ShRNA for WTAP and application thereof

Abstract

The invention relates to shRNA (short hairpin Ribonucleic Acid) aiming at a WTAP gene, which is used for knocking down the expression of the WTAP gene and regulating and controlling the functions of neutrophile granulocytes, and provides a way for treating diseases related to the neutrophile granulocytes. The invention discloses the effect of the WTAP gene on regulating and controlling the functions of the neutrophil, the proliferation of the neutrophil can be inhibited by inhibiting the expression of the WTAP in the neutrophil, the secretion of IL-1beta, IL-6 and TNF-alpha inflammatory factors is reduced, and the formation of NETs is inhibited; and a good theoretical basis and scientific basis are provided for clinically treating infection, cancers, autoimmune diseases and cardiovascular diseases through targeting the neutrophil. The shRNA sequence provided by the invention can be further developed into a clinical treatment medicine and has a wide market application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an shRNA targeting WTAP and its applications. Background Art

[0002] As the most important immune cells in the body, neutrophils play important roles in infections, cancers, autoimmune diseases, and cardiovascular diseases. Complications of severe infections, including thrombosis, acute respiratory distress syndrome, and arterial hypotension, further increase the risk of death. Previous studies have confirmed that deaths caused by these infections are related to the excessive proliferation and hyperinflammatory responses of neutrophils. In cancers, tumor cells can cause the proliferation and activation of neutrophils. Clinical studies have found that elevated levels of tumor-associated neutrophils and circulating neutrophils are closely associated with poor prognoses of cancers. In autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, primary Sjögren's syndrome, psoriasis, inflammatory bowel disease, and multiple sclerosis, neutrophils continuously proliferate and release a large amount of inflammatory factors, leading to the continuous aggravation of the diseases. In cardiovascular diseases, a large number of neutrophils secrete inflammatory factors and NETs to damage vascular endothelial cells, thus accelerating the formation of atherosclerosis. Therefore, targeted regulation of neutrophil function is of great significance for the treatment of neutrophil-related diseases such as infections, cancers, autoimmune diseases, and cardiovascular diseases.

[0003] As an important epigenetic modification, m6A methylation modification has been widely studied and confirmed as one of the most common dynamic post-transcriptional regulatory mechanisms in eukaryotic mRNAs. The methyltransferase WTAP is a key regulator of m6A methylation modification. WTAP forms a ternary complex with methyltransferases METTL3 and METTL14, precisely positioning it to specific adenine sites in target RNA molecules. In this way, WTAP not only promotes the catalytic efficiency of m6A modification but also improves the specificity of its substrates, ensuring the accuracy and selectivity of m6A modification.

[0004] However, up to now, the regulatory effect of WTAP on neutrophil function is still unclear, and there is also a lack of a sequence designed using RNA interference technology to target the WTAP gene and regulate neutrophil function, providing a way for the treatment of neutrophil-related diseases. Summary of the Invention

[0005] The purpose of the present invention is to provide an shRNA targeting the WTAP gene to inhibit WTAP mRNA or knockdown WTAP protein expression and be able to regulate neutrophil function, providing a way for the treatment of neutrophil-related diseases.

[0006] To achieve the object of the present invention, the technical solution adopted is as follows:

[0007] An shRNA targeting the WTAP gene, characterized in that the target interference sequence of the shRNA is shown as SEQ ID NO.1 or SEQ ID NO.3. The shRNA targeting the WTAP gene is used to knockdown the expression of WTAP mRNA and protein.

[0008] A lentivirus, which contains the target interference sequence of the shRNA as described above.

[0009] The present invention also provides the application of the shRNA targeting the WTAP gene or the above-mentioned lentivirus in the preparation of drugs for treating neutrophil diseases.

[0010] The treatment of neutrophil diseases is to inhibit neutrophil proliferation, reduce the secretion of inflammatory factors such as IL-1β, IL-6, and TNF-α, and inhibit the formation of neutrophil extracellular traps NETs by targeting and regulating the WTAP gene in neutrophils. The neutrophil diseases include infections, cancers, autoimmune diseases, and cardiovascular diseases. The autoimmune diseases include inflammatory bowel disease. The inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

[0011] The present invention reveals the role of the WTAP gene in regulating neutrophil function. By inhibiting the expression of WTAP in neutrophils, neutrophil proliferation can be inhibited, the secretion of inflammatory factors such as IL-1β, IL-6, and TNF-α can be reduced, and the formation of NETs can be inhibited, providing a good theoretical basis and scientific evidence for clinically treating infections, cancers, autoimmune diseases, and cardiovascular diseases by targeting neutrophils. At the same time, the present invention also provides an shRNA, which can inhibit neutrophil proliferation, reduce the secretion of inflammatory factors such as IL-1β, IL-6, and TNF-α, and inhibit the formation of NETs after being transfected into neutrophils in vitro through lentivirus packaging. If the shRNA sequence is further developed into a clinical therapeutic drug, it will have a broad market application prospect. Description of the Drawings

[0012] Figure 1 It is a Western blot of WTAP in mouse peripheral blood neutrophils in Example 1 of the present invention.

[0013] Figure 2 It is a graph of the number of neutrophils in two types of mice in Example 2 of the present invention.

[0014] Among them, Figure 2 A is WTAP fl / fl control mice; Figure 2 B is WTAP fl / fl -Ly6GCre Mouse.

[0015] Figure 3 It is the mRNA expression map of LPS-induced neutrophil inflammatory factors in Example 3 of the present invention.

[0016] Among them, Figure 3 A is the mRNA of IL-1β; Figure 3 B is the mRNA of IL-6; Figure 3 C is the mRNA of TNF-α.

[0017] Figure 4 It is the formation map of PMA-induced NETs in Example 3 of the present invention.

[0018] Among them, Figure 4 A is the mRNA of PAD4; Figure 4 B is the Western blot of PAD4 protein; Figure 4 C is the statistical chart of the gray value of the Western blot of PAD4 protein; Figure 4 D is the representative image 1 of immunofluorescence staining of neutrophils in the peripheral blood of each group of mice. Cit-H3 is red, MPO is green, DAPI is blue, scale bar = 100 μm; Figure 4 E is the representative image 2 of immunofluorescence staining of neutrophils in the peripheral blood of each group of mice. NE is red, MPO is green, DAPI is blue, scale bar = 100 μm.

[0019] Figure 5 It is the DSS-induced mouse intestinal inflammation map in Example 4 of the present invention.

[0020] Among them, Figure 5 A is the curve graph of the body weight fluctuation of mice; Figure 5 B is the curve graph of the change in the disease activity index (DAI) of mice; Figure 5 C is the colon photos of two groups of mice; Figure 5 D is the colon length graph of mice; Figure 5 E is the HE image of the paraffin section of the colon tissue of mice, scale bar = 100 μm; Figure 5 F is the histological scoring graph of the colon tissue of mice.

[0021] Figure 6 It is the mRNA expression map of peripheral blood neutrophil inflammatory factors in DSS-induced mouse intestinal inflammation in Example 4 of the present invention.

[0022] Among them, Figure 6 A is the mRNA of IL-1β; Figure 6 B is the mRNA of IL-6; Figure 6 C is the mRNA of TNF-α.

[0023] Figure 7This is the figure showing the formation of peripheral blood NETs in the intestinal inflammation of mice induced by DSS in Example 4 of the present invention.

[0024] Among them, Figure 7 A is the mRNA of PAD4; Figure 7 B is the Western blot of PAD4 protein; Figure 7 C is the statistical chart of the gray value of the Western blot of PAD4 protein; Figure 7 D is the representative image 1 of immunofluorescence staining of neutrophils in the peripheral blood of mice in each group induced by DSS, Cit-H3 (red), MPO (green), DAPI (blue), scale bar = 100 μm; Figure 7 E is the representative image 2 of immunofluorescence staining of neutrophils in the peripheral blood of mice in each group induced by DSS, NE (red), MPO (green), DAPI (blue), scale bar = 100 μm.

[0025] Figure 8 This is the expression level of WTAP after transfection of neutrophils with lentivirus in Example 5 of the present invention.

[0026] Among them, Figure 8 A is the mRNA expression level of WTAP; Figure 8 B is the Western blot of WTAP protein; Figure 8 C is the statistical chart of the gray value of the Western blot of WTAP protein.

[0027] Figure 9 This is the figure showing that shWTAP-1 lentivirus can inhibit neutrophil proliferation in Example 6 of the present invention.

[0028] Among them, Figure 9 A is the cell survival rate graph of shWTAP-1 lentivirus and the control group within 48 hours; Figure 9 B is the photo of neutrophils under the fluorescence microscope.

[0029] Figure 10 This is the mRNA expression map of LPS-induced neutrophil inflammatory factors in the HL60 cell line transfected with shWTAP-1 lentivirus in Example 7 of the present invention.

[0030] Among them, Figure 10 A is the mRNA of IL-1β; Figure 10 B is the mRNA of IL-6; Figure 10 C is the mRNA of TNF-α.

[0031] Figure 11 This is the figure showing the formation of PMA-induced NETs in the HL60 cell line transfected with shWTAP-1 lentivirus in Example 8 of the present invention.

[0032] Among them, Figure 11A is the mRNA of PAD4; Figure 11 B is the Western blot of PAD4 protein; Figure 11 C is the statistical chart of the Western blot gray value of PAD4 in HL-60 cells transfected with shWTAP-1 lentivirus; Figure 11 D is the representative image 1 of immunofluorescence staining of HL-60 cells transfected with shWTAP-1 lentivirus, Cit-H3 (red), MPO (green), DAPI (blue), scale bar = 100 μm; Figure 11 E is the representative image 2 of immunofluorescence staining of HL-60 cells transfected with shWTAP-1 lentivirus, NE (red), MPO (green), DAPI (blue), scale bar = 100 μm.

[0033] In the above figures, # represents P < 0.05, ## represents P < 0.01, ### represents P < 0.001. Detailed implementation manners

[0034] The following further describes the implementation manners of the present invention in detail with reference to the accompanying drawings.

[0035] The sources of the relevant antibodies used in this application are shown in Table 1

[0036] Table 1 Antibody names and sources

[0037]

[0038]

[0039] Since it is impossible to construct WTAP systemic deficiency mice (embryonic lethal), the neutrophil-specific WTAP deficiency mice (WTAP fl / fl -Ly6G Cre ) were obtained as follows:

[0040] WTAP fl / + mice were purchased from Cyagen Biosciences, and Ly6G Cre were purchased from Model Animal Research Center. WTAP fl / + mice were mated with Ly6G Cre mice to obtain WTAP fl / + -Ly6G Cre mice. WTAP fl / + mice were self-crossed to obtain WTAP fl / fl homozygous mice. The obtained WTAP fl / + -Ly6G Cre mice were mated with WTAP fl / fl homozygous mice, and finally WTAP fl / fl -Ly6GCre Experimental group mice.

[0041] Example 1 Purification and extraction of mouse peripheral blood neutrophils

[0042] Anesthetize WTAP using isoflurane and an ABS type small animal gas anesthesia machine fl / fl mice and WTAP fl / fl -Ly6G Cre mice, the specific method is as follows:

[0043] Collect whole blood by cardiac puncture (about 1 ml per mouse), and add it to HBSS containing 15 mM EDTA (2 ml per mouse), and gently mix immediately with a Pasteur pipette. After centrifuging the above anticoagulated blood (400 g, 10 min, room temperature), the bottom of the centrifuge tube is the red blood cell layer, and a white cell layer, namely the buffy coat, can be seen on the interface of the red blood cells. Aspirate this layer with a Pasteur pipette and resuspend it in 2 ml of HBSS containing 2 mM EDTA. Add the prepared 78%, 69% and 52% percoll separation solutions into a glass centrifuge tube in sequence. Add the separation solution to ensure a clear interface between the gradient layers, and then add the cell suspension on top of all the separation solution layers. Centrifuge at 1500 g for 30 minutes at room temperature. Finally, aspirate the neutrophils enriched at the interface of the 69% and 78% layers with a Pasteur pipette, and wash twice with HBSS to obtain purified neutrophils.

[0044] And detect WTAP using Western blot fl / fl mice and WTAP fl / fl -Ly6G Cre Expression level of WTAP in mouse peripheral blood neutrophils, the specific experimental steps are as follows:

[0045] Lyse cells with immunoprecipitation buffer and collect cell proteins on ice. Determine the protein concentration with a BCA quantification kit. After transferring the proteins to a PVDF membrane by SDS-PAGE electrophoresis, add the primary antibody PDA4 (final dilution 1:750) and incubate overnight at 4 °C, and wash repeatedly 3 times with TBST. Add the corresponding secondary antibody and incubate at room temperature for 1 h and then wash off. Perform Western blot analysis using ECL chemiluminescence detection. As Figure 1 shown, WTAP fl / fl -Ly6G Cre WTAP in mice compared with WTAP fl / fl mice was not expressed, proving that a neutrophil-specific WTAP-deficient mouse model was successfully constructed.

[0046] Example 2 Detect WTAP fl / fl mice and WTAP fl / fl ​Cre Neutrophil count in mouse peripheral blood

[0047] Collect WTAP by cardiac puncture fl / fl Mice and WTAP fl / fl -Ly6G Cre Mouse whole blood was collected and added to HBSS containing 15 mM EDTA, and gently mixed immediately with a Pasteur pipette. Take 100 μl of cell suspension resuspended in RPMI 1640 medium containing 10% FBS, add flow antibodies: CD45-FITC, CD11b-APC, and Ly6G-PE, gently vortex and mix, incubate at 4 °C in the dark for 30 min. Wash twice with pre-cooled PBS, centrifuge at 300 g for 5 min, discard the supernatant, add 150 μL of PBS and then perform flow cytometry detection. The results are as Figure 2 shown Figure 2 WTAP in A fl / fl In control mice, the neutrophil count accounted for 6.48% of the peripheral blood cell content; Figure 2 In B, the neutrophil count in neutrophil-specific WTAP-deficient mice accounted for 2.27% of the peripheral blood cell content. Compared with WTAP fl / fl control mice, the neutrophil count in neutrophil-specific WTAP-deficient mice (WTAP fl / fl -Ly6G Cre ) decreased by up to 65%. It can be seen that knocking out WTAP can inhibit neutrophil proliferation.

[0048] Example 3 WTAP fl / fl Mice and WTAP fl / fl -Ly6G Cre Neutrophils in the peripheral blood of mice were used for neutrophil inflammation model and NETs model experiments

[0049] Use LPS to construct a neutrophil inflammation model to observe the effect of WTAP on the secretion of inflammatory factors in vitro. The specific method is as follows:

[0050] The purified and extracted WTAP fl / fl Mice and WTAP fl / fl -Ly6G Cre Neutrophils in the peripheral blood of mice were seeded in 12-well plates and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) for 30 minutes, cultured in an incubator at 37 °C with 5% CO2 for 30 minutes, and then treated with 500 nm LPS for 6 hours.

[0051] After that, real-time fluorescence quantitative PCR (qPCR) experiments were performed on the above-treated neutrophils. The specific method is as follows:

[0052] Collect the cell suspension in groups with a pipette into 1.5 ml centrifuge tubes, centrifuge at 300 g for 5 minutes, discard the supernatant in the centrifuge tubes, retain the cell pellet, add an appropriate amount of Buffer RL, and vortex until there are no obvious cell clusters. Then, still according to the manufacturer's instructions, use the FastPure Cell / Tissue Total RNA Isolation Kit V2 (product number: RC112-01, Vazyme, Nanjing, China) to extract the total RNA of the cells, detect the concentration of the total RNA with a ultra-micro spectrophotometer, and store it in a -80 °C refrigerator for later use. Take an appropriate amount of RNA for reverse transcription according to the kit instructions, and use HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) to reverse transcribe the total RNA into cDNA in a nuclease-free centrifuge tube. Perform fluorescence quantitative PCR according to the kit instructions, and configure a 10 μl system in a nuclease-free EP tube. Gently mix the reagents and add them dropwise into a 96-well PCR plate, cover it, put it into a fluorescence quantitative PCR instrument, and perform the qPCR reaction according to the following conditions: ① Pre-denaturation: 95 °C for 30 seconds; ② Cycling reaction: 95 °C for 10 seconds, 60 °C for 30 seconds, cycle 40 times; ③ Melting curve: 95 °C for 15 seconds, 60 °C for 60 seconds, 95 °C for 15 seconds. Use the 2-ΔΔCt method to calculate the relative expression level of mRNA, and normalize the target gene level to the level of the internal reference gene (GAPDH). The gene sequences used are shown in Table 2.

[0053] Table 2 Gene names and primer sequences

[0054]

[0055] As Figure 3 shown, purified and extracted WTAP fl / fl control mice and neutrophils-specific WTAP-deficient (WTAP fl / fl -Ly6G Cre ) mouse peripheral blood neutrophils. After inducing a cell inflammation model with LPS, qPCR detection showed that the mRNA levels of IL-1β, TNF-α, and IL-6 in the WTAP fl / fl -Ly6G Cre mouse peripheral blood neutrophils were significantly lower than those in the control group (WTAP fl / fl ).

[0056] Thus, it can be seen that knocking out WTAP can reduce the expression of neutrophil inflammatory factors induced by LPS.

[0057] Apply PMA to construct a classical NETs model to observe the effect of WTAP on NETs formation in vitro. The specific method is as follows:

[0058] The purified and extracted WTAP fl / fl Control mice and neutrophils-specific WTAP-deficient (WTAP fl / fl -Ly6G Cre ) mouse peripheral blood neutrophils were seeded in 12-well plates and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) for 30 minutes, then cultured in an incubator with 5% CO2 at 37 °C for 30 minutes, and then treated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 4 hours. After being stimulated by PMA, neutrophils activate peptidylarginine deiminase type 4 (PAD4), which in turn induces the citrullination of histone arginine residues, leading to nuclear envelope rupture and nuclear chromatin depolymerization. After the depolymerized chromatin binds fully with antibacterial proteins such as neutrophil elastase (NE), citrullinated histone H3 (CitH3), and MPO in the cytoplasm, it is released into the extracellular environment to form NETs. Then, qPCR and Western blot experiments were performed to detect the expression of PAD4 mRNA and protein, and the specific methods and primer sequences were the same as described above. At the same time, an immunofluorescence double staining experiment was performed on PMA-treated mouse neutrophils, and the specific method was as follows: The neutrophils were fixed with 4% paraformaldehyde for 30 min, washed 3 times with sterile PBS, permeabilized with 0.1% Triton X-100 for 20 minutes, blocked with immunofluorescence blocking solution at room temperature for 1 h, washed 3 times with PBS, incubated with rabbit anti-MPO antibody (final dilution 1:100), mouse anti-NE antibody (final dilution 1:100), or rabbit anti-histone H3 antibody (final dilution 1:200) overnight at 4 °C. After washing 3 times with PBS, incubated with the corresponding immunofluorescence secondary antibody at room temperature in the dark for 1 h. DAPI (final dilution 1:2000) dye was added and incubated in the dark for 10 min for DNA detection. After washing 3 times with PBS, centrifuged to discard the supernatant, 10 μL of anti-fluorescence quenching mounting solution was added and mixed evenly, then transferred to a glass slide for mounting, and fluorescence images were obtained using a fluorescence microscope, as Figure 4 shown in D to E. Figure 4 D is a representative image of immunofluorescence staining of peripheral blood neutrophils in each group of mice, Cit-H3 (red), MPO (green), DAPI (blue), scale bar = 100 μm; Figure 4 E is a representative image of immunofluorescence staining of peripheral blood neutrophils in each group of mice, NE (red), MPO (green), DAPI (blue), scale bar = 100 μm.

[0059] As Figure 4 shown, the purified and extracted WTAPfl / fl Control mice and WTAP fl / fl -Ly6G Cre After the NETs model was induced by PMA in mouse peripheral blood neutrophils, Figure 4 As shown in A, the qPCR results showed that WTAP fl / fl -Ly6G Cre the PAD4 mRNA level in mouse peripheral blood neutrophils was significantly decreased. Figure 4 In B - C, WTAP fl / fl -Ly6G Cre the PAD4 protein level in mouse peripheral blood neutrophils was significantly decreased. Thus, knocking out WTAP could inhibit the formation of NETs induced by PMA.

[0060] Example 4 Dextran sulfate sodium (DSS)-induced intestinal inflammation in mice

[0061] WTAP fl / fl Mice and WTAP fl / fl -Ly6G Cre Intestinal inflammation was induced in mice by administering 2.5% DSS in drinking water for 7 days. Preparation method of 2.5% DSS solution: Weigh 5 g of DSS powder, dissolve it in 200 ml of sterile purified water, and stir until completely dissolved. During the DSS modeling period, the body weight, fecal traits, and blood in feces of mice were observed and recorded daily, and the three - item cumulative score was carried out according to Table 3 for the disease activity index score.

[0062] Assessment of fecal occult blood: Collect mouse feces and place them in a 96 - well plate. Add the prepared o - toluidine - glacial acetic acid solution (3:17) and 3% hydrogen peroxide solution in equal volume and mix evenly. If it turns blue - brown within 2 min, it is judged as positive.

[0063] Table 3 DAI scoring rules

[0064]

[0065] As Figure 5 shown in A - B, WTAP fl / fl -Ly6G Cre mice had smaller changes in body weight and smaller changes in disease activity index compared with WTAP fl / fl mice.

[0066] After 7 days, the colon tissue pathological score was carried out on the mice as follows:

[0067] The collected terminal colon (about 2 cm in length) was placed in 4% formaldehyde for fixation for 48 hours, followed by dehydration with an alcohol gradient, clearing with xylene, and paraffin embedding. The paraffin blocks were sectioned to a thickness of 5 μm, and the sections were immersed in water, taken out and placed in an oven at 60 °C for drying and dewaxing. After dewaxing, the washed sections were stained with hematoxylin for 5 minutes and rinsed with tap water; the sections were placed in 1% hydrochloric acid for 5 seconds and rinsed with tap water; stained with 1% eosin for 1 minute. After dehydration with an alcohol gradient, they were placed in xylene for dehydration and clearing, and mounted after air drying. Histopathological scoring: Three H&E-stained slides of the colon of each group were selected and observed and photographed under an optical microscope (200× magnification), and analyzed by a blinded pathologist according to Table 4 below.

[0068] Table 4 Histopathological Scoring Criteria

[0069]

[0070] As Figure 5 shown in C-D, representative pictures of the mouse colon and colon length are presented. Figure 5 E is a representative HE image of a paraffin section of the colon tissue, scale bar = 100 μm. As Figure 5 shown in F is the histological score of the colon tissue.

[0071] As Figure 6 shown, purified WTAP fl / fl +DSS control mice and neutrophils-specific WTAP-deficient (WTAP fl / fl -Ly6G Cre )+DSS mouse peripheral blood neutrophils were collected and purified. qPCR detection showed that the mRNA levels of IL-1β, TNF-α, and IL-6 in the peripheral blood neutrophils of WTAP fl / fl -Ly6G Cre +DSS mice were significantly lower compared with the control group (WTAP fl / fl +DSS).

[0072] Thus, knocking out WTAP can reduce the expression of neutrophil inflammatory factors in DSS-induced intestinal inflammation mice.

[0073] Purified WTAP fl / fl +DSS control mice and neutrophils-specific WTAP-deficient (WTAP fl / fl -Ly6G Cre) + Peripheral blood neutrophils of DSS-induced mice were collected, followed by qPCR and Western blot assays to detect the expression of PAD4 mRNA and protein. The specific methods and primer sequences were the same as described above. Meanwhile, immunofluorescence double staining assays were performed on the peripheral blood neutrophils collected from the DSS-induced mouse model. The specific methods and antibodies were the same as those described in Example 3.

[0074] As Figure 7 shown, after DSS-induced colonic inflammation in mice, WTAP was purified and extracted from fl / fl peripheral blood neutrophils of control mice and neutrophils-specific WTAP-deficient mice. Figure 7 A The qPCR results showed that the PAD4 mRNA level in fl / fl -Ly6G Cre peripheral blood neutrophils of mice was significantly decreased. Figure 7 B-C The PAD4 protein level in fl / fl -Ly6G Cre +DSS peripheral blood neutrophils of mice was significantly decreased. Figure 7 D Representative images 1 of immunofluorescence staining of peripheral blood neutrophils in each group of mice in the DSS-induced mouse model, Cit-H3 (red), MPO (green), DAPI (blue), scale bar = 100 μm; Figure 7 E Representative images 2 of immunofluorescence staining of peripheral blood neutrophils in each group of mice in the DSS-induced mouse model, NE (red), MPO (green), DAPI (blue), scale bar = 100 μm.

[0075] Therefore, neutrophils-specific knockout of WTAP can reduce NETs formation in DSS-induced colonic inflammation in mice.

[0076] Example 5 Construction of a stable HL60 cell line with low expression of WTAP

[0077] The neutrophil cell line HL60 cells were purchased from Sigma Aldrich (Germany). The cells were strictly tested and no mycoplasma, bacteria or fungi were detected, and they were identified by STR species identification. The cells were inoculated in RPMI-1640 complete medium (10% (v / v) FBS + 1% penicillin / streptomycin) and cultured in an incubator at 95% humidity, 37 °C and 5% CO2. Logarithmic growth phase cells were used for subsequent experiments. All cells were tested for mycoplasma contamination before culture. The cells were passaged every 2 - 3 days, and all experiments used cells with a passage number not exceeding 15 times.

[0078] 1. Plasmid construction

[0079] The gene expression of WTAP was downregulated by the knockdown protocol of plasmid GV644. Specifically, for the WTAP gene sequence, three shRNA target interference sequences were designed in this application as follows:

[0080] shWTAP-1 is shown in SEQ ID NO.1, specifically: GGTGAACTGGAACAGACTAAA.

[0081] shWTAP-2 is shown in SEQ ID NO.2, specifically: GCGACTAGCAACCAAGGAACA.

[0082] shWTAP-3 is shown in SEQ ID NO.3, specifically: GCAAGTACACAGATCTTAACT.

[0083] Among them, the blank control plasmid: GV644-scramble.

[0084] The GV644 vector was selected and digested with double enzymes AgeI and EcoRI, and the above sequences were directionally inserted into the multiple cloning site downstream of the U6 promoter.

[0085] The specific method is as follows:

[0086] ① In a 50 μL reaction system, 2 μg of high-purity GV644 plasmid DNA was mixed with FastDigest AgeI (1 μL) and EcoRI (1 μL), 10×FastDigest Buffer (5 μL) was added and ddH2O was supplemented to the final volume, and incubated at 37 °C for 30 minutes. AgeI (ACCGGT) and EcoRI (GAATTC) were used to cut the 5' and 3' ends of the MCS respectively to form complementary 5'-CCGG and 5'-AATT sticky ends;

[0087] ② The digestion efficiency was verified by 1% agarose gel electrophoresis, and it was confirmed that the linearized vector was a single band. The enzyme digestion residues were removed by silica membrane column purification, and finally a high-purity linearized GV644 vector was obtained. Next, in a 10 μL ligation system, the purified GV644 linear vector (50 ng) was mixed with the target gene fragment (the molar ratio of the inserted fragment to the vector was 3:1), T4 DNA ligase (1 μL, 400 U / μL) and 10×T4 ligation buffer (1 μL) were added, and ligated at 16 °C for 3 hours. T4 enzyme was used to catalyze the formation of phosphodiester bonds between the 5' phosphate group and the 3' hydroxyl group to achieve directional ligation;

[0088] ③ Take 5 μL of the ligation product and add it to 50 μL of chemically competent cells. Incubate on ice for 30 minutes, heat shock at 42 °C for 45 seconds, immediately incubate on ice for 2 minutes, add pre-warmed LB liquid medium, and recover at 37 °C for 1 hour to express the antibiotic resistance gene; spread the bacterial solution on LB solid medium containing ampicillin (100 μg / mL), and incubate upside down at 37 °C for 14 hours to screen for positive clones by resistance; pick single colonies for expanded culture, and verify by colony PCR and plasmid digestion, so as to identify and obtain the plasmid for knocking down WTAP expression.

[0089] 2. Lentivirus packaging

[0090] Use human embryonic kidney 293T cells as packaging cells, inoculate them into a 10 cm culture dish (at a density of about 1×10 6 / dish), and culture them in high-glucose DMEM medium (containing 10% FBS and 1% double antibiotics) at 37 °C and 5% CO2 until 80% confluence. Mix the GV644-shWTAP plasmid, packaging plasmid psPAX2, and envelope plasmid pMD2.G in a ratio of 4:3:1, dissolve them in 500 μL of serum-free Opti-MEM, add 40 μL of polyethyleneimine (PEI, 1 mg / mL, pH 7.0), let stand at room temperature for 15 minutes to form a complex, and then add it dropwise to the 293T cell culture medium and gently shake to mix well. After transfection for 6 hours, replace it with pre-warmed complete medium and continue to culture for 48 hours. After 48 hours, collect the supernatant containing lentivirus particles, filter it through a 0.45 μm filter membrane to remove cell debris, centrifuge at 20,000×g at 4 °C for 2 hours, discard the supernatant, resuspend the lentivirus precipitate with 1 / 100 volume of PBS (containing 5% glycerol), aliquot and store at -80 °C. Infect 293T cells with serial dilutions of the lentivirus solution, and detect the proportion of copGFP-positive cells by flow cytometry 48 hours later. All lentivirus titers are ≥1×10 8 TU / mL.

[0091] 3. Cell transfection to construct a stable HL60 cell line with low expression of WTAP

[0092] Culture HL-60 cells to the logarithmic growth phase, collect the cells and adjust the cell concentration to about 2×10 5 / ml. During the infection process, mix the lentivirus supernatant with HL-60 cells and add an infection enhancer. To improve the infection efficiency, use a flat-angle rotor centrifuge to centrifuge at 1800 rpm at room temperature for 90 minutes. Co-culture the cell-virus mixture for 72 hours, change the culture medium as needed during this period to ensure cell viability, add puromycin (2 μg / mL) for screening for 72 hours to remove uninfected cells. After infection, use an inverted fluorescence microscope to observe the cell infection efficiency and confirm that it reaches about 80%.

[0093] The expression levels of WTAP in cells transfected with lentiviruses carrying three groups of shRNA target interference sequences were detected using qPCR and Western Blot techniques. The detection method was the same as above, and the gene sequences are shown in Table 5. As Figure 8 shown in A, the mRNA levels of WTAP in HL60 cells decreased after transfection with each lentivirus, and the decrease in shWTAP-1 was the lowest. Protein immunoblot analysis was performed on the protein levels of WTAP in cells after transfection with each lentivirus. As Figure 8 shown in B-C, the lentiviruses shWTAP-1 and shWTAP-3 had better interference efficiency.

[0094] Table 5 Gene names and primer sequences

[0095]

[0096] Example 6 CCK-8 experiment on the HL60 cell line transfected with shWTAP-1 lentivirus

[0097] Cells from the HL60 cell line transfected with shWTAP-1 lentivirus and the empty-transfected shWTAP-NC HL60 cell line were respectively taken and seeded in 48-well plates at a density of 2×10 3 / well. The CCK-8 reagent was diluted with the culture medium at a ratio of 1:10, and 100 μl of the diluted CCK-8 solution was added to each well. The mixture was incubated at 37 °C for 2 hours. The absorbance was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader. As Figure 9 shown in A, the proliferation activity of the stably transfected HL60 cell line (shWTAP-1) with WTAP knockdown was significantly reduced compared to the control cells (shWTAP-NC). As Figure 9 shown in B, after 48 hours, the number of stably transfected HL60 cell lines with WTAP knockdown was significantly reduced. Compared with the control group, # P<0.05, ### P<0.001.

[0098] This indicates that the WTAP lentivirus can inhibit neutrophil proliferation.

[0099] Example 7 LPS-induced neutrophil inflammation model of the HL60 cell line transfected with shWTAP-1 lentivirus

[0100] After successful transfection of the shWTAP-1 neutrophil cell line, it was treated with DMSO (1.25%) for 5 days to differentiate HL-60 cells into neutrophil-like cells. Subsequently, the differentiated HL-60 cells were seeded in 6-well plates and treated with 500 nM LPS for 4 hours to induce a neutrophil inflammation model. Then, qPCR experiments were performed on the above-treated neutrophils. The specific operation method was the same as in Example 3, and the different gene sequences are shown in Table 6

[0101] Table 6 Gene names and primer sequences

[0102]

[0103]

[0104] The experimental results are as Figure 10 shown in A - C. In the cell inflammation model induced by LPS, the qPCR detection results of the stably transfected HL60 cell line with WTAP knockdown showed that the mRNA levels of IL-1β, TNF-α, and IL-6 were significantly decreased compared with those of the control group cells (A - C).

[0105] Thus, at the in vitro cell level, the WTAP lentivirus can reduce the expression of neutrophil inflammatory factors induced by LPS.

[0106] Example 8 PMA-induced NETs model of the HL60 cell line transfected with shWTAP-1 lentivirus

[0107] After the successful transfection of HL-60 cells with shWTAP-1 lentivirus, the cells were treated with DMSO (1.25%) for 5 days to differentiate HL-60 cells into granulocyte-like cells. Subsequently, the differentiated HL-60 cells were seeded in 6-well plates and treated with 100 nM PMA for 3 - 4 hours to induce NETs formation. Then, qPCR, Western blot, and immunofluorescence double staining experiments were performed on the above-treated neutrophils. The specific methods were the same as those in Example 3, and the different gene sequences are shown in Table 7. The results are as Figure 11 shown in D - E.

[0108] Table 7 Gene names and primer sequences

[0109]

[0110] As Figure 11 shown in A, qPCR detection showed that the PAD4 mRNA level in HL-60 transfected with shWTAP-1 lentivirus was significantly decreased. As Figure 11 shown in B - C, in the PMA-induced NETs model, the PAD4 protein level in the stably transfected HL-60 cell line with shWTAP-1 lentivirus was significantly decreased compared with that of the control group.

[0111] Thus, at the in vitro cell level, transfection of neutrophils with the WTAP lentivirus can reduce the formation of PMA-induced NETs.

[0112] In summary, targeting and regulating the WTAP gene in neutrophils can inhibit neutrophil proliferation, reduce the secretion of inflammatory factors such as IL-1β, IL-6, and TNF-α, and inhibit the formation of NETs. After transfecting neutrophils with lentivirus prepared by the shRNA sequences such as SEQ ID NO.1 or SEQ ID NO.3, neutrophil proliferation can be inhibited, the secretion of inflammatory factors such as IL-1β, IL-6, and TNF-α can be reduced, and the formation of NETs can be inhibited.

[0113] Obviously, the above-mentioned embodiments of the present invention are only examples for clearly explaining the technical solutions of the present invention, rather than limitations on the specific implementation manners of the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the claims of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. An shRNA targeting the WTAP gene, characterized in that: The target interference sequence of the shRNA is shown in SEQ ID NO.1 or SEQ ID NO.

3.

2. The shRNA against the WTAP gene according to claim 1, characterized in that: The shRNA targeting the WTAP gene is used to inhibit WTAP mRNA or knockdown the expression of WTAP protein.

3. A lentivirus, characterized in that: The lentivirus contains the target interference sequence of the shRNA as described in claim 1.

4. Application of the WTAP gene, characterized in that: The application refers to the application in the preparation of drugs for treating neutrophil diseases.

5. The application according to claim 4, wherein: The treatment of neutrophil diseases is to inhibit neutrophil proliferation by targeting and regulating the WTAP gene in neutrophils, reduce the secretion of inflammatory factors such as IL-1β, IL-6, and TNF-α, and inhibit the formation of neutrophil extracellular traps NETs.

6. The application according to claim 5, characterized in that: Use the shRNA targeting the WTAP gene as described in claim 1 or the lentivirus as described in claim 3 for the preparation of drugs for treating neutrophil diseases.

7. The application according to claim 6, wherein: The neutrophil diseases include infections, cancers, autoimmune diseases, and cardiovascular diseases.

8. The application according to claim 7, characterized in that: The autoimmune diseases include inflammatory bowel disease.

9. The application according to claim 8, wherein: The inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

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