR amplification. qPCR reactions were performed using a SYBR Green MasterMix (Roche, Germany) under the following conditions: 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute, for 40 cycles. The primers used are listed in Table 1. Data are presented in 2... -ΔΔCT Quantitative analysis was performed. GAPDH was used as an internal reference.

[0034] Table 1. Primer sequences

[0035]

[0036] The results are as follows Figure 1 As shown in Figure C, MSU inhibited the expression of LINC02224 in THP-1 macrophages in a concentration-dependent manner.

[0037] Example 2 of the present invention relates to the effect of LINC02224 overexpression or silencing on the expression of inflammatory factors in MSU-induced THP-1 macrophages. The specific experimental steps are as follows:

[0038] (1) Cell transfection

[0039] The pcDNA3.1 / Hygro(+) mammalian expression vector was used to construct the LINC02224 overexpression vector, with the empty plasmid serving as a negative control for overexpression (NC). siLINC02224-1 and siLINC02224-2 were synthesized by Sangon Biotech, with siNC serving as a negative control. Cells were loaded at 3 × 10⁻⁶ cells / year. 5 The plasmids or siRNAs were seeded in 6-well plates and incubated overnight at 37°C in a CO2 incubator. Then, the plasmids or siRNAs and Lipofectamine 3000 transfection reagent were diluted with Opti-MEM medium. The diluted plasmids or siRNAs were mixed with the diluted Lipofectamine 3000 transfection reagent and added to the cells for incubation for 24 hours.

[0040] (2) Cell grouping

[0041] Cells were divided into 6-7 groups: control group, MSU group, NC group, LINC02224 overexpression group, siNC group, siLINC02224-1 group, and siLINC02224-2 group. Control group cells were cultured normally, while cells in the other groups were induced with 200 μg / mL MSU for 24 hours. The latter five groups were transfected with NC, LINC02224 overexpression plasmids, siNC, siLINC02224-1, and siLINC02224-2, respectively, before MSU induction.

[0042] QRT-PCR was performed according to the method in Example 1(3), and the results are as follows: Figure 2 As shown in Figure A, MSU inhibited the expression of LINC02224. Compared with the NC group, LINC02224 expression increased after cells were transfected with the LINC02224 overexpression plasmid. Compared with the siNC group, LINC02224 expression decreased in the siLINC02224-1 and siLINC02224-2 groups. Since siLINC02224-2 had better interference efficiency, it was used in subsequent experiments.

[0043] (3) ELISA detection of inflammatory factors in cell extracts

[0044] ELISA was performed according to the method in Example 1(2). The results are as follows: Figure 2As shown in BD, MSU promoted the levels of IL-1β, IL-6, and TNF-α in cells. Compared with the NC group, the levels of IL-1β, IL-6, and TNF-α were decreased after cells were transfected with the LINC02224 overexpression plasmid. Compared with the siNC group, the levels of IL-1β, IL-6, and TNF-α were increased in the siLINC02224 group.

[0045] (4) Detection of mRNA levels of inflammatory factors in cells by QRT-PCR

[0046] QRT-PCR was performed according to the method in Example 1(3), and the results are as follows: Figure 3 As shown in AC, MSU promoted the levels of IL-1β, IL-6, and TNF-α in cells. Compared with the NC group, the levels of IL-1β, IL-6, and TNF-α were decreased after cells were transfected with the LINC02224 overexpression plasmid. Compared with the siNC group, the levels of IL-1β, IL-6, and TNF-α were increased in the siLINC02224 group.

[0047] Finally, it should be noted that the above-described embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and not to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments within the technical scope disclosed in the present invention, or make equivalent substitutions for some of the technical features; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Application of a reagent for detecting the expression level of long non-coding LINC02224 in gout; the reagent is used to prepare auxiliary diagnostic agents for gout.

2. The application of the reagent for detecting the expression level of long non-coding LINC02224 in gout as described in claim 1, characterized in that, The reagents include: oligonucleotide probes that specifically recognize LINC02224; or primers that specifically amplify LINC02224.

3. The application of the reagent for detecting the expression level of long non-coding LINC02224 in gout as described in claim 1, characterized in that, The gout auxiliary diagnosis uses a real-time fluorescence quantitative detection reagent.

4. The application of the reagent for detecting the expression level of long non-coding LINC02224 in gout as described in claim 3, characterized in that, The real-time fluorescence quantitative detection reagent contains primer sequences for real-time fluorescence quantitative detection of LINC02224 expression: Upstream primer: 5'-ACAAGCGGTGCCTGAGCC-3'; Downstream primer: 5'-CCGACTCTCCTACACATCCACAG-3'.

5. Use of overexpression vectors containing the LINC02224 gene in the preparation of drugs for treating gout.

6. A medication for treating gout, characterized in that, This includes medically acceptable vectors and overexpression vectors containing the LINC02224 gene.

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