Application of miR-29a-3p in targeting PLSCR4 and inhibiting vascular inflammation and evaluation method of miR-29a-3p

Abstract

The invention discloses application of miR-29a-3p in targeting PLSCR4 and inhibiting vascular inflammation and an evaluation method of miR-29a-3p, and relates to the technical field of biomedicine.The method comprises the following steps that miR-29a-3p simulant or inhibitor is transfected to human umbilical vein endothelial cells and then stimulated with TNF-alpha, inflammatory response is evaluated through qRT-PCR, Western blot, immunofluorescence, Transwell migration, mononuclear cell chemotaxis and scanning electron microscope technologies, and therefore the inflammatory response is evaluated. According to the present invention, the PLSCR4 is identified as the miR-29a-3p direct target through the dual luciferase reporter gene experiment, the overexpression or knockout effect of the PLSCR4 is evaluated through Annexin V-PI dyeing and flow cytometry, the in vivo model of the mouse is established, the intravenous injection of the miR-29a-3p agonist is performed, and the lung tissue inflammation is analyzed by using the immunohistochemical method; the invention discloses that miR-29a-3p inhibits the activation of NLRP3 inflammasomes by down-regulating PLSCR4, so that the CASP1 mediated GSDMD cutting and subsequent cell pyroptosis pore formation are inhibited.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of miR-29a-3p in targeting PLSCR4 and inhibiting vascular inflammation. BACKGROUND

[0002] Vascular endothelial cells form a continuous monolayer lining the vascular lumen, acting as a key interface between circulating blood components and underlying vascular tissue. These specialized cells regulate vascular tone through paracrine signaling, maintain hemostasis through anticoagulant mechanisms, and maintain vascular integrity through intercellular tight junction complexes. Endothelial dysfunction, often induced by adverse stimuli, plays a key role in the pathogenesis of cardiovascular diseases, including atherosclerosis and hypertension. In response to inflammatory stimuli such as lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), and oxidized low-density lipoprotein, endothelial cells are activated and secrete pro-inflammatory cytokines, including interleukin-18 (IL-18) and interleukin-1 beta (IL-1 beta). In addition, they produce chemotactic factors such as monocyte chemoattractant protein-1 (MCP-1), promoting monocyte migration and subsequent differentiation into macrophages within the vascular wall. In addition, endothelial cells express adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), which mediate monocyte adhesion to the endothelial surface. At the same time, the disruption of tight junctions between endothelial cells increases vascular permeability and reduces the expression of tight junction proteins, promoting immune cell infiltration into tissues and exacerbating local inflammation.

[0003] Recent studies have highlighted that TNF-alpha induces vascular inflammation by triggering endothelial pyroptosis, a lytic form of programmed cell death characterized by extensive cytokine release. Experimental evidence suggests that TNF-alpha activates endothelial pyroptosis through a sequential activation of the NLRP3 / CASP1 / GSDMD signaling cascade. Specifically, TNF-alpha promotes the assembly of the NLRP3 inflammasome, leading to the proteolytic activation of caspase-1 (CASP1). This protease simultaneously processes pro-inflammatory cytokines (IL-1 beta / IL-18) and cleaves gasdermin D (GSDMD), producing a pore-forming N-terminal fragment (GSDMD-NT). Oligomerization of GSDMD-NT forms supramolecular pores in the plasma membrane, promoting the release of inflammatory mediators.

[0004] Integration of GSDMD-NT into the membrane requires dynamic remodeling of the phospholipid bilayer, a process regulated by phospholipid flippase 4 (PLSCR4). Recent biochemical analyses have shown that PLSCR4 catalyzes the ATP-independent bidirectional transmembrane redistribution of phosphatidylserine (PS) and phosphatidylethanolamine (PE), a key step in amplifying inflammatory signals.

[0005] MicroRNAs (miRNAs) are established as major regulators of cardiovascular pathophysiology, modulating gene expression through their sequence-specific post-transcriptional regulation. These non-coding RNAs finely tune cellular processes, such as proliferation and activation of inflammatory pathways, by binding to complementary regions of target mRNAs, causing translational repression or transcript degradation. Notably, the miR-29 family (miR-29a-3p, miR-29b-3p, miR-29c-3p) is involved in the dual regulation of extracellular matrix remodeling and innate immune response, positioning it as a central mediator of cardiometabolic disorders, but its potential to modulate vascular inflammation remains questionable. SUMMARY

[0006] The purpose of the present application is to make up for the shortcomings of the prior art, and to provide the application of miR-29a-3p in targeting PLSCR4 and inhibiting vascular inflammation, and to provide a new path for the precise diagnosis and treatment of vascular inflammation.

[0007] The present application provides the following technical solutions to solve the above technical problems:

[0008] The present application provides the application of miR-29a-3p in targeting PLSCR4.

[0009] The present application also provides the application of miR-29a-3p in inhibiting vascular inflammation.

[0010] In one possible design, miR-29a-3p inhibits the NLRP3-mediated pyroptosis pathway by targeting PLSCR4 to relieve vascular inflammation.

[0011] The present application also provides a drug for targeting PLSCR4, which at least comprises miR-29a-3p and a pharmaceutically acceptable carrier, diluent or excipient.

[0012] In one possible design, the administration form at least includes one of the following: direct blending administration, liposome encapsulation administration, nanoparticle encapsulation administration, aptamer-mediated targeted administration, antibody-mediated targeted administration, and administration through a hydrogel complex administration system.

[0013] The present application also provides a method for reducing the expression of PLSCR4 in target cells, which is carried out by administering miR-29a-3p.

[0014] The present application also provides a drug for inhibiting vascular inflammation, comprising miR-29a-3p and a pharmaceutically acceptable carrier, diluent or excipient.

[0015] In one possible design, the administration form includes at least one of the following: direct co-mixing administration, liposome-encapsulated administration, nanoparticle-encapsulated administration, aptamer-mediated targeted administration, antibody-mediated targeted administration, and administration through a hydrogel complex administration system.

[0016] The application also provides a method for inhibiting vascular inflammation, comprising administering miR-29a-3p to a subject in need thereof.

[0017] The application also provides a method for verifying the effect of miR-29a-3p targeting PLSCR4 and inhibiting vascular inflammation, comprising the following steps:

[0018] After transfecting the miR-29a-3p overexpression or inhibitor into human umbilical vein endothelial cells and stimulating with TNF-α, the inflammatory response is evaluated by qRT-PCR, Western blot, immunofluorescence, Transwell migration, monocyte chemotaxis, and scanning electron microscopy technology, the PLSCR4 is identified as a direct target of miR-29a-3p by dual luciferase reporter gene experiment, and the overexpression or knockout effect of PLSCR4 is evaluated by Annexin V-PI staining and flow cytometry, and a mouse in vivo model is established, the miR-29a-3p agonist is intravenously injected, and the lung tissue inflammation is analyzed by immunohistochemical method.

[0019] Compared with the prior art, the application of the siRNA targeting LOC124906852 in tumors has the following beneficial effects:

[0020] In the present application, we explored the molecular mechanism of miR-29a-3p alleviating TNF-α-induced endothelial inflammation by directly targeting PLSCR4, using multi-omics methods and combining functional verification of cell and animal models. Our findings reveal that miR-29a-3p inhibits the activation of NLRP3 inflammasome by down-regulating PLSCR4, thereby inhibiting CASP1-mediated GSDMD cleavage and subsequent pyroptosis pore formation. These results reveal a new miR-29a-3p / PLSCR4 / NLRP3 regulatory axis, providing key mechanistic insights into endothelial homeostasis and identifying a promising therapeutic target for inflammatory vascular diseases. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0022] Figure 1 Figure for the expression of IL-18, IL-1β, MCP-1, ICAM-1 and VCAM-1 genes under the stimulation of TNF-α in the embodiment of the present application, wherein A: the potential binding site of miR-29a-3p and PLSCR4 predicted in the Seed region; B: PLSCR4 is determined to be the target gene of miR-29a-3p by the detection of dual luciferase reporter experiment;

[0023] Figure 2 Figure for the influence of overexpression and inhibition of miR-29a-3p on the migration ability of EA.hy926 cells in the embodiment of the present application, wherein A: the influence of overexpression and inhibition of miR-29a-3p on the migration ability of EA.hy926 cells is detected by Transwell cell migration experiment; B: the quantitative analysis of A; Figure 2

[0024] Figure 3 Figure for the influence of overexpression and inhibition of miR-29a-3p on the migration ability of EA.hy926 cells in the embodiment of the present application, wherein A: the influence of overexpression and inhibition of miR-29a-3p on the migration ability of EA.hy926 cells is detected by Transwell cell migration experiment; B: the quantitative analysis of A; Figure 3

[0025] Figure 4 Figure for the influence of overexpression and inhibition of miR-29a-3p on the adhesion ability of EA.hy926 cells in the embodiment of the present application, wherein A: the influence of overexpression and inhibition of miR-29a-3p on the adhesion ability of EA.hy926 cells is detected by adhesion experiment; B: the quantitative analysis of A; Figure 4

[0026] Figure 5 Figure for the influence of overexpression and inhibition of miR-29a-3p on the chemotaxis ability of EA.hy926 cells in the embodiment of the present application, wherein A: the influence of overexpression and inhibition of miR-29a-3p on the chemotaxis ability of EA.hy926 cells is detected by monocyte chemotaxis; B: the quantitative analysis of A; Figure 5

[0027] Figure 6 Figure for the differential gene heat map of transcriptome in the embodiment of the present application;

[0028] Figure 7 Figure for the expression of PLSCR4 protein directly targeted by miR-29a-3p in EA.hy926 cells in the embodiment of the present application, wherein A: the potential binding site of miR-29a-3p and PLSCR4 predicted in the Seed region; B: PLSCR4 is determined to be the target gene of miR-29a-3p by the detection of dual luciferase reporter experiment;​​​​

[0029] Figure 8 Figures for proving that miR-29a-3p targets PLSCR4 in qRT-PCR and Western blot experiments in embodiments of the present application, wherein A: qRT-PCR experiment proves that miR-29a-3p targets PLSCR4; B: Western blot experiment proves that miR-29a-3p targets PLSCR4;

[0030] Figure 9 Figures for effects of overexpression and knockdown of PLSCR4 on secretion of proinflammatory cytokines, chemokines and adhesion molecules by EA.hy926 cells in embodiments of the present application, wherein A: qRT-PCR detects effects of overexpression and knockdown of PLSCR4 on secretion of proinflammatory cytokines by EA.hy926 cells; B: qRT-PCR detects effects of overexpression and knockdown of PLSCR4 on secretion of chemokines and adhesion molecules by EA.hy926 cells; C: Western blot experiment proves that knockdown of PLSCR4 can also down-regulate expression of adhesion molecules high-expression ICAM-1 and VCAM-1;

[0031] Figure 10 Figures for effects of overexpression and knockdown of PLSCR4 on secretion of pyroptosis factors by EA.hy926 cells in embodiments of the present application, wherein A: qRT-PCR detects effects of knockdown of PLSCR4 on secretion of pyroptosis factors by EA.hy926 cells; B: qRT-PCR detects effects of overexpression of PLSCR4 on secretion of pyroptosis factors by EA.hy926 cells; C: Western blot experiment detects expression amount of pyroptosis factor protein; D: scanning electron microscope image observes cell pyroptosis;

[0032] Figure 11 Figure for observing cell membrane PS exposure by Annexin-V-PI fluorescence double staining experiment in embodiments of the present application;

[0033] Figure 12 Figure for observing expression of three kinds of tight junction proteins by EA.hy926 cells by immunofluorescence experiment in embodiments of the present application. DETAILED DESCRIPTION

[0034] In order to further illustrate the technical means and effects adopted by the present application to achieve the predetermined object, the specific embodiments, structures, features and effects according to the present application are described in detail below with reference to the accompanying drawings and preferred embodiments.

[0035] In at least one embodiment, the present application provides an application of miR-29a-3p in targeting PLSCR4.

[0036] In at least one embodiment, the present application also provides an application of miR-29a-3p in inhibiting vascular inflammation.

[0037] In at least one embodiment, the present application also provides an application of miR-29a-3p in inhibiting vascular inflammation.

[0038] In at least one embodiment, the present application also provides an application of miR-29a-3p in inhibiting vascular inflammation.

[0039] In at least one embodiment, the present application also provides a drug for targeting PLSCR4, at least comprising miR-29a-3p and a pharmaceutically acceptable carrier, diluent or excipient.

[0040] In at least one embodiment, the present application also provides a drug for targeting PLSCR4, at least comprising miR-29a-3p and a pharmaceutically acceptable carrier, diluent or excipient.

[0041] In at least one embodiment, the present application also provides a method for reducing the expression of PLSCR4 in target cells, which is carried out by administering miR-29a-3p.

[0042] In at least one embodiment, the present application also provides a drug for inhibiting vascular inflammation, comprising miR-29a-3p and a pharmaceutically acceptable carrier, diluent or excipient.

[0043] In at least one embodiment, the present application also provides a drug for inhibiting vascular inflammation, comprising miR-29a-3p and a pharmaceutically acceptable carrier, diluent or excipient.

[0044] In at least one embodiment, the present application also provides a method for inhibiting vascular inflammation, which comprises administering miR-29a-3p to a subject in need thereof.

[0045] In at least one embodiment, the present application also provides a method for verifying the effect of miR-29a-3p in targeting PLSCR4 and inhibiting vascular inflammation, comprising the following steps:

[0046] After transfection of miR-29a-3p overexpression or inhibitor into human umbilical vein endothelial cells and stimulation with TNF-a, inflammatory response was evaluated by qRT-PCR, Western blot, immunofluorescence, Transwell migration, monocyte chemotaxis and scanning electron microscopy technology, dual luciferase reporter gene experiment identified PLSCR4 as a direct target of miR-29a-3p, and the effect of PLSCR4 overexpression or knockout was evaluated by Annexin V-PI staining and flow cytometry, and a mouse in vivo model was established, intravenous injection of miR-29a-3p agonist and analysis of lung tissue inflammation by immunohistochemical method.

[0047] The verification method provided by the application specifically comprises the following steps:

[0048] 1.1 Cell lines and cell culture

[0049] Human umbilical vein endothelial cells (EA.hy926) and THP-1 monocytes were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and used for in vitro studies. Cells were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM; Procell) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U / mL) and incubated at 37°C in a humidified atmosphere containing 5% CO2. Prior to subsequent experiments, EA.hy926 cells were treated with recombinant human tumor necrosis factor-a (TNF-a) at a concentration of 10 ng / mL (R&D Systems, Minneapolis, MN, USA; Catalog No. 210-TA) for 5 hours.

[0050] 1.2 Real-time polymerase chain reaction

[0051] Total RNA was extracted from EA.hy926 cells using TRIzol TM Reagent (Invitrogen, Carlsbad, CA, USA; Catalog No. 15596026). RNA integrity was verified by 1.5% agarose gel electrophoresis, ensuring that the RNA integrity number (RIN) was greater than 8.0. First-strand complementary DNA (cDNA) synthesis used 1 pg of total RNA using Superscript TM II Reverse Transcriptase (Catalog No. 18064014; Invitrogen) following the manufacturer's protocol. In 480 System (Roche, Basel, Switzerland) using SYBR Green PCR Master Mix (Cat. No.: 1725270; Bio-Rad, Hercules, CA, USA). Thermal cycling conditions included an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing / extension at 60 °C for 1 min. Gene expression levels were normalized to beta-actin (ACTB) using the 2^(-ΔΔCt) method. Primer sequences were designed to span exon-exon junctions and are detailed in Table 1. TM Green PCR Master Mix (Cat. No.: 1725270; Bio-Rad, Hercules, CA, USA). Thermal cycling conditions included an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing / extension at 60 °C for 1 min. Gene expression levels were normalized to beta-actin (ACTB) using the 2^(-ΔΔCt) method. Primer sequences were designed to span exon-exon junctions and are detailed in Table 1.

[0052] Table 1 Primer sequences

[0053]

[0054] 1.3 Western blot

[0055] After treatment, EA.hy926 cells were washed with ice-cold PBS three times and lysed in RIPA buffer (Cat. No. P0013B; Beyotime Biotechnology, Shanghai, China) supplemented with phosphatase inhibitors (Cat. No. 04906837001; Roche) and protease inhibitors (Cat. No. 4693159001; Roche) following the manufacturer’s instructions. The lysates were centrifuged at 12,000 x g for 15 min at 4 °C, and the protein concentration was quantified using the BCA method (Cat. No. 23225; Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Protein samples (30 pg per lane) were denatured in Laemmli buffer at 95 °C for 5 min, separated by 10% SDS-PAGE, and transferred to a PVDF membrane (0.45 pm; Cat. No. IPFL00010; Millipore) by wet transfer at 100 V for 90 min. The membrane was blocked with 5% skim milk powder in TBST at room temperature for 1 h, and then incubated with primary antibodies overnight at 4 °C: ICAM-1 (1:2000; Cat. No. TU295218; Abmart), VCAM-1 (1:2000; Cat. No. TU295218; Abmart), NLRP3 (1:1000; Cat. No. T55651; Abmart), cleaved caspase-1 (1:1000; Cat. No. M025280; Abmart), gasdermin D (1:1000; Cat. No. PU224937; Abmart), claudin-1 (1:1000; Cat. No. TA0127; Abmart), ZO-1 (1:5000; Cat. No. 82870-1-RR; Proteintech), occludin (1:10,000; Cat. No. 27260-1-AP; Proteintech), and b-actin (1:20,000; Cat. No. 66009-1-Ig; Proteintech). After washing with TBST, the membrane was incubated with HRP-conjugated secondary antibodies (1:20,000; Cat. No. SA00001-2; Proteintech) for 1 h at room temperature. Protein bands were detected using ECL Prime (Cat. No. RPN2232; Cytiva) and imaged with an Amersham Imager 600 (GE Healthcare). Density measurement analysis was performed using ImageJ v1.53e (NIH), and the levels of target proteins were normalized to b-actin.

[0056] 1.4 EA.hy926 transfection

[0057] EA.hy926 cells using riboFECT TM CP transfection reagent (Cat. No. C10511-1; RiboBio) was used to transfect MicroON hsa-miR-29a-3p mimic (100 nM; RiboBio Co., Ltd., Guangzhou, China) or micriOFF hsa-miR-17-3pinhibitor (Anti-29a-3p, 100 nM; RiboBio) according to the manufacturer's instructions. After transfection, cells were cultured in complete DMEM medium supplemented with 10% fetal bovine serum (FBS) for 24 hours before subsequent experiments. For PLSCR4 silencing, cells were transfected with PLSCR4-specific siRNA (100 pmol; XIANGHENG BioTech, Beijing, China) via Lipofectamine. TM Transfection with 8000 (Cat. No. C11507; Invitrogen, Carlsbad, CA, USA) for 24 hours was followed by an additional 48 hours of culture in complete medium. PLSCR4 overexpression was achieved via lentiviral transduction at a multiplicity of infection (MOI) of 40, a viral titer of 6 × 10^8 transduction units per mL (TU / mL; OBiO Technology, Shanghai, China), in the presence of 8 μg / mL polybrene (Cat. No. H9268; Sigma-Aldrich) for 12 hours, followed by 48 hours of culture in complete medium. The expression levels of hsa-miR-29a-3p and PLSCR4 mRNA were quantified by real-time quantitative polymerase chain reaction (RT-qPCR), and PLSCR4 protein expression was assessed by Western blot analysis to confirm the efficiency of the transfection procedure.

[0058] 1.5 Transwell migration experiment

[0059] Transwell migration assays were performed using EA.hy926 cells. Briefly, 5 × 10⁴ cells were seeded per well into wells with an 8 μm diameter. The upper chamber contained 500 μL of serum-free DMEM. The lower chamber was filled with 600 μL of complete DMEM supplemented with 10 ng / mL TNF-a (R&D Systems). After incubation at 37 °C in a 5% CO2 atmosphere for 14 hours, the cells that did not migrate on the upper surface of the membrane were gently removed using a cotton swab. The migrated cells were fixed with 4% paraformaldehyde (Beyotime, Cat. No. P0099) for 15 minutes at 25 °C, stained with 0.1% crystal violet (Sigma-Aldrich, Cat. No. C0775) for 10 minutes, and washed with PBS three times. Images of three random fields per insert were acquired using a Nikon Eclipse Ti2 inverted microscope at 40x magnification. Cell quantification was performed using ImageJ software v1.53e (NIH). Data are presented as mean ± standard deviation of three independent experiments.

[0060] 1.6 Monocyte chemotaxis experiment

[0061] In the monocyte chemotaxis experiment, THP-1 monocytes were labeled with 5 μM CellTracker TM CMFDA (Cat. No. 40721ES50; Yeasen Biotechnology, Shanghai, China) for 30 minutes at 37 °C. After labeling, the cells were washed with PBS twice and resuspended in complete medium. Previously treated EA.hy926 cells were seeded in a 6-well plate at a density of 2 x 10^5 cells / well and cultured until 90% confluence. Labeled THP-1 cells (1 x 10^5 cells / well) were added to the endothelial cell monolayer and co-cultured for 4 hours under physiological conditions (37 °C, 5% CO2). Non-adherent cells were removed by washing with PBS three times (5 minutes per wash). Adherent monocytes were fixed with 4% paraformaldehyde (Beyotime, Cat. No. P0099) for 15 minutes at 25 °C, followed by PBS washing to remove residual fixative. Images of three random fields per well were acquired using a Nikon Eclipse Ti2 inverted fluorescence microscope at 40x magnification using the GFP filter set. CMFDA-positive cells were quantified using ImageJ software v1.53e (NIH), and data were normalized to the control group (mean ± SEM, n = 3).

[0062] 1.7 Immunofluorescence detection

[0063] EA.hy926 cells were seeded at 50-60% confluence in 35mm glass-bottom confocal culture dishes (NEST, catalog number 801002). After transfection and treatment with TNF-α (10 ng / mL), cells were washed twice with PBS (pH 7.4) and fixed with 4% paraformaldehyde (Beyotime, catalog number P0099) at 25°C for 15 minutes. The fixed cells were permeabilized with PBS containing 0.3% Triton X-100 (Beyotime, catalog number ST795) at 25°C for 20 minutes. After washing three times with PBS (5 minutes each time), non-specific binding sites were blocked for 1 hour at 25°C with PBS containing 10% fetal bovine serum (Gibco, catalog number 16140071). Cells were then incubated overnight at 4°C with primary antibody diluted in blocking buffer. After washing three more times with PBS (5 minutes each time), the samples were incubated with secondary antibody at 25°C for 2 hours. Nuclear counterstaining was performed using DAPI (1 μg / mL; Sigma-Aldrich, catalog number D9542). Imaging was performed using a Leica TCS SP8 laser confocal microscope at 40x objective (NA = 1.4). Three fields of view were randomly selected for each sample, with consistent laser power and gain parameters. Fluorescence intensity was quantitatively analyzed using ImageJ v1.53e (NIH).

[0064] 1.8 Dual-luciferase reporter gene assay

[0065] To verify the interaction between miR-29a-3p and PLSCR4, pmiR-RB-REPORT, which contains the 3' untranslated region (3'UTR) of human PLSCR4 mRNA and the predicted miR-29a-3p binding site, was used. TM Vector (Raybro, Guangzhou, China). Mutants were constructed by replacing the seed sequence (GGTGCT) in the PLSCR4 3'UTR with CCACGA via site-directed mutagenesis. Lipofectamine was used. TM HEK293T cells were co-transfected with 100 ng of wild-type / mutant plasmid and 50 nM miR-29a-3p agonist (agomir) or negative control (NC) using 8000 (Thermo Fisher Scientific). 24 hours after transfection, [the cells were then analyzed]. The Luciferase assay system (Promega) was used to measure the luciferase activity of fireflies and kidney luciferase. During data analysis, the kidney luciferase activity was normalized to the firefly luciferase activity.

[0066] 1.9 Quantification of mature miRNAs

[0067] Using TRIzol TM Total RNA was extracted from EA.hy926 cells using reagents (Thermo Fisher Scientific). miDETECT ATrack was used.TM miRNA qRT-PCR kit (RiboBio, Cat# C10712-1) was used to detect the expression level of mature miRNA according to the manufacturer’s instruction. U6 small nuclear RNA (snRNA) was used as the internal control gene.

[0068] 1.10 ELISA method for detecting cytokines

[0069] The commercial ELISA kit (Yisheng Biotech, Wuhan, China) was used to quantify the concentration of human interleukin-18 (IL-18) and interleukin-1β (IL-1β) in cell supernatant according to the manufacturer’s instruction. The absorbance was measured at 450 nm wavelength using a microplate reader (BioTek Instruments).

[0070] 1.11 RNA sequencing analysis

[0071] To compare the gene expression profile of EA.hy926 cells overexpressing miR-29a-3p and control-treated cells, total RNA was extracted using TRIzol Reagent (Invitrogen, Cat# 10296-010). After confirming the RNA integrity number (RIN) > 8.0 using Agilent 2100 Bioanalyzer, the library was prepared using NEBNext® Ultra II RNA Library Prep Kit for Illumina® and sequenced on an Illumina NovaSeq 6000 platform at Beijing Annovogen Company Limited. TM Ultra TM II RNA Library Prep Kit for Illumina®. Sequencing was performed on an Illumina NovaSeq 6000 platform at Beijing Annovogen Company Limited.

[0072] 1.12 Scanning electron microscopy

[0073] Logarithmic growth phase cells were collected, washed twice with PBS (pH 7.4; Thermo Fisher Scientific), and digested with 0.25% trypsin-EDTA (Gibco TM ) for 3 minutes at 37°C. The cell pellet was collected by centrifugation (300 x g, 5 minutes), fixed with 2.5% glutaraldehyde (Electron Microscopy Sciences) in 0.1M phosphate buffer (pH 7.4) for 12 hours at 4°C. Then, the sample was dehydrated by gradient ethanol (50%, 70%, 80%, 90%, 100%) and replaced with each step after centrifugation (1000 x g, 5 minutes). The sample was dried by liquid CO2 critical point (Leica EM CPD300), coated with 10 nm gold-palladium layer (Quorum Q150TES), and finally placed on an aluminum stub for observation using a field emission scanning electron microscope (Hitachi SU8010) at an acceleration voltage of 5-15 kV and a working distance of 5-10 mm. The magnification images were collected at 1,000x to 50,000x.

[0074] 1.13 Annexin V-PI fluorescent double staining experiment​

[0075] Annexin V-PI kit (Yisheng Biotech) was used. Annexin V, a calcium-dependent phospholipid binding protein, can specifically bind to phosphatidylserine (PS) on the outer membrane of cells; iodinated propidium (PI) staining indicates cell membrane integrity. EA.hy926 cells were seeded in 6-well plates to the optimal state, and after discarding the culture medium, PBS was washed. Add 500 μL of 1 x Annexin V binding buffer, then add 5 μL of FITC-labeled Annexin V reagent and 5 μL of PI reagent, incubate at room temperature for 15-20 minutes. After washing with Annexin V binding buffer, observe under a fluorescence microscope, randomly select three fields of view for imaging, and quantify Annexin V-positive cells by ImageJ v1.53e.

[0076] 1.14 Flow cytometry

[0077] Annexin V-PI kit (Yisheng Biotech) was used. After treatment, EA.hy926 cells were centrifuged (1000 rpm, 5 minutes) and the supernatant was discarded, and resuspended and washed with PBS. After centrifugation again, the cells were resuspended with 500 μL of 1 x Annexin V binding buffer, 5 μL of Annexin V-FITC and 5 μL of PI reagent were added, and after gentle vortex, incubated at room temperature for 15-20 minutes. Use Beckman Coulter flow cytometer for detection, and quantify Annexin V-positive cells by FlowJo software.

[0078] 1.15 Immunohistochemical detection

[0079] Fresh mouse lung tissue was fixed with 4% paraformaldehyde, dehydrated and paraffin-embedded, and then sectioned. The sections were deparaffinized and hydrated, and the antigen was repaired with 0.01 M citrate buffer (pH 6.0) at high temperature and high pressure. After cooling to room temperature and washing with PBS, 3% H2O2 was used to block endogenous peroxidase, and serum was used to block non-specific binding. The sections were incubated with the primary antibody (1:200 dilution, Abmart) at 4°C overnight, washed with PBS, and then incubated with biotin-labeled secondary antibody (1:500 dilution, CST) at 37°C for 30 minutes, followed by reaction with horseradish peroxidase-labeled streptavidin (1:500 dilution, Jackson ImmunoResearch) at room temperature for 20 minutes. DAB color development, hematoxylin counterstaining, dehydration and mounting, and then observed under a microscope, and quantified the antigen expression by ImageJ software.

[0080] 1.16 Experimental animals

[0081] All animal experiments were approved by the Medical Ethics Committee of Qinghai University and followed the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Twenty 7-week-old male C57BL / 6 mice were purchased from Sibeifu Biotechnology Co., Ltd. (Henan, China) and housed in a 12-hour light-dark cycle environment with free access to food and water, and the temperature and humidity were controlled at 22 ± 2°C and 50% ± 5%, respectively. After adaptive feeding, the mice were randomly divided into three groups (n = 6): control group, TNF-α group, and miR-29a-3p agonist + TNF-α group. The miR-29a-3p agonist (RiboBio) was synthesized based on mmu-miR-29a-3p (miRBase: MIMAT0000535). On days 0, 3, and 6, the mice were injected with saline or the agonist (20 nmol / mouse) via the tail vein, once every three days for a total of three doses; from days 6 to 9, the mice were injected with recombinant mouse TNF-α protein (30 μg / kg body weight, R&D Systems) or saline intraperitoneally, once daily for four days. After the last injection, the mice were fasted for 12 hours and then euthanized under pentobarbital anesthesia.

[0082] 1.17 Data statistics

[0083] SPSS 28.0 and GraphPad Prism 10.0 were used for analysis. Measurement data were expressed as mean ± standard deviation, and independent sample t-test was used for comparison between groups, and one-way analysis of variance (ANOVA) was used for multiple comparisons, P < 0.05 was considered statistically significant.

[0084] 2. Results

[0085] 2.1 miR-29a-3p inhibits the expression of pro-inflammatory factors and adhesion molecules induced by TNF-α

[0086] TNF-α stimulation can up-regulate the expression of pro-inflammatory factors and adhesion molecules in endothelial cells. The expression levels of various inflammation-related molecules in EA.hy926 cells treated with TNF-α were detected by qRT-PCR, and the results showed that compared with the untreated control group, TNF-α stimulation significantly up-regulated the mRNA expression levels of pro-inflammatory cytokines (IL-8, IL-1β), chemokines (MCP-1), and adhesion molecules (ICAM-1, VCAM-1) in EA.hy926 cells, with statistically significant differences (P < 0.05), as shown in Figure 1 A and Figure 1B, *** represents P < 0.001 compared with the control group, the above results confirm that TNF-α can effectively activate EA.hy926 cells and induce them into an inflammatory state, showing extensive pro-inflammatory mediator release and enhanced leukocyte adhesion ability. The microRNA expression profile was analyzed, and the qRT-PCR detection results showed that under the condition of TNF-α stimulation, the expression level of miR-29a-3p in EA.hy926 cells was significantly down-regulated (P < 0.05), as shown in Figure 2 *** represents P < 0.001 compared with the control group, indicating that TNF-α can negatively regulate the expression of miR-29a-3p. In summary, miR-29a-3p may reduce endothelial inflammation by down-regulating pro-inflammatory factors (IL-18 / IL-1β), chemotactic factors (MCP-1), and adhesion molecules (ICAM-1 / VCAM-1).

[0087] 2.2 miR-29a-3p restores endothelial cell migration, reduces monocyte chemotaxis, and maintains intercellular tight junctions

[0088] During the inflammatory process, pro-inflammatory factors can impair endothelial cell migration function. To explore the effect of miR-29a-3p on endothelial cell migration, we evaluated the effect of TNF-α stimulation by in vitro migration experiment. As expected, miR-29a-3p had a significant regulatory effect on TNF-α-induced EA.hy926 cell migration function. Through Transwell migration experiment detection, it was found that under the condition of TNF-α stimulation, overexpression of miR-29a-3p could significantly promote the migration ability of EA.hy926 cells, which had a statistically significant difference compared with the control group (P < 0.05); on the contrary, after inhibiting the expression of miR-29a-3p, the cell migration ability was significantly weakened (P < 0.05), as shown in Figure 3 *** represents P < 0.001 compared with the control group, indicating that miR-29a-3p enhances endothelial cell migration under inflammatory conditions.

[0089] miR-29a-3p has a significant regulatory effect on the adhesion function of EA.hy926 cells. Under the condition of TNF-α stimulation, through in vitro adhesion experiment detection, it was found that overexpression of miR-29a-3p could significantly enhance the adhesion ability of EA.hy926 cells, which had a statistically significant difference compared with the control group (P < 0.05); while inhibiting the expression of miR-29a-3p, the cell adhesion ability was significantly reduced (P < 0.05), as shown in Figure 4 A and Figure 4B, *** represents P < 0.001 compared with the control group, the above results confirm that miR-29a-3p can effectively restore the adhesion function of endothelial cells damaged in the inflammatory environment, indicating that it plays an important role in maintaining the stability of vascular endothelial structure and anti-inflammatory protection.

[0090] Chemokines (such as MCP-1) and adhesion molecules (ICAM-1 / VCAM-1) promote monocyte chemotaxis and adhesion in inflammation. To clarify the effect of miR-29a-3p on monocyte chemotaxis, we evaluated the chemotactic ability of endothelial cells under TNF-α stimulation by in vitro monocyte adhesion experiment. TNF-α enhances monocyte adhesion, but miR-29a-3p overexpression significantly reduces THP-1 monocyte adhesion by 74.28%, and inhibition enhances 89.15%, as shown in Figure 5 A and 5B, *** represents P < 0.001 compared with the control group, suggesting that miR-29a-3p may reduce TNF-α-induced inflammation by inhibiting monocyte-endothelial cell adhesion.

[0091] 2.3 miR-29a-3p directly targets PLSCR4

[0092] To identify the potential target of miR-29a-3p, we performed transcriptome sequencing on EA.hy926 cells overexpressing miR-29a-3p. Analysis showed that PLSCR4 was the most significantly down-regulated among the differentially expressed genes, as shown in Figure 6 , suggesting that it may be a target of miR-29a-3p. To verify the direct targeting, we first predicted the binding site of the seed region of miR-29a-3p to PLSCR4, as shown in Figure 7 A, and verified by dual luciferase reporter gene detection: co-transfection of wild-type PLSCR4 plasmid with miR-29a-3p significantly reduced luciferase activity, while mutation of the binding site eliminated this inhibition and increased activity by 10.56%, as shown in Figure 7 B, ns represents no statistical significance, *** represents P < 0.001 compared with the control group, confirming that PLSCR4 is a direct target of miR-29a-3p. Overexpression of miR-29a-3p down-regulates PLSCR4 mRNA by about 60.05%, as shown in Figure 8 A, *** represents P < 0.001 compared with the control group, and the protein level is also reduced by 45.05%, as shown in Figure 8 B, *** represents P < 0.001 compared with the control group. In summary, miR-29a-3p functions by directly targeting PLSCR4.

[0093] 2.4 PLSCR4 reduces TNF-α-induced endothelial cell inflammation by inhibiting pyroptosis

[0094] To verify whether the anti-inflammatory effect of PLSCR4 knockdown is the same as that of miR-29a-3p overexpression, we quantified the pro-inflammatory factors IL-18 and IL-1β in the culture medium of EA.hy926 cells by ELISA.

[0095] The results showed that the levels of IL-18 and IL-1β were significantly reduced by 43.54% and 22.54%, respectively, after PLSCR4 knockdown, while they were increased by 34.87% and 27.2%, respectively, after overexpression, as shown in Figure 9 A, compared with the control group, *** represents P < 0.001. Meanwhile, the quantitative analysis of VCAM-1 and ICAM-1 in the lysate of EA.hy926 cells showed that PLSCR4 knockdown reduced their expression by 36.18% and 61.61%, respectively, while overexpression increased them by 103.55% and 43.51%, respectively, as shown in Figure 9 B and Figure 9 C, compared with the control group, *** represents P < 0.001, suggesting that PLSCR4 knockdown can mimic the anti-inflammatory effect of miR-29a-3p overexpression.

[0096] PLSCR4 gene as a key factor in regulating pyroptosis and inflammatory response in vascular endothelial cells, specifically by inhibiting the expression of PLSCR4 to inhibit TNF-α-induced pyroptosis and inflammatory response in EA.hy926 cells, thus providing a new target and strategy for the prevention and treatment of related inflammatory diseases. The qRT-PCR detection analysis showed that under the condition of TNF-α stimulation, compared with the control group, siRNA-mediated PLSCR4 gene knockdown (siPLSCR4) significantly down-regulated the mRNA expression levels of pyroptosis-related factors in EA.hy926 cells, including Caspase-1, Caspase-1-P20, Caspase-4 and GSDMD-NT, with statistically significant differences (P < 0.05); on the contrary, PLSCR4 overexpression significantly up-regulated the expression levels of the above pyroptosis-related factors (P < 0.05), as shown in Figure 10 A and Figure 10 B, compared with the control group, *** represents P < 0.001. The results of Western blot experiment were consistent with those of qRT-PCR detection, indicating that the expression level of PLSCR4 was positively correlated with the protein expression of Caspase-1, Caspase-1-P20, Caspase-4 and GSDMD-NT, further verifying the regulatory effect of PLSCR4 on the pyroptosis pathway, as shown in Figure 10C. Compared with the control group, *** represents P < 0.001. The results of scanning electron microscopy showed that compared with the control group, the siPLSCR4 group had rich and uniform microvilli structure, and no typical pyroptosis bubble was formed; while the PLSCR4 overexpression group showed obvious pyroptosis morphological characteristics, including cell membrane rupture, cell collapse and various sizes of perforation structure, suggesting that PLSCR4 promotes TNF-α-induced cell pyroptosis, as shown in Figure 10 D.

[0097] In view of the fact that PLSCR4 mainly mediates the transmembrane flipping of charged phospholipids (such as phosphatidylserine PS and phosphatidylethanolamine PE), we evaluated the effect of PLSCR4 knockdown on PS exposure by Annexin V-PI double staining. Annexin V, as a calcium-dependent phospholipid binding protein, can specifically recognize PS outside. The data showed that under the stimulation of TNF-α, the transmembrane flipping of PS in PLSCR4-knockdown EA.hy926 cells was reduced by 44.6%, while overexpression was increased by 44.44%, as shown in Figure 11 .

[0098] PLSCR4 has a new use as a target for regulating the barrier function of vascular endothelial cells, specifically by inhibiting the expression of PLSCR4 to restore or up-regulate the expression of tight junction proteins in EA.hy926 cells, thereby improving endothelial barrier function. For the regulation of cell adhesion by inflammation, we performed a transfection experiment. The results of Western blot experiment showed that in the TNF-α-induced EA.hy926 cell model, after knockdown of PLSCR4, the protein expression levels of three key tight junction proteins, ZO-1, Occludin and Claudin-5, were significantly up-regulated, which had statistical significance compared with the control group. At the same time, the results of immunofluorescence staining showed that the continuity of ZO-1, Occludin and Claudin-5 in the membrane boundary of the siPLSCR4 treatment group was significantly enhanced, and the fluorescence intensity was increased, suggesting that the intercellular connection structure was reconstructed and restored, as shown in Figure 12 .

[0099] In summary, PLSCR4 knockdown inhibits PS transmembrane flipping, damages GSDMD-NT membrane targeting, and thus inhibits TNF-α-induced cell pyroptosis, ultimately reducing endothelial cell inflammation.

[0100] The above merely describes the preferred embodiments of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content to obtain equivalent embodiments with equivalent changes, as long as the changes or modifications do not deviate from the technical solution of the present application. Any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application still belongs to the scope of the technical solution of the present application.

Claims

1. Use of miR-29a-3p on targeting PLSCR4.

2. Use of miR-29a-3p on inhibiting vascular endothelial inflammation.

3. Use of miR-29a-3p for the inhibition of vascular inflammation according to claim 2, characterized in that, miR-29a-3p relieves vascular inflammation by targeting PLSCR4 to inhibit NLRP3-mediated pyroptosis pathway.

4. A medicament for targeting PLSCR4, characterized by, At least comprising miR-29a-3p and a pharmaceutically acceptable carrier, diluent or excipient.

5. The medicament for targeting PLSCR4 according to claim 4, characterized by, The administration forms thereof at least include one of the following: direct blending administration, liposome wrapping administration, nanoparticle wrapping administration, aptamer-mediated targeted administration, antibody-mediated targeted administration and administration through hydrogel complex administration system.

6. A method of reducing the expression of PLSCR4 in a target cell, characterized in that, The method is performed by administering miR-29a-3p.

7. A medicament for inhibiting vascular inflammation, characterized by comprising a compound of the formula (I) as an active ingredient. Comprising miR-29a-3p and a pharmaceutically acceptable carrier, diluent or excipient.

8. The medicament for inhibiting vascular inflammation according to claim 7, characterized by, The administration forms thereof at least include one of the following: direct blending administration, liposome wrapping administration, nanoparticle wrapping administration, aptamer-mediated targeted administration, antibody-mediated targeted administration and administration through hydrogel complex administration system.

9. A method of inhibiting vascular inflammation, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-8. The method comprises administering miR-29a-3p to a subject in need thereof. 10.A method for verifying the effect of miR-29a-3p targeting PLSCR4 on inhibiting vascular inflammation, characterized in that, The method comprises the following steps: After transfecting miR-29a-3p overexpression or inhibitor into human umbilical vein endothelial cells and stimulating with TNF-α, evaluate the inflammatory response by qRT-PCR, Western blot, immunofluorescence, Transwell migration, monocyte chemotaxis and scanning electron microscopy technology, identify PLSCR4 as a direct target of miR-29a-3p by dual luciferase reporter gene experiment, and evaluate the effect of PLSCR4 overexpression or knockout by Annexin V-PI staining and flow cytometry, while establishing a mouse in vivo model, intravenously injecting miR-29a-3p agonist and analyzing lung tissue inflammation by immunohistochemical method.

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