Interfering RNA of PLK1 and application thereof

Abstract

The application discloses an interfering RNA of PLK1 and application thereof, and belongs to the technical field of biological medicines. The application discloses application of a reagent for knocking down PLK1 in preparation of a medicine for preventing and treating pseudoallergy, wherein the reagent comprises siRNA for interfering with PLK1 gene expression, and the nucleotide sequence of the siRNA is shown as SEQ ID NO: 5-6. Through in-vitro cell experiments, it is proved that the expression level of the knocked-down PLK1 can improve morphological changes of RBL-2H3 cells in degranulation induced by C48 / 80, reduce cell apoptosis, and reduce HA and beta-HEX release amount, and has an obvious improvement effect on pseudoallergy; it is proved that PLK1 has a significant regulation effect on pseudoallergy, and provides a theoretical basis and experimental basis for exploring a potential mechanism and a treatment method of pseudoallergy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an interfering RNA of PLK1 and application thereof. BACKGROUND

[0002] With the change of living environment and lifestyle, allergic reactions induced by various factors have become one of the important problems threatening human health, seriously affecting people's physical and mental health, and causing a huge burden on people's quality of life and the whole society. Allergic reactions are divided into immediate type I allergic reactions and delayed type II, III, IV allergic reactions, which are related to the immune regulation of the human body. In addition to these allergic reactions mediated by the immune system, there is a kind of adverse reaction in clinical practice whose symptoms are similar to type I allergic reactions but is not immune-mediated, and its mechanism is not related to the immune pathway. This kind of reaction is called pseudoallergic reaction (pseudoallergic reactions), also known as non-immune hypersensitivity reaction.

[0003] Pseudoallergic reactions are different from IgE-mediated type I allergic reactions. Type I allergic reactions involve the participation of the immune pathway, while pseudoallergic reactions do not involve the immune system, and do not cause antigen-antibody reactions, but directly stimulate mast cells (MCs) to degranulate, prompting them to release histamine and other inflammatory factors, causing the production of allergic-like symptoms. MC is the most critical effector cell of pseudoallergic reactions. When receiving external stimulation, MC first releases stored cytokines and inflammatory mediators, and further secretes newly synthesized inflammatory mediators as the disease progresses, inducing MCs to degranulate and causing the production of allergic-like symptoms.

[0004] Polo-like kinase (PLK) is a serine / threonine protein kinase widely expressed in eukaryotic cells. PLK includes five family members, namely PLK1 (Polo-like kinase 1), PLK2, PLK3, PLK4, and PLK5. The main difference between PLK family members is the number of C-terminal polo-box domains (PBD), so their functions are different. Among them, PLK1 plays an important role in regulating cell division, maintaining genomic stability, spindle assembly, mitosis, and DNA damage response. Several studies have confirmed that PLK1 acts as an oncogene, which is highly expressed in various primary tumor tissues such as breast cancer, gastric cancer, liver cancer, prostate cancer, colorectal cancer, and esophageal cancer. However, there is no research report on the regulation of pseudoallergic reactions by PLK1. SUMMARY

[0005] The application aims to provide an interfering RNA of PLK1 and application thereof to solve the problems in the prior art.

[0006] To achieve the above object, the application provides the following solutions.

[0007] The application provides application of a reagent for knocking down PLK1 in preparation of a medicine for preventing and treating pseudoallergy, wherein the reagent comprises siRNA for interfering with expression of the PLK1 gene.

[0008] Further, the nucleotide sequence of the siRNA is shown as SEQ ID NO: 5-6.

[0009] Further, the siRNA plays a role in treating pseudoallergy by improving morphological changes of mast cell degranulation, reducing cell apoptosis, and reducing release amounts of histamine and beta-hexosaminidase of cells.

[0010] The application also provides a medicine for preventing and treating pseudoallergy, comprising siRNA for interfering with expression of the PLK1 gene.

[0011] Further, the nucleotide sequence of the siRNA is shown as SEQ ID NO: 5-6.

[0012] Further, the dosage form of the medicine is a tablet, a capsule, a granule, an injection or a spray.

[0013] Further, the medicine is taken orally or in a non-gastrointestinal form.

[0014] The application also provides an interfering RNA of PLK1, wherein the interfering RNA has a nucleotide sequence shown as SEQ ID NO: 5-6.

[0015] The application discloses the following technical effects:

[0016] The application is verified by in-vitro cell experiments, and it is confirmed that knocking down the expression level of PLK1 can improve morphological changes of RBL-2H3 cell degranulation induced by C48 / 80, reduce cell apoptosis, and reduce release amounts of HA and beta-HEX, and has a significant improvement effect on pseudoallergy. The application confirms that PLK1 has a significant regulation effect on pseudoallergy, and provides a theoretical basis and experimental evidence for exploring potential mechanisms and treatment methods of pseudoallergy. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only relate to some embodiments of the present application, and for those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0018] Figure 1 Fig. 4 is a fluorescence image of 150 nM RNA Oligo siRNA transfection (bar = 100 μm); (A) bright field; (B) fluorescence;

[0019] Figure 2 Fig. 5 is a result of PLK1 quantitative PCR, **p < 0.01 compared with the normal group;

[0020] Figure 3 Fig. 6 is a result of PLK1 protein expression (n = 3); (a) Western Blot detection of PLK1 knockdown; (b) gray scale analysis result, **p < 0.01 compared with the normal group;

[0021] Figure 4 Fig. 7 is a neutral red staining result of the influence of PLK1 knockdown on RBL-2H3 cell degranulation (bar = 100 μm);

[0022] Figure 5 Fig. 8 is a result of the influence of PLK1 knockdown on the release of β-Hex by RBL-2H3 cells, **p < 0.01 compared with the normal group; ##p < 0.01 compared with the model group;

[0023] Figure 6 Fig. 9 is a result of the influence of PLK1 knockdown on the release of histamine by RBL-2H3 cells, **p < 0.01 compared with the normal group; ##p < 0.01 compared with the model group;

[0024] Figure 7 Fig. 10 is a result of the influence of PLK1 knockdown on RBL-2H3 cell apoptosis (bar = 200 μm);

[0025] Figure 8 Fig. 11 is a result of the influence of PLK1 knockdown on RBL-2H3 cell apoptosis, **p < 0.01 compared with the normal group; ##p < 0.01 compared with the model group. DETAILED DESCRIPTION

[0026] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.

[0027] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentrations, dosage amounts, and the like, it is to be understood that each intervening value, to the upper and lower limits of the ranges is also specifically included within the scope of the present application. The upper and lower limits of these intervening values are also specifically included within the scope of the present application. These smaller ranges are not insubstantial.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0029] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0030] With respect to the use of "comprising", "including", "containing", "having" and "ensing" and the like, these terms are used in the sense of "open" terms (i.e., in the sense of "including, but not limited to").

[0031] The MRGPRX2 receptor on the surface of mast cells (MCs) is found to be an important receptor for inducing pseudoallergy. MRGPRX2 is a low-selectivity receptor, which enables it to interact with a variety of ligands, such as C48 / 80, opioid drugs, nerve blockers and the like compounds. Compound C48 / 80 is a synthetic condensation polymer of n-methyl-p-methoxyphenethylamine and formaldehyde, which has degranulation activity in differentiated MCs, mature MCs, LAD2 MCs and CD341 cell-derived MCs, and is often used to study the degranulation of MCs in humans and rodents. RBL-2H3 is a rat basophilic granulocyte, which can simulate the physiological and pathological responses related to MCs, and contains a large number of basophilic granules in the cytoplasm. When stimulated by antigens or non-antigen substances, it can release histamine, β-amino hexosidase, interleukin, TNF-α and the like. Therefore, RBL-2H3 cells have become a classic cell model for studying degranulation reaction instead of MCs. In this study, compound C48 / 80 is used to induce RBL-2H3 cells to construct a cell degranulation model, which simulates the process of mast cell degranulation, and is used to study the mechanism of pseudoallergy.

[0032] In the early stage of the application, genes related to pseudoallergy were screened by transcriptome sequencing. RBL-2H3 cells were degranulated by C48 / 80 induction, and RNA was extracted for transcriptome sequencing to obtain differential genes. GO and KEGG enrichment analysis was performed to screen significantly enriched signaling pathways. By screening the differential fold of genes related to the enriched pathways and combining with literature data, PLK1 was finally selected as the research target gene. The expression of PLK1 was verified by qPCR and Western blotting techniques, and it was found that the expression level of PLK1 in C48 / 80-induced RBL-2H3 cells was significantly increased, indicating that PLK1 was closely related to pseudoallergy. Therefore, the role of PLK1 in regulating pseudoallergy was studied and verified, as follows.

[0033] Example 1 Effect of knocking down PLK1 gene on pseudoallergy

[0034] 1 Experimental reagents and instruments

[0035] 1.1 Experimental materials

[0036] The RNAi sequence of knocking down PLK1 gene, PLK1-siRNA-1, PLK1-siRNA-2, PLK1-siRNA-3, and the negative control sequence NC, NC-FAM were synthesized by Gen-Pharm Pharmaceutical Technology Co., Ltd.

[0037] 1.2 Experimental reagents

[0038] Table 1

[0039]

[0040] 2 Experimental methods

[0041] 2.1 siRNA-PLK1 transfection and verification of transfection efficiency

[0042] 2.1.1 siRNA-PLK1 transfection

[0043] The transfection reagent was GP-transfect-Mate, and 150nm NC-FAM sequence was transfected. The transfection effect was observed under a fluorescence microscope 6h after transfection. The siRNA sequence is shown below.

[0044] PLK1 siRNA-1:

[0045] GGUGCAUCAUGUAUACCUUTT (SEQ ID NO: 1);

[0046] AAGGUAUACAUGAUGCACCTT (SEQ ID NO: 2);

[0047] PLK1 siRNA-2:

[0048] CCGAGGAUCCUGCUUGCAUTT (SEQ ID NO: 3);

[0049] AUGCAAGCAGGAUCCUCGGTT (SEQ ID NO: 4);

[0050] PLK1 siRNA-3:

[0051] GCACACGCAGCGCCAUCAUTT (SEQ ID NO: 5);

[0052] AUGAUGGCGCUGCGUGUGCTT (SEQ ID NO: 6);

[0053] Negative Control:

[0054] UUCUCCGAACGUGUCACGUTT (SEQ ID NO: 7);

[0055] ACGUGACACGUUCGGAGAATT (SEQ ID NO: 8);

[0056] Negative Control FAM:

[0057] UUCUCCGAACGUGUCACGUTT (SEQ ID NO: 9);

[0058] ACGUGACACGUUCGGAGAATT (SEQ ID NO: 10).

[0059] (1) Trypsin digestion of logarithmic growth phase RBL-2H3 cells, count 3 x 10 5 cells were inoculated in a 6-well plate, MEM complete culture medium 2 mL / well, cultured in a 37°C incubator. Set up normal group: no treatment; negative control group: transfection of NC-siRNA sequence; siRNA group: transfection of PLK1 siRNA-1, PLK1 siRNA-2, PLK1 siRNA-3; 3 replicate wells per group.

[0060] (2) Place the GP-transfect-Mate transfection reagent at room temperature, and mix gently before use.

[0061] (3) Add 193 μL of serum-free medium to a 1.5 mL enzyme-free centrifuge tube, slowly add 7 μL of GP-transfect-Mate transfection reagent, mix gently with a pipette, and stand at room temperature for 5 min.

[0062] (4) Take another 1.5 mL centrifuge tube without enzyme, add 190 μL serum-free medium, slowly drop 10 μL siRNA (100 nM), gently mix with pipette, and stand at room temperature for 5 min.

[0063] (5) Drop GP-transfect-Mate medium mixture into siRNA medium mixture, gently mix with pipette, stand at room temperature for 20 min, and immediately transfect.

[0064] (6) Take the six-well plate pre-seeded from the incubator, observe the cell density under microscope, and aspirate the original medium, and replace it with 1.6 mL fresh medium per well.

[0065] (7) Add 400 μL transfection mixture to the well, and the final system is 2 mL. After adding, gently shake the six-well plate to mix the mixture evenly.

[0066] (8) Incubate the cells at 37°C, and replace the complete medium after 6 h.

[0067] 2.1.2 Quantitative PCR detection of PLK1 expression

[0068] (1) Extraction of total RNA and reverse transcription

[0069] Collect the cells transfected for 36 h in 2.1.1, extract RNA by using cell tissue RNA extraction kit (Takara RNAisoPlus), and reverse the RNA to cDNA according to the steps of FastQuant cDNA first strand synthesis kit (PrimeScript RT reagent Kit with gDNA Eraser). The above kits are purchased from Takara Company. TM RT reagent Kit with gDNA Eraser). The above kits are purchased from Takara Company.

[0070] (2) Amplification of PLK1 gene

[0071] According to the mRNA full sequence of the gene, the primers are designed as shown in the following table:

[0072] Table 2

[0073]

[0074] PCR reaction system: 2x Flash Hot Start MasterMix (Dye) 25 μL, Forward Primer, 10 μM 1 μL, Reverse Primer, 10 μM 1 μL, Template cDNA 2 μL, ddH2O up to 25 μL.

[0075] The reaction system was configured on ice, and the PCR reaction conditions were set as follows: 98°C, 10 s; 55°C, 5 s; 72°C, 10 s; 30 cycles. The product was detected by 1.5% agarose gel, and the gel electrophoresis imaging instrument was photographed.

[0076] 2.1.3 Western blot detection of PLK1 protein expression

[0077] The cells transfected for 72 h in 2.1.1 were collected for protein expression detection. The detection steps are as follows:

[0078] (1) Protein extraction

[0079] The six-well plate was taken out from the 37°C incubator, and the operation was performed after alcohol disinfection. The old culture medium was discarded, 150 μL of cell lysis solution was added to each well of the six-well plate, and the pipette gun was repeatedly blown and transferred to a 1.5 mL centrifuge tube. Ice bath for 10 min, 4°C 12000g centrifugation for 5 min, the supernatant was taken to a new centrifuge tube, and stored at -20°C.

[0080] (2) Protein quantification

[0081] The BCA protein quantification method was used to detect the concentration of the extracted protein, and the protein concentration of each group was adjusted according to the measurement results.

[0082] (3) Western blotting

[0083] (a) Protein denaturation treatment. Add one-fourth volume of SDS-PAGE protein loading buffer to the sample, boil at 100°C for 5 to 10 min, centrifuge after cooling to room temperature, and store at -20°C.

[0084] (b) Electrophoresis. Prepare 10% separation gel and 5% concentrated gel, add 1x electrophoresis buffer to the electrophoresis tank, and add 20 μg of protein per well. Electrophoresis program: concentrated gel 80V constant voltage for 30 min; separation gel 120V constant voltage for 90 min.

[0085] (c) Transfer. Place the gel, filter paper, and PVDF membrane in the transfer buffer for 10 min, then place them in the transfer instrument for membrane transfer. The transfer conditions are 22V constant voltage for 45 min.

[0086] (d) Blocking. Block the protein with 5% skimmed milk powder, and shake at room temperature for 2 h.

[0087] (e) Primary antibody incubation. After blocking, take out the PVDF membrane, wash with 1x TBST for three times, each for 10 min, and incubate the primary antibody at 4°C overnight.

[0088] (f) Secondary antibody incubation. After the primary antibody incubation, the PVDF membrane was washed with 1x TBST for 10 min, 3 times. The secondary antibody was incubated at room temperature for 1 h. After the incubation, the PVDF membrane was washed with 1x TBST for 10 min, 3 times.

[0089] (g) Development. After the washing, the ECL luminescence solution was prepared, and the luminescence solution was evenly dropped on the PVDF membrane, and was placed for 2-3 min. Then, the PVDF membrane was placed in the imager for photographing and observation.

[0090] 2.2 Effect of PLK1 knockdown on the morphology of RBL-2H3 cells

[0091] The logarithmic growth period RBL-2H3 cells were trypsinized, and 10 5 cells were counted and inoculated in a 24-well plate at 1 mL / well of MEM complete medium. The normal group was added with DMSO, the model group was added with C48 / 80, the knockdown group was transfected with siRNA-PLK1 sequence and added with C48 / 80, and the negative control group was transfected with NC sequence and added with C48 / 80. Each group had 3 replicate wells. After 36 h of transfection, 200 μL of complete medium was added to the model group, and the C48 / 80 concentration was 25 μg / mL. The normal group was added with complete medium containing the same volume of DMSO. After 30 min, the reaction was terminated in an ice box. Neutral red dye was added, and the operation was performed according to the instructions. The transfection steps of the knockdown group and the negative control group are as follows:

[0092] (1) siRNA-PLK1 RNA Oligo and siRNA-NC RNA Oligo were taken at 3.75 μL / well, and were diluted to 50 μL (150 nM) with serum-free medium and mixed.

[0093] (2) GP-transfect-Mate transfection reagent was taken at 2 μL / well, and was diluted to 50 μL with serum-free medium and mixed.

[0094] (3) The GP-transfect-Mate-medium mixture was added to the diluted plasmid-medium mixture, mixed, and incubated at room temperature for 20 min.

[0095] (4) 100 μL of the transfection reagent / plasmid mixture was evenly and slowly added to the pre-placed serum-free medium to form 500 μL of medium.

[0096] (5) After incubation in a constant temperature incubator for 6 h, the complete medium was replaced.

[0097] 2.3 Effect of PLK1 knockdown on the release of β-Hex by RBL-2H3 cells

[0098] The logarithmic growth period RBL-2H3 cells were trypsinized, and the cell count was 8 x 10 4Cells were seeded per well in 24-well plates. Cells were divided into four groups: normal group (DMSO added); model group (C48 / 80 added); knockdown group (transfected with siRNA-PLK1-3, C48 / 80 added); negative control group (transfected with NC-siRNA sequence, C48 / 80 added); and blank wells were also included. Each group had 3 replicates. The culture medium was changed 12 h after transfection. 24 h later, except for the normal group and blank wells, the model group was added 200 μL of complete culture medium with a C48 / 80 concentration of 25 μg / mL, and the normal group was added complete culture medium containing an equal volume of DMSO. The reaction was stopped after 10 min by placing the plates on ice. The supernatant was transferred to centrifuge tubes and centrifuged at 4℃, 2500 rpm for 6 min. The supernatant was then collected. 0.1% Triton X-100 was added to the blank wells, and the cells were lysed for 10 min. Parameter settings: Centrifuge at 4℃, 11000 rpm for 6 min, and use the supernatant of cell lysis buffer from blank wells as the total enzyme supernatant. Add 50 μL of supernatant and 50 μL of chromogenic solution to each 96-well plate, let stand for 1 h, then add 200 μL of stop solution. Detect the OD value at 405 nm and record the value. The β-Hex release rate is calculated as follows:

[0099] β-Hex release rate (%) = (OD value of experimental group supernatant - OD value of blank well supernatant) / (OD value of total enzyme wells - OD value of blank well supernatant) × 100%

[0100] 2.4 Effect of PLK1 knockdown on histamine release from RBL-2H3 cells

[0101] RBL-2H3 cells in logarithmic growth phase were digested with trypsin, and 8 × 10⁸ cells were seeded in 24-well plates. 4 Cells per well, grouped as in 2.3. 12 h after knockdown transfection, the culture medium was changed. 24 h later, except for the normal group, each group was given 200 μL of complete culture medium with a C48 / 80 concentration of 25 μg / mL. The normal group was given complete culture medium containing an equal volume of DMSO. Observe under a microscope after 30 min, and stop the reaction after 10 min by placing the container on ice. Collect the supernatant, centrifuge at 3000 rpm for 5 min at 4℃, and proceed according to the histamine detection kit procedure.

[0102] 2.5 Effect of PLK1 knockdown on RBL-2H3 cell apoptosis

[0103] Apoptotic changes in RBL-2H3 cells during degranulation were observed using AO / EB double staining. Logarithmically growing RBL-2H3 cells were digested with trypsin and seeded in 24-well plates at 8 × 10⁸ cells / wells. 4Cells were grouped as in 2.2. After 12 h of transfection, the medium was replaced, and 24 h later, 200 μL of complete medium was added to each group, with C48 / 80 at a concentration of 25 μg / mL, and complete medium was added to the normal group, containing the same volume of DMSO. After 30 min, the reaction was observed under a microscope, and after 10 min, the reaction was terminated in an ice box. The AO / EB kit was used to detect apoptosis, and 300 cells in 5 randomly selected fields were observed and recorded under a fluorescence microscope. The apoptosis rate was calculated as follows:

[0104] Apoptosis rate (%) = (apoptotic cells) / (normal cells + necrotic cells) x 100%.

[0105] 2.6 Statistical analysis

[0106] SPSS 22.0 software was used for statistical analysis, and the data results were expressed as mean ± standard deviation. Single-factor analysis of variance was used for pairwise comparison between multiple samples, and P < 0.05 was considered statistically significant.

[0107] 3 Experimental results

[0108] 3.1 Verification of PLK1 transfection effect

[0109] 3.1.1 Verification of siRNA transfection efficiency

[0110] The transfection efficiency was verified using NC-FAM with fluorescence. As shown in Figure 1 , at a concentration of 150 nM NC-FAM, obvious fluorescence appeared in the cells, indicating good transfection efficiency. Therefore, a concentration of 150 nM was finally selected for gene transfection.

[0111] 3.1.2 PLK1 quantitative PCR results

[0112] The RNA extraction concentration and quality are shown in Table 3, and A260 / A280 is between 1.9 and 2.2, indicating good quality of extracted RNA. The quantitative PCR results are shown in Figure 2 , and the knockdown effect of PLK-siRNA-3 is the most significant, so PLK-siRNA-3 is selected for subsequent experiments.

[0113] Table 3

[0114]

[0115]

[0116] 3.1.3 PLK1 protein expression results

[0117] WB was used to verify PLK1 protein expression, and the results are shown in Figure 3As shown, PLK1-siRNA-3 can significantly inhibit the expression of PLK1, and the degree of reduction is significantly better than that of the other two siRNAs, which is consistent with the results of quantitative PCR. Therefore, PLK1-siRNA-3 was selected as the siRNA to knock down PLK1.

[0118] 3.2 Effect of PLK1 knockdown on RBL-2H3 cell morphology

[0119] like Figure 4 As shown, after neutral red staining, the normal RBL-2H3 cells had smooth edges, a normal spindle shape, and uniform red granules inside the cells. Compared with the normal group, the model group and the negative control group after C48 / 80 stimulation showed obvious swelling, deformation, and uneven staining. After reducing PLK1 expression with siRNA, the swelling of the cells was significantly improved, and the neutral red staining inside the cells was more uniform. The results indicate that reducing PLK1 expression can significantly alleviate the morphological changes of RBL-2H3 cells during C48 / 80-induced degranulation.

[0120] 3.3 Effect of PLK1 knockdown on β-Hex release from RBL-2H3 cells

[0121] The results are as follows Figure 5 As shown, compared with the normal group, the β-Hex release rate of the model group and the negative control group was significantly increased. However, after reducing PLK1 expression, the β-Hex release rate of the knockdown group was significantly decreased, which was significantly different from the model group. This suggests that PLK1 reduction may reduce the release of mast cell inflammatory mediators.

[0122] 3.4 Effect of PLK1 knockdown on histamine release from RBL-2H3 cells

[0123] Histamine is one of the main mediators of mast cell degranulation; detecting histamine release allows for observation of mast cell degranulation. Results are as follows: Figure 6 As shown, compared with the normal group, the histamine release in the model group and the negative control group was significantly increased, while the histamine release was significantly reduced after PLK1 expression was decreased, indicating that the degranulation of mast cells was inhibited.

[0124] 3.5 Effect of PLK1 knockdown on RBL-2H3 cell apoptosis

[0125] After AO / EB staining, normal surviving cells should exhibit green fluorescence and normal morphology, while apoptotic cells should exhibit red fluorescence and appear round. Results are as follows: Figure 7 As shown, cells in the normal group all exhibited green fluorescence and showed no morphological abnormalities. Compared with the normal group, the model group showed obvious red fluorescence and significantly increased apoptosis. However, the decrease in PLK1 expression significantly improved this situation. Figure 8 ).

[0126] 4Result analysis

[0127] Small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA, non-coding RNA. The concept of siRNA was discovered in 1998, which is a naturally occurring defense mechanism formed by endonuclease cutting double-stranded RNA into small fragments of RNA with specific sequences to resist the invasion of foreign genes. These siRNAs can bind to the mRNA expressed by foreign genes and induce the degradation of the corresponding mRNA. Since the discovery of siRNA, it has been widely used to silence the RNA of various genes. Because it can silence genes related to any disease, this method is simple to operate and has lower requirements than the CRISPR / CAS9 gene knockout method, and is a commonly used experimental method for studying gene function.

[0128] RBL-2H3 cells, as a classic cell for studying pseudoallergy, have been proven to be feasible and effective by multiple studies. RBL-2H3 cells contain abundant basophilic granules. Under normal conditions, RBL-2H3 cells are irregularly fusiform. After being stimulated by antigens or non-antigen substances, they release various inflammatory mediators, swell rapidly, and the intracellular granules are unevenly distributed. Staining RBL-2H3 cells with neutral red dye shows that C48 / 80-induced RBL-2H3 cells exhibit obvious swelling and uneven coloring. After knocking down the PLK1 gene, the abnormal cell morphology is significantly reduced, indicating that reduced PLK1 expression reduces the release of inflammatory mediators in RBL-2H3 cells and has a significant improvement effect on pseudoallergy.

[0129] RBL-2H3 cells contain various inflammatory mediators in the cytoplasm, which can release histamine, β-Hex, interleukins, and other substances under external stimulation. Histamine and β-Hex are important markers of MCs degranulation. Histamine is an organic nitrogen compound produced by decarboxylation of histidine and is widely distributed in MCs of tissues such as skin, lungs, and intestinal mucosa. When inflammation and allergic reactions occur, histamine is released to cause vasodilation, tissue edema, and other symptoms. This experiment found that the release of β-Hex and histamine in RBL-2H3 cells was significantly reduced after reducing PLK1 expression, indicating that reduced PLK1 expression can inhibit cell degranulation. AO / EB fluorescence staining is a classic method for detecting apoptosis. AO / EB can be used to identify changes in cell membranes during apoptosis, allowing for a direct view of cell apoptosis with the advantages of simplicity, intuitiveness, and low cost. AO / EB staining shows that reduced PLK1 expression can inhibit C48 / 80-induced RBL-2H3 cell apoptosis.

[0130] In summary, the present application proves that knocking down the expression level of PLK1 can improve the morphological changes of RBL-2H3 cells induced by C48 / 80, reduce cell apoptosis, and reduce the release amount of HA and beta-HEX, and has a significant improvement effect on pseudoallergy. The present application proves that PLK1 has a significant regulatory effect on pseudoallergy, and provides a theoretical basis and experimental evidence for exploring the potential mechanism and treatment method of pseudoallergy.

[0131] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. Use of an agent that knocks down PLK1 in the manufacture of a medicament for preventing and treating pseudoallergy, characterized in that, The agent comprises siRNA interfering with PLK1 gene expression; The nucleotide sequence of the siRNA is shown as SEQ ID NO: 5-6.

2. Use according to claim 1, characterized in that, The siRNA plays a role in treating pseudoallergy by improving the morphological changes of mast cell degranulation, reducing cell apoptosis, and reducing the release amount of histamine and beta-hexosaminidase of cells.

3. A medicament for preventing pseudoallergic reactions, characterized by, The agent comprises siRNA interfering with PLK1 gene expression; The nucleotide sequence of the siRNA is shown as SEQ ID NO: 5-6.

4. The medicament according to claim 3, characterized in that, The dosage form of the medicine is tablet, capsule, granule, injection or spray.

5. The medicament according to claim 3, characterized in that, The administration method of the medicine is oral or non-gastrointestinal administration.

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