CN110846402B

CN110846402B - Application of hsa-circ-0004287 as therapeutic target in preparation of medicine for treating atopic dermatitis

Info

Publication number: CN110846402B
Application number: CN201911082737.1A
Authority: CN
Inventors: 周玉峰; 杨兰; 韩晓; 张彩艳
Original assignee: Childrens Hospital of Fudan University
Current assignee: Childrens Hospital of Fudan University
Priority date: 2019-11-07
Filing date: 2019-11-07
Publication date: 2022-10-21
Anticipated expiration: 2039-11-07
Also published as: CN110846402A

Abstract

The invention discloses application of hsa-circ-0004287 as a therapeutic target in preparing a medicament for treating atopic dermatitis. The invention collects peripheral blood mononuclear cells of atopic dermatitis patients and normal people, and carries out circRNA chip analysis, and hsa-circ-0004287 is screened out from the peripheral blood mononuclear cells and has obvious high expression in atopic dermatitis patients. In THP1 cells, hsa-circ-0004287 can remarkably reduce the phosphorylation level of p38 protein so as to inhibit the expression of downstream inflammation mediators induced by LPS, thereby exerting certain inflammation inhibition effect. Our studies showed that hsa-circ-0004287 is significantly highly expressed in atopic dermatitis patients and significantly inhibits inflammation caused by LPS-induced macrophage M1 polarization, suggesting that hsa-circ-0004287 may be a new target for intervention therapy of atopic dermatitis.

Description

Application of hsa-circ-0004287 as therapeutic target in preparation of medicine for treating atopic dermatitis

Technical Field

The invention belongs to the field of biological medicines, and relates to application of hsa-circ-0004287 as a treatment target in preparation of a medicine for treating atopic dermatitis.

Background

Atopic Dermatitis (AD), also known as allergic Dermatitis, is a chronic recurrent, inflammatory, pruritic skin disease with clinical manifestations mainly of chronic skin inflammation, impaired skin barrier function, pruritus (1-3). With age and population, the incidence of AD varies from 2% to 20% (4-5). The disease is lingering and has great influence on the physical and mental health and the life quality of the patients. Furthermore, it is most noted that many AD patients show symptoms of other allergic diseases such as asthma and allergic dermatitis with the delay of disease conditions, and serious patients are at risk of death (6). The etiology and pathogenesis of the disease are complex, and various factors such as heredity, immunity, infection, environment, spirit and the like are involved, so that the disease is also a main reason for ineffective treatment of AD clinically. At present, many studies have demonstrated that abnormal immune responses are a key component in the pathogenesis of AD, involving mast cells, eosinophils, basophils, macrophages, lymphocytes and other immune cells, as well as cytokines and inflammatory factors secreted by these immune cells (7-9).

Studies have shown that macrophages are closely associated with the pathogenesis of AD, and a large number of macrophage accumulations are seen in both acute and chronic skin inflammation in AD patients (9). Macrophages, a functionally diverse group of immune cells, are functionally dependent on the activation state, wherein M1-type macrophages activated by LPS, IFN-gamma and the like enhance inflammatory responses by releasing large amounts of inflammatory mediators such as IL-1 beta, TNF-alpha and the like, while M2-type macrophages activated by IL-4 and IL-13 and involved in immune regulation and repair mainly secrete cytokines such as CD206, CD209, CD163 and the like (10). During the acute inflammatory phase of AD, macrophages exhibit a M2 dominated polarization state, whereas during the chronic phase they exhibit a M1 dominated polarization state, and as the disease progresses in AD, macrophages may exhibit a mixed polarization state with both M1/M2 present (9). Therefore, the research on the polarization regulation mechanism of the macrophage has very important significance for researching the pathogenesis, diagnosis and treatment and prevention measures of AD.

In recent years, non-coding RNA (non-coding RNA) has become a hotspot for studies on regulation of gene expression in genomes. Circular RNA (circular RNA) is a circular non-coding RNA molecule formed by covalently combining a 3 'end and a 5' end after reverse splicing, is widely present in various biological cells, and has the characteristics of stable structure, conserved sequence, specific expression of cells or tissues and the like (11-12). The circRNA is involved in the regulation of important life processes such as cell differentiation, ontogeny and the like on multiple levels, and the regulation of the circRNA plays an extremely important role in the regulation process of various human diseases, and has the potential of becoming a new detection marker of the diseases in the future. Recent studies have shown that circRNA is also involved in the development of some immunological diseases. For example, studies have found that the cyclic RNA CDR1as affects insulin secretion and islet beta cell turnover in diabetic patients by blocking miR-7 function in islet cells (13). However, the effect of circRNA on macrophage polarization and its role in AD is poorly understood.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides application of hsa-circ-0004287 as a therapeutic target in preparing a medicament for treating atopic dermatitis.

The invention also aims to provide the application of hsa-circ-0004287 as a drug screening target in preparing the drug for treating atopic dermatitis.

The purpose of the invention can be realized by the following technical scheme:

the application of hsa-circ-0004287 as a therapeutic target in preparing anti-inflammation drugs.

Application of hsa-circ-0004287 as a therapeutic target in preparing a medicament for treating atopic dermatitis.

Application of hsa-circ-0004287 as a drug screening target in preparing a drug for treating atopic dermatitis.

Use of hsa-circ-0004287 or a medicament which promotes the expression of hsa-circ-0004287 in the manufacture of a medicament for the treatment of atopic dermatitis.

Has the advantages that:

according to the invention, the circRNA expression spectrum of differential expression is obtained by carrying out circRNA chip analysis on PBMCs of AD patients and normal people, and the expression level of circ287 in the PBMCs of the AD patients is found to be up-regulated; through further experimental verification, we find that has-circ-0004287 with remarkably high expression in AD can remarkably inhibit expression of downstream inflammation mediators caused by macrophage M1 polarization induced by LPS by down-regulating phosphorylation level of MAPK-p38, so as to play a role in inflammation inhibition; therefore, the hsa-circ-0004287 can be used as a therapeutic target to be applied to the preparation of the medicine for treating the atopic dermatitis; or can be used as a drug screening target for screening drugs for inhibiting the treatment of atopic dermatitis.

Drawings

Abnormal expression of circRNA in PBMCs from AD patients. (A) Separating PBMCs of AD patients, extracting RNA and carrying out circRNA chip analysis; (B) Abnormally high expression of circ287 was demonstrated by expression levels in PBMCs in AD and normal humans.

Figure 2 circ287 looping validation. (A) sequencing of circ287 across the splice site. (B) RNase R protection test for circ287 and its parent gene MALAT 1. (C) LPS stimulated the nuclear and cytoplasmic distribution ratio of circ287 compared to unstimulated.

Figure 3circ287 inhibits macrophage M1-type polarization. (A) Adding PMA into a human THP1 cell line to induce and differentiate the PMA into macrophages, adding IFN-I, LPS, IFN-gamma and Pam3CSK4, collecting RNA at different time points, and carrying out qPCR to detect the change of the expression level of the circ 287; (B) (C) in induced macrophages, siRNA was used to reduce the expression level of circ287 followed by stimulation of cells with LPS, qPCR was used to measure the expression level of LPS-induced macrophage polarization gene and western blotting was used to measure changes in the phosphorylation level of p 38.

Detailed Description

The general test methods in the following examples are as follows:

1. cell culture and transfection

The human THP1 cell line was cultured in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) under 37 ℃ cell culture conditions containing 5% CO 2.

siRNA and NC sequences from circ287 (200 nM, gima, shanghai) were transfected into cells using Lipofectamine RNAiMAX reagent (Invitrogen); cells were stimulated with LPS (100 ng/ml) for 3 hours 48 hours post transfection.

RNA extraction, reverse transcription and real-time fluorescent quantitative PCR

Intracellular total RNA was extracted by the Trizol (Invitrogen) method. Reverse transcription of RNA was performed using PrimeScript RT reagent kit (TaKaRa), 10ul of sample addition system, reverse transcription reaction conditions of 37 ℃ C. 15min and 85 ℃ C. 5s. For real-time fluorescent quantitative PCR

Premix Ex Taq ^TM II (Takara) kit, then using Roche 480 instrument machine. Use of beta-actin as an internal reference Gene, 2 ^-ΔΔCT Analytical methods the data were analyzed.

3. Immunoblotting experiment (western blotting)

Total protein was extracted by RIPA Buffer (Thermo) lysis method, and the total protein was quantified by BCA reagent (Bio-Rad) and then denatured at 100 ℃ for 10 minutes. SDS-PAGE was prepared at 10% for protein electrophoresis, and the loading was 20. Mu.g, and the electrophoresis time was 90 minutes. The PVDF membrane (Millipore) was used for the membrane conversion process, and the membrane conversion time was 90 minutes. The blocking solution was blocked for one hour at room temperature using a TBST solution containing 5% BSA and 0.1% Tween-20. Followed by overnight incubation with beta-tubulin, p-p38 and p-p65 (CST Corp.) primary antibody at 4 ℃. The next day, membranes were washed three times for 10 minutes each with TBST solution containing 0.1% Tween-20. Thereafter, the corresponding secondary antibody (CST Co.) was incubated at room temperature for 1 hour. Finally, the membrane was washed three times with TBST solution, and developed with ECL luminescence solution (Thermo Co.).

Example 1 screening for multiple highly expressed circRNAs in PBMCs of AD patients

We screened multiple circular RNAs with differential expression by collecting PBMCs cells from AD patients and performing high-throughput chip analysis of circRNA (FIG. 1A), wherein circ287, circRNA-A, circRNA-B and circRNA-C (FIG. 1B) with up-regulated expression levels are found, andbase:Sub>A single verification is performed, which reveals that the circRNA in PBMCs is very likely to participate in the generation and development process of AD.

Example 2

We designed primers spanning the splicing site of circ287 and amplified by PCR in THP1 cells, and the sequence spanning the splicing site was obtained after sequencing the product (FIG. 2A).

RNA was extracted from 5. Mu.g of THP1 cells, and both the RNase R-treated and untreated groups were placed in a 37 ℃ water bath for 20 minutes. The remaining RNA in both sets of samples was then purified with phenol-chloroform (5, sigma company); subsequent RNA extraction and qRT-PCR were as described previously. The RNase R protection experiment demonstrated that circ287 is more resistant to degradation by RNase R than its parent gene MALAT1 (fig. 2B).

Nuclear and cytoplasmic isolation assay nuclear and cytoplasmic isolation kit (CWBiotech); subsequent RNA extraction and qRT-PCR were as described previously. The results indicated that LPS stimulation increased the enucleation of circ287 (figure 2C), suggesting that circ287 may play a role in LPS-induced macrophage inflammation.

Example 3circ287 significantly inhibits LPS-induced macrophage M1 polarization

THP1 cells were plated in 24-well plates at a density of 120,000cells/well and then stimulated with PMA (500 ng/ml) for 48h, total RNA was collected after stimulation with actinomycin D (0.25. Mu.g/ml) for 0h,3h,6h,12h,24h, respectively, followed by RNA extraction and qRT-PCR as described above.

We added PMA to human monocyte THP1 cells to induce differentiation into macrophages, and stimulated IFN-I, LPS, IFN-. Gamma.and Pam3CSK4 to find that the expression level of circ287 is regulated by these stimuli (FIG. 3A).

Further using siRNA to reduce the expression of circ287, then LPS stimulates macrophages, and qPCR detection shows that after reducing the expression of circ287, the expression level of M1 type polarization-related genes (IL-6, TNF-alpha and IL-1 beta) induced by LPS can be obviously promoted (FIG. 3B).

By immunoblotting to detect changes in phosphorylation levels of the corresponding proteins of the NF-. Kappa.B and MAPK pathways downstream of TLR4, we verified that knock-down of circ287 could significantly up-regulate the phosphorylation level of LPS-activated p38 (FIG. 3C), which revealed that circ287 plays a negative regulatory role in M1-type polarization of macrophages.

Claims

1. Application of a medicament for promoting expression of hsa-circ-0004287 in preparing a medicament for treating atopic dermatitis.