CN114990229B - Basophil activation related biomarker and application thereof - Google Patents

Abstract

The invention relates to the technical field of transcriptomics, and discloses a biomarker related to basophil activation and application thereof. The biomarker related to basophil activation is miRNA-519e-5p, and the nucleotide sequence is shown in SEQ ID No. 1. The biomarker is applied to a basophil activation detection preparation, and is also applied to a basophil activation inhibitor. The invention provides a new way and a powerful molecular basis for developing the research of detecting the basophil activation and related functions, and has profound significance and popularization.

Description

Basophil activation related biomarker and application thereof

Technical Field

The invention relates to the technical field of transcriptomics, in particular to a biomarker related to basophil activation and application thereof.

Background

The function of basophils (basophils) in innate immunity and adaptive immunity is receiving increasing attention, and the role in immune regulation is also a focus of scientific research. Activation is the key to the biological function of basophils, and the research on the activation mechanism of basophils has progressed from classical IgE pathway-mediated activation to multi-pathway-mediated activation such as interleukin-33 and thymic stromal lymphopoietin. Basophils are activated by a number of signals, including cytokines, antigen-antibody complexes, proteases, parasitic antigens, viral antigens, toll-like receptor agonists and complement components, which bind to receptors such as immunoglobulin receptors, TLRs, chemokine receptors (CCRs, CXCRs) and cytokine receptors (IL-3R, IL-33R, TSLPR, IL-18R) present on the surface of basophils, thereby activating basophils. After basophils are activated, a series of effector molecules such as histamine, leukotriene C4, antimicrobial peptides, immunoregulatory cytokines (IL-4/IL-6, etc.) are produced. These effector molecules can further exert the biological functions of basophils by direct or indirect means, and play a crucial role in innate immunity and adaptive immunity.

It has been found that a variety of pathways can mediate basophil activation in different disease states. However, there are two main approaches to inhibiting IgE-mediated basophil activation at present: the first scheme is that Anti-IgE monoclonal antibody (such as Omalizumab) removes IgE in peripheral blood circulation by combining free IgE and mIgE on the surface of IgE + B cells after class transition, so that basophil activation mediated by the binding of the IgE with a high affinity receptor Fc epsilon RI is reduced, rapid degranulation after the basophil activation is reduced, and release of active substances such as histamine and LTC4 is reduced, and the Anti-IgE monoclonal antibody is used for treating allergic diseases such as bronchial asthma and chronic urticaria. However, this solution still has disadvantages: non-specific, igE antibodies that bind all but "allergen" specific IgE antibodies; can only clear free IgE, has no effect on IgE bound with cells, and is not suitable for treating diseases after basophils are activated. The second approach is directed to the development of chemical small molecule drugs that inhibit basophil activation. Mainly takes a series of key kinases (Lyn, syk, BTK, PI3K, AKT, PKC, FK-BP, KDR, KIT, FLT3 and the like) of downstream signal molecules of an IgE mediated basophil activation signal channel as targets. At present, the chemical small molecule drugs are still in the development stage and cannot be applied to clinic.

mirnas are a class of non-coding single-stranded RNA molecules of about 20-24 nucleotides in length encoded by endogenous genes. mirnas have important regulatory effects on the body in physiological and pathological states. In recent years, studies on miRNA have mainly focused on the mechanism of regulation of gene expression at the post-transcriptional level, and miRNA can bind to the 3' non-coding region (3 ' untranslated regions,3' UTR) of mRNA by the principle of base complementarity, and then degrade mRNA or inhibit translation of mRNA, thereby regulating gene expression. Mirnas have been reported to regulate approximately 1/3 of the human genes, including at least 60% of the human protein-encoding genes.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a biomarker related to basophil activation and application thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides a biomarker related to basophil activation, wherein the biomarker is miRNA-519e-5p.

The expression of miRNA-519e-5p in the activated basophils is reduced. The miRNA-519e-5p is derived from human chromosome 19, and the nucleotide sequence of the miRNA-519e-5p is shown in SEQ ID No. 1.

In a second aspect, the invention provides a primer of the biomarker associated with basophil activation, wherein the nucleotide sequence of the primer is shown as SEQ ID No. 2.

In a third aspect, the invention applies said biomarker or said primer in a basophil activation detection formulation. The level of basophil activation can be regulated or identified by modulating or detecting the expression level of the miRNA-519e-5p in the test sample.

As a preferred embodiment of the application of the invention, the detection agent is a reagent for detecting the expression level of the biomarker based on gene chip detection or real-time fluorescent quantitative PCR.

In a fourth aspect, the invention uses the biomarker in an inhibitor of basophil activation. So that the expression level of the miRNA-519e-5p in the activated basophils is obviously reduced compared with the normal control expression level.

As a preferred embodiment of the application of the invention, the inhibitor comprises miR-519e-5p imic.

In a fifth aspect, the invention provides a basophil activation detection system, which is a kit, a chip or a detection platform; the system comprises the biomarker or the primer. So that the system can regulate or identify the activation level of basophil by regulating or detecting the expression level of miRNA-519e-5p.

In a sixth aspect, the present invention provides a basophil activation inhibition system, wherein the system is a kit, a chip or a detection platform; the system comprises miR-519e-5p imic. So that the system can inhibit basophil activation by up-regulating the expression level of miRNA-519e-5p.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention firstly provides that the biomarker miRNA-519e-5p is related to the activation of basophils. The miRNA-519e-5p disclosed by the invention is reduced in expression in activated basophils, can be used for identifying the activation level of the basophils, and can inhibit the activation of the basophils by up-regulating the expression level of the miRNA-519e-5p.

2. The miRNA-519e-5p is a novel biomarker, is different from the traditional biomarker, is stable, minimally invasive, easy to detect and accurate in quantification, and can greatly improve the sensitivity and specificity of basophil activation identification.

3. The invention provides a new way and a powerful molecular basis for developing the research of detecting the basophil activation and related functions, and has profound significance and popularization.

Drawings

FIG. 1 is a graph of the clusters of differentially expressed miRNAs screened for control (non-activated Ba) and anti-IgE pathway-mediated activation (activated Ba) in example 1.

Figure 2 is the qPCR validation results for differentially expressed mirnas in example 1.

FIG. 3 is a graph showing that the expression level of basophil miR-519e-5p in systemic lupus erythematosus patients (SLE patients) is significantly reduced compared to that in a normal human control group (health controls) in example 2.

FIG. 4 shows that the expression of basophil miR-519e-5p is remarkably increased after miR-519e-5p is transfected in example 3.

FIG. 5 shows that the proportion of IL-4 positive basophils is remarkably reduced after miR-519e-5p imimic is transfected in example 3.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following examples. It should be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The materials, reagents and the like used are commercially available unless otherwise specified.

Example 1: activated and non-activated basophil miRNA microarray sequencing

1. Negative selection of basophils (Ba)

(1) Collecting a specimen: collecting 60-80 ml of peripheral venous blood of a volunteer or 8ml of peripheral venous blood of an SLE patient by using an EDTA (ethylene diamine tetraacetic acid) anticoagulation tube, and treating the blood in 2h as much as possible;

(2) Negative selection of basophils: placing EDTA anticoagulated peripheral venous blood into a 15ml centrifuge tube, adding HetaSep ^TM Solution(HetaSep ^TM Solution, anticoagulation =1:5, mixing well and centrifuging (90 Xg, 5min,20 ℃); standing at room temperature for 10min to precipitate erythrocytes; the plasma was then collected in a 50ml centrifuge tube and 4 volumes of plasma EasySep added ^TM Buffer, mix well and centrifuge (120 Xg, 10min,20 ℃) to remove platelets; using EasySep ^TM Buffer adjusted cell density to 5X 10 ⁷ Cells/ml, placed in a 5ml flow tube. Adding 50 μ l of EasySep Human Basophil Enrichment Cocktail in Basophil negative selection kit into each ml of cell suspension, mixing uniformly, and incubating at room temperature for 7min; add EasySep ^TM Adjusting the volume of Buffer to 2.5ml, gently blowing and mixing, and putting EasySep ^TM In the Magnet pole, stand at room temperature for 3miAfter n, pouring the cell suspension into a new flow tube in a slight inclination manner, then placing the new flow tube into a magnetic pole, and pouring out the enriched basophilic granulocyte suspension after placing for 3 min; detecting the activity of basophilic granulocytes by trypan blue dye, and performing the next experiment when the cell activity reaches more than 97 percent; FCM detects basophils of CD203c + CD123+, and the purity of the cells reaches more than 95%, and then the next experiment is carried out.

(3) Activation and culture of basophils: the enriched cells are divided into two groups, the Anti-IgE group is activated by Anti-Human IgE (epsilon-chain specific), the working concentration is 1 mu g/mL, the Control group is added with PBS with the same volume, the number of the enriched basophils is counted by using a cell counting plate, and the cell concentration is adjusted to be 5.0 multiplied by 10 ⁵ Per ml, X-VIVO, maintenance of basophil activity with 5% (v/v) exosome-free serum, 5ng/ml IL-3 ^TM 15Medium，37℃，5％CO ₂ Culturing in an incubator.

Sequencing of miRNA microarrays

(1) Extraction and quality inspection of total RNA: experiments are divided into 2 groups, 3 biological repeats are set, control groups (non-activated Ba) are Control 1, control 2 and Control 3, and corresponding Anti-IgE pathway mediated activation groups (activated Ba) are Anti-IgE 1, anti-IgE 2 and Anti-IgE 3. Extracting total RNA of basophils by using a Trizol method. The RNA concentration of each sample was measured using a Nanodrop ND-1000 instrument (Thermo Fisher Scientific, waltham, MA, USA) and expressed as OD ₂₆₀ /OD ₂₈₀ The value was used as an index of RNA purity. RNA integrity was checked using denaturing agarose gel electrophoresis.

(2) Construction of miRNA expression profile: chip maps were obtained using Agilent Feature Extraction software (v11.0.1.1) and read to obtain the raw data. Quantile normalization and subsequent data processing were performed on the raw data using GeneSpring GX v12.1 software (Agilent Technologies). Raw data were normalized and further analyzed by screening for high quality probes (at least 3 of 6 samples were labeled as Detected). Statistically significant differentially expressed miRNAs between the two groups of samples were screened by volcano plots. The screening standard of the differentially expressed genes between the two groups of samples is as follows: FC is more than or equal to 2.0 and P is less than or equal to 0.05. The larger the FC value difference factor, the larger the difference between the samples of Control group (non-activated Ba) and Anti-IgE group (activated Ba), and the smaller the P value, the higher the reliability of the difference gene.

The clustering chart of differential expression miRNA screened by a control group (non-activated Ba) and an anti-IgE pathway mediated activation group (activated Ba) is shown in figure 1. Selecting differentially expressed miRNAs: miR-6721-5p, miR-6126, miR-892b, miR-466, miR-519e-5p, miR-6129, miR-4710 and miR-30c-1-3p.

RT-qPCR validation of differentially expressed miRNAs

(1) And (3) miRNA reverse transcription reaction:

Mir-X miRNA First-Strand Synthesis Kit (Takara Cat.638313) from Takara was used.

(1) According to the kit instructions, the miRNAs reverse transcription reaction system (Table 1) was prepared on ice, and each gene was provided with 3 duplicate wells. (2) Mixing, centrifuging, and reverse transcribing. (3) Reaction procedure: the first stage, reverse transcription reaction, 37 ℃,60min; in the second stage, the reverse transcriptase was inactivated at 85 ℃ for 5min. (4) The product was stored at-80 ℃.

TABLE 1 preparation of reverse transcription reaction systems for miRNAs

Reagent	Volume of use
		mRQ Enzyme Mix	1.25μl
mRQ Buffer(2×)	5.0μl
		RNA sample (0.25. Mu.g-8. Mu.g)	3.75μl
Total	10.0μl

(2) Designing and synthesizing a primer:

the primer is synthesized by Shanghai, the internal reference U6 is matched with the miRNA reverse transcription kit, and the human internal reference GAPDH is purchased from Shanghai. As shown in table 2.

TABLE 2 MiRNAs primer sequences

Note: forward: forward Sequence.

(3) Detecting the relative expression quantity of the differential miRNAs by fluorescent quantitative PCR:

TB GreenTM Premix Ex TaqTM II (Tli RNaseH Plus) kit (Takara No. RR820A) supplied by Takara was used.

(1) According to the kit instructions, a fluorescent quantitative PCR reaction system (Table 3) was prepared on ice, and 3 duplicate wells were provided for each gene. (2) Reaction procedure: first stage, pre-denaturation (1 cycle), 94 ℃,30s; the second stage, qPCR reaction (40 cycles), 95 ℃,5s denaturation; annealing and extending at 60 ℃ for 20 s. (3) And (3) data analysis: the Light Cycler 480II PCR instrument can automatically analyze and calculate the Ct value of each gene of each sample. This experiment was run through 2 ^-ΔΔCt The method calculates relative expression amount of target gene, and corrects the initial amount of sample mainly by housekeeping gene U6 and GAPDH ^Δ Ct (experimental group) = Ct (experimental group target gene) -Ct (experimental group reference gene); ^Δ ct (control group) = Ct (control group target gene) -Ct (control group internal reference gene); ^ΔΔ Ct＝ ^Δ ct (Experimental group) - ^Δ Ct (control).

TABLE 3 preparation of fluorescent quantitative PCR reaction system

Reagent	Amount of the composition used	Final concentration
			TB Green Premix Ex Taq II(2×)	5.0μl	1×
Forward Primer(10μM)	0.4μl	0.4μM
			Reverse Primer(10μM)	0.4μl	0.4μM
cDNA template	1.0μl	/
			RNase-Free DEPC H 2 O	3.2μl	/
Total	10.0μl	/

The qPCR verification result of the miRNA with differential expression (shown in figure 2) shows that the expression level of miR-519e-5p is reduced after basophils are activated. The base sequence of miR-519e-5p is shown in SEQ ID No. 1.

Example 2: application of miRNA-519e-5p in preparation of basophil activation detection preparation

To further analyze the biological functions of the differentially expressed miRNAs, the databases TargetScan7.1 and mirdbV6 were used for prediction, and target mRNAs of the differentially expressed miRNAs were screened by a threshold (the screening threshold of TargetScan7.1 is a historical Weighted Context + + Score < -0.3, the screening threshold of total Context + + Score < -0.3 is Score ≧ 70), the intersecting target mRNAs of the two datasets were taken, and these mRNAs were subjected to KEGG pathway enrichment analysis. Wherein the target NRAS of miR-519e-5p is enriched on the Fc epsilon RI signal path.

Abnormal activation of basophils in the state of systemic lupus erythematosus is primarily IgE pathway-mediated. Based on the real-time fluorescent quantitative PCR assay, further verified in the disease model, the expression level of miR-519e-5p in basophilic granulocytes of systemic lupus erythematosus patients (SLE Patients) was found to be significantly reduced (21 cases, respectively) compared to the Healthy control group (health controls) (as shown in FIG. 3).

Therefore, the level of basophil activation can be identified by detecting the expression of the miRNA-519e-5p in the test sample. miRNA-519e-5p can be used for preparing a basophil activation detection preparation.

Example 3: application of miRNA-519e-5p in preparation of basophil activation inhibitor

1. Cell transfection

(1) Cell plating: basophilic granulocytes (Ba) were cultured at 5X 10 per well ⁵ The individual cells were seeded in 24-well plates;

(2) Preparation of miRNA-RFectSP mixture:

(1) 50nmol of miR-519e-5p mimic and 50nmol of mimic NC are respectively diluted with 50 μ l of Opti-EME medium; (2) mu.l RFectSP was diluted with 100. Mu.l Opti-MEM medium. Mixing, and incubating at room temperature for 5min; (3) after incubation for 5min, respectively and evenly mixing 50 mul of miR-519e-5p mic diluent and 50 mul of mic NC diluent with 50 mul of RFectSP diluent (the total volume is 100 mul), and incubating for 15-20 min at room temperature;

(3) Adding the mixture to the cultured cells:

(1) adding 100 μ l of the mixture into the culture holes corresponding to the cell groups, slightly shaking the culture plate for 5min, and mixing uniformly; (2) 5% CO at 37% ₂ The culture box is used for culturing for 24 hours. After the culture is finished, collecting cells and extracting RNA for later use, and then verifying the transfection efficiency by RT-qPCR; if cell function detection is carried out, after 24h of culture, liquid is changed, anti-IgE stimulation is added, the culture is continued for 24h, and then FCM detection is carried out.

(4) Anti-IgE stimulation: changing the solution 24 hours before the final step of transfection, adding Anti-IgE solution and continuing to culture for 24 hours; mu.l BFA/Monensin mix (250X) was added to 500. Mu.l of cell suspension 8h before the end of the culture.

(5) Fluorescence labeling of basophils;

(6) Detection of intracellular cytokines: (1) fixing and penetrating the membrane: adding 250 μ l of Fixation/Permeabilization into each tube, and incubating for 20min at 4 ℃ in the dark; 500. Mu.l of 1 XPerm/Wash were added to each tube ^TM Centrifuging (300 g multiplied by 6 min) at room temperature (15 ℃ -25 ℃), removing supernatant, and repeating for 2 times; (2) adding an intracellular antibody: 200 μ l of 1 XPerm/Wash ^TM Resuspending the cells, then adding 5 μ l each of Anti-Human IL-4-PE-Cy7/Anti-Human IL-13-FITC antibodies, and incubating for 30min in the dark on ice (instruction 20-60 min); (3) FCM detection: each tube was washed 1 time with 3ml PBS, centrifuged (300 g X6 min) at room temperature (15-25 deg.C), the supernatant removed, and the cells resuspended in 300. Mu.l PBS were added for detection on the machine.

2. After miR-519e-5p mimic transfection, the expression level of basophil miR-519e-5p is remarkably increased (shown in figure 4; the number of cases is respectively 5).

The results also show that the proportion of IL-4 positive basophils is reduced after miR-519e-5p is up-regulated (as shown in FIG. 5; the number of cases is 15 respectively).

Therefore, the up-regulation of miR-519e-5p can inhibit Anti-IgE mediated basophil activation, and can be used for preparing a basophil activation inhibitor and a related kit, chip or detection platform.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Guangdong medical university subsidiary hospital

<120> basophil activation related biomarker and application thereof

<130> 2022

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<170> PatentIn version 3.5

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Claims (4)

1. The application of a primer for detecting a biomarker in preparing an IgE pathway mediated basophil activation detection preparation is characterized in that the biomarker is miRNA-519e-5p; the nucleotide sequence of the miRNA-519e-5p is shown in SEQ ID No. 1.

2. The use of claim 1, wherein the nucleotide sequence of the primer is as shown in SEQ ID No. 2.

3. The use of claim 1, wherein the detection agent is a reagent for detecting the expression level of the biomarker based on gene chip detection or real-time fluorescent quantitative PCR.

The application of the miR-519e-5p imic in the preparation of the IL-4 positive basophil activation inhibitor is characterized in that the nucleotide sequence of the miRNA-519e-5p is shown in SEQ ID No. 1.

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