A miRNA with anti-allergic activity and its application

Abstract

The present invention discloses a miRNA with anti-allergic activity and its application, wherein the miRNA is let‑7d‑5p. The present invention establishes a DNP‑IgE / BSA-induced RBL‑2H3 cell allergy model, detects the expression level of let‑7d‑5p in the cell model before and after curcumin treatment, and confirms for the first time that let‑7d‑5p is involved in the anti-allergic effect of curcumin; in addition, transfection of let‑7d‑5p mimics can significantly reduce the release of β‑HEX in the DNP‑IgE / BSA-induced RBL‑2H3 cell allergy model, while transfection of let‑7d‑5p inhibitors can reverse the decrease in the release of β‑HEX in the DNP‑IgE / BSA-induced RBL‑2H3 cell allergy model caused by curcumin. Therefore, let‑7d‑5p can be used to prepare anti-allergic drugs and can also be used to evaluate the anti-allergic ability of curcumin compounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and specifically relates to a miRNA with anti-allergic activity and application thereof, wherein the miRNA is let-7d-5p. Background Art

[0002] Food allergy is a systemic immune response mediated by the immune system that is detrimental to the body. IgE-mediated food allergy is the most common allergic reaction. Patients will experience abdominal pain, diarrhea, nausea, vomiting, and even anaphylactic shock in severe cases, which has a certain risk of death. The number of people with food allergies increases year by year, which not only causes a serious economic burden on people but also seriously affects people's quality of life. Curcumin (Cur), as a hydrophobic polyphenol compound, is a food functional factor with multiple functions such as anti-inflammatory and antioxidant. Studies have found that curcumin can inhibit the release of allergic mediators such as histamine, β-HEX, TNF-α and IL-4, but how to quickly and accurately evaluate curcumin's inhibition of allergic reactions in the early stage remains to be established.

[0003] MicroRNA (miRNA) is a class of endogenous non-coding small RNA with regulatory functions found in eukaryotes, with a size of about 18 to 24 nucleotides. As an important post-transcriptional regulatory factor, it mainly inhibits gene expression or degrades it by binding to the 3′UTR of the target gene mRNA, thereby exerting its negative regulatory function. Previous studies by our team have shown that let-7d-5p mediates the antioxidant effect of curcumin, but whether let-7d-5p can be used as a molecular diagnostic marker for curcumin to reduce allergic reactions has not yet been reported.

[0004] RBL-2H3 cells are a cell line commonly used in in vitro experimental research. They have stable and single characteristics and are easy to culture in vitro. They express high-affinity IgE receptors on their surface and can be used for allergen identification, diagnosis, and potential immunotherapy analysis. When the body is exposed to allergens for the first time, the FcεRI receptors on the surface of RBL-2H3 cells bind to IgE and sensitize the body. When the allergen enters the body again, the FcεRI receptors cross-link with the IgE-antigen complex, inducing RBL-2H3 cells to activate and degranulate, releasing inflammatory mediators such as β-hexosaminidase (β-HEX), histamine, and chemokines, thereby causing allergic symptoms. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-allergic miRNA and its application in view of the above problems.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention first provides a miRNA, wherein the miRNA is let-7d-5p, and the nucleotide sequence thereof is 5′-AGAGGUAGUAGGUUGCAUAGUU-3′ (U represents uracil).

[0008] The present invention also provides the use of the above let-7d-5p in the preparation of antiallergic drugs;

[0009] Wherein, the antiallergic drug refers to an agent that increases the expression of let-7d-5p;

[0010] The reagent contains an effective dose of let-7d-5p mimetic.

[0011] The present invention also provides a method for evaluating the anti-allergic ability of a let-7d-5p mimetic, the method comprising the following steps:

[0012] 1) Establish a DNP-IgE / BSA-induced RBL-2H3 cell allergy model, transfect NC mimic, and detect the amount of β-HEX released, denoted as A;

[0013] 2) In the DNP-IgE / BSA-induced RBL-2H3 cell allergy model, let-7d-5p mimics were transfected and the amount of β-HEX released was detected, denoted as a;

[0014] 3) The β-HEX release in uninduced normal RBL-2H3 cells was set as 1.0, and the anti-allergic ability of let-7d-5p mimics was calculated according to the following formula:

[0015] Anti-allergic ability R = |Aa| / 1.0×100%.

[0016] The present invention also provides an application of the above-mentioned let-7d-5p in evaluating the anti-allergic ability of curcuminoid compounds, and the application specifically comprises: treating a system expressing let-7d-5p with a candidate curcuminoid compound; detecting the expression of let-7d-5p in the system; if the expression of let-7d-5p increases, it indicates that the candidate curcuminoid compound is a potential substance with anti-allergic ability.

[0017] The present invention also provides a kit for evaluating the anti-allergic ability of curcumin compounds, wherein the kit comprises a reagent for detecting the expression level of let-7d-5p.

[0018] The beneficial effects of the present invention are:

[0019] The present invention establishes a DNP-IgE / BSA-induced RBL-2H3 cell allergy model, detects the expression of let-7d-5p in the cell model before and after curcumin treatment, and confirms for the first time that let-7d-5p is involved in the anti-allergic regulatory effect of curcumin; in addition, transfection of let-7d-5p mimics can significantly reduce the release of β-HEX in the DNP-IgE / BSA-induced RBL-2H3 cell allergy model, and transfection of let-7d-5p inhibitors can reverse the decrease in the release of β-HEX in the DNP-IgE / BSA-induced RBL-2H3 cell allergy model caused by curcumin. Therefore, let-7d-5p can be used to prepare anti-allergic drugs and can also be used to evaluate the anti-allergic ability of curcumin compounds. While expanding the application direction of let-7d-5p, the present invention also provides a basis for the establishment of a method for evaluating the anti-allergic ability of curcumin compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Effect of curcumin on the release of β-HEX in RBL-2H3 cell allergy model. ## P < 0.01, compared with the control group, ** P < 0.01, compared with the IgE+BSA group, n=3.

[0021] Figure 2 : Relative expression level of let-7d-5p in the RBL-2H3 cell allergy model treated with curcumin. ## P < 0.01, compared with the control group, ** P < 0.01, compared with the IgE+BSA group, n=3.

[0022] Figure 3 :Effect of let-7d-5p on the release of β-HEX in RBL-2H3 cell allergy model. ## P < 0.01, compared with the control group, ** P < 0.01, compared with the NC mimic group, n=3.

[0023] Figure 4 :The effect of let-7d-5p inhibitor on the release of β-HEX in the RBL-2H3 cell allergy model after curcumin treatment. The "+" in the figure indicates that the corresponding substance was added; "-" indicates that the corresponding substance was not added. The group treated with DMSO after transfection with NC inhibitor was used as the negative control. ** P < 0.01, comparison between let-7d-5p inhibitor transfection group, curcumin treatment group and negative group; **P < 0.01, comparison between the let-7d-5p inhibitor-transfected and curcumin-treated groups and the curcumin-treated group, n = 3. DETAILED DESCRIPTION

[0024] The technical scheme of the present invention is further described below by specific examples. However, the present invention is not limited to the following examples. The raw materials used in the present invention can be purchased on the market or synthesized by methods known in the art.

[0025] Experimental samples and reagents:

[0026] RBL-2H3 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; curcumin (CAS: 458-37-7) was purchased from Sigma, USA; DNP-IgE was purchased from Sigma, USA; DNP-BSA was purchased from Biosearch, USA; NC mimic, NC inhibitor, let-7d-5p mimic, and let-7d-5p inhibitor were purchased from Guangzhou Ruibo Biotechnology Co., Ltd.; TRIzol was purchased from Thermo Fisher Scientific, USA; miRcute enhanced miRNA cDNA first-strand synthesis kit and miRcute enhanced miRNA fluorescence quantitative detection kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; benchtop solution was purchased from Beijing Solebow Technology Co., Ltd. Among them, let-7d-5p mimic is a let-7d-5p analog, a chemically synthesized mature miRNA double-stranded protein, which can enhance the function of endogenous let-7d-5p; let-7d-5p inhibitor is a let-7d-5p inhibitor, a chemically modified mature miRNA single-stranded protein, which can reduce the regulatory effect caused by let-7d-5p in cells.

[0027] Example 1 Construction of RBL-2H3 cell allergy model to detect the effect of curcumin on β-HEX release

[0028] RBL-2H3 cells were grown at 1.5×10 5 The cells were inoculated at a density of 100 μL / mL in a 48-well plate (200 μL per well) and divided into the following 4 treatment groups:

[0029] ①Control group: 200 μL MEM medium was added to each well and cultured at 37 ℃ for 18 h. After discarding the supernatant, 200 μL benchtop medium was added to each well and cultured at 37 ℃ for another 1 h.

[0030] ②IgE+BSA group: 2 μL 200 ng / mL DNP-IgE was added to each well and incubated at 37°C for 18 h. Then, the residual DNP-IgE was washed away with benchtop solution, and 2 μL 500 ng / mL DNP-BSA and 2 μL DMSO were added to each well and incubated at 37°C for another 1 h.

[0031] ③5 (Cur 5μM) group: 2 μL of 200 ng / mL DNP-IgE was added to each well and cultured at 37°C for 18 h. Then, the residual DNP-IgE was washed away with benchtop solution, and 2 μL of 0.5 mM curcumin and 2 μL of 500 ng / mL DNP-BSA were added to each well and cultured at 37°C for 1 h.

[0032] ④10 (Cur 10μM): Add 2 μL of 200 ng / mL DNP-IgE to each well and incubate at 37°C for 18 h. Then use benchtop solution to wash away the residual DNP-IgE, and then add 2 μL of 1 mM curcumin and 2 μL of 500 ng / mL DNP-BSA to each well and continue to incubate at 37°C for 1 h.

[0033] After incubation, take the supernatant into a 1.5 mL EP tube, centrifuge at 4000 g for 5 min, add 250 μL / well of cell lysate to the 48-well plate, and shake for 5 min. Take 25 μL of supernatant and cell lysate respectively and add them to the 96-well plate, add 100 μL of 1.2 mM β-HEX substrate solution to each well, incubate at 37 °C for 30 min, and use a microplate reader to detect the fluorescence value under the conditions of excitation wavelength of 360 nm and emission wavelength of 450 nm. Calculate the amount of β-HEX released, β-HEX released (%) = supernatant fluorescence value / (supernatant fluorescence value + lysate fluorescence value).

[0034] The results show that ( Figure 1 ), compared with the control group, the β-HEX release in the IgE+BSA group increased significantly, indicating that the model was successfully established; compared with the IgE+BSA group, the β-HEX release after 5 μM and 10 μM curcumin decreased significantly in a concentration-dependent manner. The above results show that curcumin has the effect of inhibiting allergic reactions.

[0035] Example 2 Detection of let-7d-5p expression in RBL-2H3 cell allergy model

[0036] RBL-2H3 cells were grown at 8×10 5 The cells were inoculated at a density of 1000 μL / mL in a 12-well plate (1000 μL per well) and divided into the following 4 treatment groups:

[0037] ①Control group: 1000 μL MEM medium was added to each well and cultured at 37 ℃ for 18 h. After discarding the supernatant, 1000 μL benchtop medium was added to each well and cultured at 37 ℃ for another 1 h.

[0038] ②IgE+BSA group: 10 μL of 200 ng / mL DNP-IgE was added to each well and incubated at 37°C for 18 h. Then, the residual DNP-IgE was washed away with benchtop solution, and 10 μL of 500 ng / mL DNP-BSA and 10 μL of DMSO were added to each well and incubated at 37°C for another 1 h.

[0039] ③5 (Cur 5μM) group: 10 μL of 200 ng / mL DNP-IgE was added to each well and cultured at 37°C for 18 h. Then, the residual DNP-IgE was washed away with benchtop solution, and 10 μL of 0.5 mM curcumin and 10 μL of 500 ng / mL DNP-BSA were added to each well and cultured at 37°C for 1 h.

[0040] ④10 (Cur 10μM): Add 10 μL of 200 ng / mL DNP-IgE to each well and incubate at 37°C for 18 h. Then use benchtop solution to wash away the residual DNP-IgE, and then add 10 μL of 1 mM curcumin and 10 μL of 500 ng / mL DNP-BSA to each well and continue to incubate at 37°C for 1 h.

[0041] (1) Primer design

[0042] Based on the sequence of let-7d-5p (5′- AGAGGUAGGUUGCAUAGUU-3′, U represents uracil

[0043] ) Design primers. The tailing primer sequences designed are as follows:

[0044] Upstream primer of let-7d-5p: 5′-GGCCAGAGGTAGTAGGTTGCATAGTT-3′.

[0045] Downstream primer of let-7d-5p: 5′-GGCCGCTGTCAACGATACGCTACCGT-3′.

[0046] (2) Detection of let-7d-5p expression level

[0047] TRIzol Reagent was used to extract total RNA from RBL-2H3 cells. The miRcute enhanced miRNA cDNA first-strand synthesis kit was used for reverse transcription to synthesize the first-strand cDNA. The miRcute enhanced miRNA fluorescence quantitative detection kit was used to detect the expression level of let-7d-5p in the RBL-2H3 cell allergy model on an ABI 7300 real-time fluorescence quantitative PCR instrument. The let-7d-5p real-time fluorescence quantitative PCR expression detection used U6 (the upstream primer of U6 is: 5′-GGCGCAAATTCGTGAAGCGTTCCATA -3′, the downstream primer of U6 is: 5′- GGCCGCTGTCAACGATACGCTACCGT-3′) as the internal reference. The real-time fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 95.0 ℃ for 5 min; followed by 40 cycles of amplification, each cycle consisting of denaturation at 95.0 ℃ for 5 s and amplification at 60.0 ℃ for 31 s; then the reaction was terminated at 95.0 ℃ for 15 s, 60.0 ℃ for 1 min, 95.0 ℃ for 15 s, and 60.0 ℃ for 15 s.

[0048] Adoption 2 -△△Ct The relative expression of let-7d-5p was calculated by the method, where: △△Ct = △Ct (experimental) - △Ct (control), △C = Ct (let-7d-5p) - Ct (U6). Figure 2 As shown, compared with the IgE+BSA group, the expression of let-7d-5p in cells was significantly increased after curcumin treatment, that is, curcumin promoted the expression of let-7d-5p.

[0049] Example 3 Effect of let-7d-5p on β-HEX release in RBL-2H3 cell allergy model

[0050] 1×10 cells were seeded per well of a 48-well plate. 5RBL-2H3 cells were transfected when the cell density reached about 70%. First, the lipo2000 transfection reagent was diluted to a concentration of 20 ng / mL with Opti-MEM serum-free medium, and the let-7d-5p mimic was diluted to a concentration of 400 nM with Opti-MEM serum-free medium. After standing for 5 minutes, the two solutions were mixed at a volume ratio of 1:1, and stood at room temperature for 20 minutes. The mixed solution was evenly added to the cell culture plate at 200 μL per well, and incubated at 37 ° C for 24 hours, and 2 μL of 200 ng / mL IgE was added to each well for further incubation for 18 hours, and then 2 μL of 500 ng / mL DNP-BSA was added to each well for further incubation for 1 hour. The sample was collected to detect the amount of β-HEX released (the method was the same as in Example 1). NC mimic was used as a control.

[0051] The evaluation formula for the anti-allergic ability of let-7d-5p mimics is: R=|Aa| / 1.0×100%; where R is the anti-allergic ability; A is the β-HEX release amount of the NC mimic group in the RBL-2H3 cell allergy model; a is the β-HEX release amount of the let-7d-5p mimic group in the RBL-2H3 cell allergy model; the β-HEX release amount in the supernatant of the control group was set to 1.0.

[0052] The results show that ( Figure 3 ), the β-HEX release amount of NC mimic transfected samples in the RBL-2H3 cell allergy model was recorded as A, which increased by 70% compared with the control group; the β-HEX release amount of let-7d-5p mimic group in the RBL-2H3 cell allergy model was recorded as a, which increased by 50% compared with the control group; the anti-allergic ability of let-7d-5p mimic R = |Aa| / 1.0×100% = 20%.

[0053] It can be seen from this example that transfection of let-7d-5p mimics can significantly reduce the release of β-HEX in the DNP-IgE / BSA-induced RBL-2H3 cell allergy model.

[0054] Example 4 let-7d-5p mediates curcumin to reduce β-HEX levels in RBL-2H3 cell allergy model

[0055] 1×10 cells were seeded per well of a 48-well plate. 5RBL-2H3 cells were transfected when the cell density reached about 70%. First, the lipo 2000 transfection reagent was diluted to a concentration of 20 ng / mL with Opti-MEM serum-free medium, and the let-7d-5p inhibitor was diluted to a concentration of 400 nM with Opti-MEM serum-free medium. After standing for 5 minutes, the two solutions were mixed at a volume ratio of 1:1, and stood at room temperature for 20 minutes. The mixed solution was evenly added to the cell culture plate at 200 μL per well, and incubated at 37 °C for 24 hours. After that, 2 μL of 200 ng / mL IgE was added to each well and incubated for another 18 hours. The treatment method is as follows:

[0056] ①DMSO group: Add 200 μL benchtop solution (containing 2 μL 500 ng / mL DNP-BSA and 2 μL DMSO) to the wells transfected with NC inhibitor and let-7d-5p inhibitor and continue to co-culture for 1 h.

[0057] ②Cur 10μM group: Add 200μL benchtop solution (containing 2 μL 500 ng / mL DNP-BSA and 2 μL 1mM curcumin) to the wells transfected with NC inhibitor and let-7d-5p inhibitor and continue to co-culture for 1 h.

[0058] The samples were collected to detect the β-HEX release (the method was the same as in Example 1). The β-HEX release in the cell supernatant of the group transfected with NC inhibitor and added with DMSO was used as a negative control.

[0059] The results show that ( Figure 4 ), compared with the negative control, after transfection with let-7d-5p inhibitor, the release of β-HEX in the DNP-IgE / BSA-induced RBL-2H3 cell allergy model increased by 23% ( p < 0.01); however, after curcumin treatment, the release of β-HEX in the DNP-IgE / BSA-induced RBL-2H3 cell allergy model decreased by 10% ( p < 0.01). Compared with the curcumin-treated group, the amount of β-HEX released in the DNP-IgE / BSA-induced RBL-2H3 cell allergy model induced by transfection of let-7d-5p inhibitor and curcumin treatment was significantly increased ( p < 0.01).

[0060] It can be seen from this example that the let-7d-5p inhibitor reversed the decrease in β-HEX release in the DNP-IgE / BSA-induced RBL-2H3 cell allergy model caused by curcumin.

[0061] In summary, the present invention proposes for the first time the application of let-7d-5p in anti-allergy. In the DNP-IgE / BSA-induced RBL-2H3 cell allergy model, curcumin reduced the amount of β-HEX released in a concentration-dependent manner and significantly increased the ratio of let-7d-5p / U6; after transfection of let-7d-5p mimics, the amount of β-HEX released in the DNP-IgE / BSA-induced RBL-2H3 cell allergy model was significantly reduced; after transfection of let-7d-5p inhibitors, the decrease in the amount of β-HEX released in the DNP-IgE / BSA-induced RBL-2H3 cell allergy model caused by curcumin could be reversed. Therefore, let-7d-5p can be used to prepare anti-allergic drugs and can also be used to evaluate the anti-allergic efficacy of curcumin compounds. While expanding the application direction of let-7d-5p, the present invention also provides a basis for the establishment of a method for evaluating the anti-allergic ability of curcumin.

[0062] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. The use of let-7d-5p mimetic in the preparation of antiallergic drugs, characterized in that: The nucleotide sequence of Let-7d-5p is 5′-AGAGGUAGUAGGUUGCAUAGUU-3′; the allergy refers to the RBL-2H3 cell allergy induced by DNP-IgE / BSA.

2. Application of let-7d-5p in evaluating the anti-allergic ability of curcuminoids, characterized in that: The nucleotide sequence of let-7d-5p is 5′-AGAGGUAGUAGGUUGCAUAGUU-3′; a system expressing let-7d-5p is treated with a candidate curcuminoid compound, and the expression of let-7d-5p in the system is detected. If the expression of let-7d-5p increases, it indicates that the candidate curcuminoid compound is a potential substance with anti-allergic ability; the system expressing let-7d-5p refers to a DNP-IgE / BSA-induced RBL-2H3 cell allergy model; the curcuminoid compound is curcumin.

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