Application of macrophage exosome molecules induced by fusobacterium nucleatum

Abstract

The invention discloses application of macrophage exosome molecules induced by fusobacterium nucleatum, and belongs to the technical field of molecular biology. The nucleotide sequence of the macrophage exosome molecule induced by fusobacterium nucleatum is 5 '-ACUGGCUAGGGAAAAUGAUUGGAU-3', and the macrophage exosome molecule can be applied to preparation of a preparation for inducing VSMC cells to be converted into macrophage-like phenotypes, can regulate and control the atherosclerosis process, and provides a new thought and theoretical basis for further research of exosome in the atherosclerosis progress process.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to the application of macrophage exosome molecules induced by Fusobacterium nucleatum. Background Art

[0002] In recent years, the study of microbial-host interactions has attracted increasing attention. F.nucleatum ) is a Gram-negative anaerobic bacterium that resides in the periodontal microecological environment and plays a synergistic pathogenic role in a variety of diseases such as periodontitis, atherosclerosis (AS) and intestinal inflammation. Periodontitis is an independent and significant risk factor for AS and plays an important role in the occurrence and development of cardiovascular diseases. Studies have found that F.nucleatum As one of the oral bacteria detected in AS plaques, its detection rate is directly related to the severity of periodontitis. F.nucleatum It can promote the progression of AS lesions in ApoE- / - mice, leading to increased macrophage (mø) infiltration within plaques, inflammation, and imbalanced lipid metabolism. Macrophages are a cell type enriched in AS plaques. However, lineage tracing studies have shown that approximately 60% of macrophages in AS plaques may be derived from vascular smooth muscle cells (VSMCs) through phenotypic transformation. CD68+ macrophages expressing the VSMC-specific marker α-smooth muscle actin (α-SMA) have been observed in human and mouse AS plaques, confirming their VSMC, rather than myeloid, origin. This suggests that during the transformation process, VSMCs acquire macrophage characteristics and begin to express macrophage markers such as CD68, assuming macrophage functions and thereby exacerbating AS progression.

[0003] Exosomes (Exosomes) are extracellular nanoscale vesicles secreted by a variety of cells into various biological fluids. They contain a variety of molecules, such as miRNAs, mRNAs, lncRNAs, proteins, and lipids, mediating intercellular communication and serving as important vectors for transmitting intercellular information within the AS microenvironment. Studies have shown that exosomes can regulate the biological functions of VSMCs and promote the progression of AS by delivering miRNAs or proteins. Upon stimulation, macrophages secrete large quantities of exosomes, accounting for the vast majority of exosomes in plaques. Studies have shown that macrophage exosomes are important mediators of the phenotypic transformation of VSMCs in AS. VSMCs migrate from the tunica media to the intima, transforming into a macrophage-like phenotype, and then phagocytizing lipids to form foam cells, exacerbating AS. Macrophage-derived exosomes promote VSMC phenotypic transformation by activating the c-Jun / AP-1 signaling pathway and triggering the expression of MMP-2 in VSMCs via the JNK and p38 pathways, both of which collectively accelerate AS progression.

[0004] Defects and shortcomings of existing technologies: Periodontitis is a factor that promotes the occurrence and development of AS, and its key pathogens F.nucleatum Whether it can affect plaque stability by regulating macrophage exosomal miRNA to participate in the phenotypic transformation of VSMCs into a macrophage-like phenotype, has not been found. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides the application of macrophage exosome molecules induced by Fusobacterium nucleatum to provide periodontal pathogens F.nucleatum Application of the exosomal miRNA molecule hsa-miR-664a-5p produced by infected macrophages in promoting the phenotypic transformation of VSMCs.

[0006] In order to achieve the above-mentioned object, the technical solution adopted by the present invention is: to provide the use of macrophage exosome molecules induced by Fusobacterium nucleatum in the preparation of a preparation for inducing VSMC cells to transform into a macrophage-like phenotype, wherein the nucleotide sequence of the macrophage exosome molecules induced by Fusobacterium nucleatum is 5'-ACUGGCUAGGGAAAAUGAUUGGAU-3'.

[0007] Furthermore, the Fusobacterium nucleatum is ATCC25586.

[0008] Furthermore, exosome-producing macrophages were induced from human monocytes.

[0009] Furthermore, the preparation is a preparation for regulating the progression of atherosclerosis.

[0010] The beneficial effects of the present invention are: the present invention analyzes for the first time F.nucleatumThe effect of the exosomal miRNA molecule hsa-miR-664a-5p secreted by the in vitro inflammatory model established by infection with dTHP-1 on the biological functions of VSMCs clearly illustrates the potential role of hsa-miR-664a-5p in the proliferation, migration and phenotypic transformation of vascular smooth muscle cells; the exosomal miRNA molecule hsa-miR-664a-5p participates in the phenotypic transformation of VSMCs and thus affects the progression of AS, which can provide new ideas and theoretical basis for further research on the role of exosomes in the progression of AS. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Transmission electron microscopy was used to identify the uninfected and F.nucleatum Morphological characteristics of exosomes in macrophages of the infection group, scale bar = 100 nm; A is Ex, B is Fn -Ex; Figure 2 For NTA detection of exosomes Ex and Fn -Ex particle size and concentration (dilution factor = 1:1000); where A is the particle size of Ex, B is Fn -Ex is the particle size, C is the concentration of exosomes; Figure 3 These are the protein immunoblot images and statistical graphs of exosome marker proteins CD9 and TSG101; A is the protein immunoblot image, and B is the statistical graph of the protein immunoblot results; Figure 4 The results of the BCA protein assay were obtained by testing different MOIs (0, 25, 50, 75, 100, and 200). F.nucleatum Effects on the amount of exosomes released by macrophages; Figure 5 The differentially expressed miRNAs in exosomes; A is a barplot, and B is a volcano plot; Figure 6 Cluster diagram of differentially expressed miRNAs in exosomes; Figure 7 KEGG functional analysis of differentially expressed miRNAs target genes; Figure 8 The expression level of hsa-miR-664a-5p in cells was detected by qRT-PCR; A is the level in dTHP-1 cells, and B is the level in VSMC cells; Figure 9 CCK8 was used to detect the effect of hsa-miR-664a-5p on VSMC cell proliferation; Figure 10To detect the effect of hsa-miR-664a-5p on VSMC cell migration ability; A and C are the results of Transwell migration assays in different groups (magnification 10X), and B and D are the corresponding statistical results of the Transwell migration assay; Figure 11 To detect the effect of hsa-miR-664a-5p on VSMC cell migration ability; A and C are the results of the scratch test in different groups (scale = 100 μm), B and D are the corresponding statistical results of the scratch test; Figure 12 qRT-PCR detection of the expression levels of related cell markers after VSMC cells were treated with hsa-miR-664a-5p; A and B are VSMC contraction markers α-SMA and SM22α, and phenotypic conversion to macrophage-like marker CD68; C and D are inflammatory factors MCP-1, MMP-2, and IL-1β; Note: * indicates P < 0.1, ** indicates P < 0.01, *** indicates P < 0.001, and ***** indicates P < 0.0001. DETAILED DESCRIPTION

[0012] The specific implementation methods of the present invention are described in detail below with reference to the embodiments.

[0013] The human monocytic cell line THP-1, human aortic vascular smooth muscle cells (T / GHAVSMC), and Fusobacterium nucleatum strains used in the present invention are F.nucleatum ATCC25586 were provided by the National Key Laboratory of Oral Disease Prevention and Treatment, Sichuan University.

[0014] Example 1 F.nucleatum Culture and preparation of bacterial suspension (1) Preparation of BHI culture medium Add 7.4 g of brain heart infusion, 1 g of yeast extract YEAST, and 40 μg of vitamin K to 200 mL of ultrapure water, dissolve and mix thoroughly, and sterilize by high-pressure steam.

[0015] (2) F.nucleatum nourish Take out from -20℃ refrigerator F.nucleatum ATCC 25586 strain was quickly thawed in a water bath. A small amount of frozen culture was inoculated into BHI culture medium near an alcohol lamp in a clean bench. The culture was placed in an anaerobic jar and passaged every other day.

[0016] (3) F.nucleatum Suspension preparation First, yes F.nucleatumAfter Gram staining to confirm that there was no contamination, the cells were centrifuged at 4000 rpm for 5 min. The resulting precipitate was washed three times with sterile PBS buffer and resuspended. The OD value of the resuspended bacterial solution was measured by spectrophotometer under visible light conditions at 580 nm. When OD = 0.8, the bacterial solution concentration was 1 × 10 9 CFU / mL.

[0017] Example 2 Culture, passage, cryopreservation and thawing of human monocytic cell line THP-1 (1) Cell culture and passage Human monocytic THP-1 cells were grown in suspension in complete culture medium (RPMI 1640 medium with 10% FBS and 1% penicillin-streptomycin) and observed every other day. When the cell density reached 80%-90%, the cell culture medium was collected and centrifuged at low speed at room temperature (800 rpm for 5 minutes) to obtain a cell pellet. The cells were resuspended in fresh complete culture medium and passaged at a 1:2 or 1:3 ratio.

[0018] (2) Cell cryopreservation When the density of human monocytic THP-1 cells reaches 80%-90%, collect the cell culture medium and centrifuge at low speed at room temperature. Centrifuge at 800 rpm for 5 minutes. Carefully discard the old culture medium and resuspend the cells in 1 mL of freezing solution (90% serum + 10% DMSO). Place the cells in a cryovial and immediately insert them into a cell program cooling box. Store in a -80°C refrigerator overnight and transfer to a liquid nitrogen tank the next day.

[0019] (3) Cell recovery Ensure 37°C warm water or a water bath is readily available. Remove the frozen cells from the liquid nitrogen tank and thaw them in a 37°C water bath. Once the cells are completely thawed, quickly transfer them to a 15mL centrifuge tube containing 1mL of complete culture medium. Centrifuge at room temperature for 5 minutes at 800 rpm to pellet the cells. Carefully discard the supernatant and resuspend the cells in 1mL of fresh complete culture medium. After gently pipetting to evenly distribute the cells, transfer the cells to an appropriately sized cell culture flask containing the appropriate amount of fresh complete culture medium. Finally, place the culture flask in a constant temperature cell incubator (37°C, 5% CO2).

[0020] Example 3 Induce THP-1 cells to differentiate into macrophages (dTHP-1) When the density of human mononuclear THP-1 cells reached 80%-90%, the cell culture medium was collected and centrifuged at low speed at room temperature, centrifuged at 800 rpm for 5 minutes, and the old culture medium was carefully discarded. The cell pellet was resuspended in fresh complete culture medium and the cell concentration was adjusted to 1×10 7 / mL, phorbol methyl paraformaldehyde (PMA) was added at a concentration of 100 ng / mL and cultured for 48 h. The cell status was observed under a microscope. Successful differentiation was indicated by the complete attachment of successfully induced dTHP1 cells to the wall.

[0021] Example 4 Build F.nucleatum In vitro co-culture inflammation model infected with dTHP-1: (1) F.nucleatum Subculture 1-2 days in advance to allow the cells to be in the exponential growth phase; (2) Wash dTHP1 cells three times with sterile PBS buffer to remove residual antibiotics; (3) The exponential growth period F.nucleatum After centrifugation at 4000 rpm for 5 minutes at room temperature, the cell pellet was washed three times with sterile PBS buffer and resuspended in serum-free medium. Bacteria were added to dTHP1 cells at a multiplicity of infection (MOI, bacteria:cell) of 50 (no bacteria were added in the control group). The cells were then incubated in a constant temperature incubator (37°C, 5% CO2) for 48 hours. The cell supernatant and cell pellet were collected for subsequent experiments.

[0022] Example 5 Infected / uninfected F.nucleatum Macrophage-derived exosomes ( Fn -Ex / Ex) extraction and identification: (1) Extraction of exosomes: The cell supernatant collected in Example 4 was used to extract and separate exosomes by ultracentrifugation.

[0023] The specific experimental steps are as follows: the cell supernatant is centrifuged at 300 × g for 10 minutes, the supernatant is centrifuged again at 2000 × g for 10 minutes, the supernatant is filtered through a 0.22 μm sterile filter, and the collected supernatant is centrifuged at 10,000 × g for 30 minutes. The supernatant is centrifuged again at 100,000 × g for 70 minutes. The resulting pellet is washed three times with pre-chilled sterile PBS buffer and centrifuged again at 100,000 × g for 70 minutes. The resulting pellet is the exosomes. All the above centrifugation operations must be performed in a pre-chilled 4°C centrifuge.

[0024] (2) Transmission electron microscopy observation and identification of exosome morphological characteristics Take 30 μL of fresh exosomes extracted in step (1) (referring to those that have not been frozen), add an equal amount of 4% paraformaldehyde to resuspend, take 5-10 μL and drop it onto a dry copper grid, and let it dry for 30 minutes; then gently wash it three times with sterile PBS buffer, dry it, and add a 2.5% glutaraldehyde solution to fix it for 5-10 minutes; gently wash it three times with sterile water and dry it, add uranyl acetate to develop color for 5 minutes; finally, absorb the remaining liquid with filter paper, dry it at room temperature for 10 minutes, and then observe the exosomes under a transmission electron microscope at 80 kV.

[0025] The results of transmission electron microscopy observation of exosomes are as follows Figure 1 As shown, the uninfected group and F.nucleatum Transmission electron microscopy revealed that the exosomes from macrophages in the infection group were small disc-shaped with a diameter ranging from 30 to 200 nm, which meets the definition of exosomes.

[0026] (3) Nanoparticle tracing analysis of exosome particle size and concentration Take 100 μL of fresh exosomes extracted in step (1) (not frozen), dilute them with appropriate amount of ultrapure water in a proportional gradient, inject them into the detection hole through a 1 mL syringe, adjust the dilution ratio according to the particle count reported by the machine, and finally record the data report detected by the machine.

[0027] Test results such as Figure 2 As shown, the particle size of the two groups of exosomes is 30-200nm, which meets the definition. Compared with uninfected macrophages, in the same volume of culture medium, F.nucleatum The exosome secretion concentration of the infected group was approximately 1.6 times that of the uninfected group.

[0028] (4) Detection of exosome marker proteins by immunoblotting ① Sample preparation: Lyse and dilute the prepared exosomes, add loading buffer, boil, and place in a -20℃ refrigerator for later use; ② Glue preparation: After confirming that the gel preparation device is clean and leak-free, prepare 12% SDS-PAGE separation gel and 5% SDS-PAGE stacking gel according to the preparation method. Insert the comb at a 45° angle from the edge and let it stand at room temperature for 20 minutes to allow the gel to solidify. ③ Sample loading: Carefully remove the comb and add 30µL of exosome sample to each well. Run the electrophoresis at a constant voltage of 150V for the entire process. ④ Cutting the membrane: Cut the PVDF membrane according to the size of the separation gel and the protein pre-stained marker, and activate it with methanol for 30 seconds; ⑤ Transfer: Transfer the membrane according to the "sandwich method" at a constant current of 200 mA / 90 min; ⑥ Blocking: Block the membrane with 1×TBST blocking solution containing 2 wt% BSA on a shaker at room temperature for 1-2 h; ⑦ Wash the membrane: After blocking, wash the membrane three times with 1×TBST buffer on a shaker at room temperature for 15 minutes each time. ⑧ Incubate with primary antibody: dilute the primary antibody with 1×TBST diluent containing 2 wt% BSA, incubate the PVDF membrane with the diluted primary antibody, and incubate on a shaker in a 4°C refrigerator overnight; ⑨ Wash the membrane: The next day, remove the membrane and wash it three times with 1× TBST buffer on a shaker at room temperature, each time for 15 minutes. ⑩ Incubation with secondary antibody: Dilute the secondary antibody with 1×TBST diluent containing 2 wt% BSA, incubate the PVDF membrane with the diluted secondary antibody, and incubate on a shaker at room temperature for 90 min; ⑪ Wash the membrane: Wash the membrane three times with 1×TBST buffer on a shaker at room temperature, 15 min each time; ⑫ Exposure: Mix equal volumes of ECL reaction solution A and solution B, add the luminescent solution onto the membrane, and expose to capture images.

[0029] The protein immunoblotting images and statistical results of exosome marker proteins CD9 and TSG101 are shown in Figure 2. Figure 3 As shown, both groups of exosomes expressed specific marker proteins CD9 and TSG101, compared with the uninfected group, F.nucleatum The protein expressions of CD9 and TSG101 in exosomes of the infected group were significantly increased.

[0030] (5) Quantification of exosomal proteins using the BCA protein quantification kit (Biyuntian, Code No. P0012S) To determine the promotion F.nucleatum The MOI of bacteria that produces the most exosomes when infected with dTHP-1 was used to detect the total protein content of the extracted exosomes. F.nucleatum dTHP-1 exosomes from the infection group were added with 50 μL of protein lysis buffer (RIPA: protease inhibitor PMSF = 100:1) for 5 minutes, and then the BCA working solution (reagent A:B = 50:1) was prepared according to the instructions. The standard solution was carefully pipetted and mixed. The standard solution was added to a 96-well plate as required and made up to 20 μL with PBS buffer. 20 μL of 20-fold diluted protein sample was added to the sample well, and then 200 μL of BCA working solution was added to all wells. The plates were placed in a constant temperature cell incubator (37°C, 5% CO2) and incubated for 30 minutes. The absorbance value of each well was then detected using a microplate reader (wavelength A562), and the protein concentration of the sample was calculated according to the standard curve.

[0031] The results of BCA protein assay are as follows Figure 4 As shown, different MOI (0, 25, 50, 75, 100, 200) F.nucleatumAll infection groups could significantly stimulate exosome secretion. When MOI=50, dTHP-1 secreted the most exosomes after 48h of co-culture.

[0032] Example 6 Human aortic vascular smooth muscle cells (T / G HAVSMC) culture, passaging, cryopreservation and thawing: (1) Cell culture and passage Human aortic vascular smooth muscle cells (T / G HAVSMC) were cultured in complete medium (DMEM high-glucose medium with 10% FBS and 1% penicillin-streptomycin) in a constant-temperature cell incubator (37°C, 5% CO2) and observed every other day. When the cell density reached 80%-90%, the old medium was carefully discarded, and the cells were gently washed three times with sterile PBS buffer. 1 mL of trypsin was added for approximately 30 seconds. After gently tapping the culture flask, cells were observed to become rounded, with enlarged intercellular spaces and a small number of cells detaching from the bottom of the flask under a light microscope. Immediately add 6-8 mL of fresh complete medium to terminate the digestion. Gently pipette the cells to completely dislodge them. Recover the cells and centrifuge them at low speed at room temperature (800 rpm for 5 minutes). The old medium was discarded, and the cells were resuspended in fresh complete medium and passaged at a 1:2 ratio.

[0033] (2) Cell cryopreservation When the cell density of human aortic vascular smooth muscle cells (T / G HAVSMC) reaches 80%-90%, discard the old culture medium, gently wash three times with sterile PBS buffer, add 1 mL of trypsin to digest for about 30 seconds, gently tap the culture bottle, and find under an optical microscope that the cell morphology becomes rounded and blunt, the intercellular space becomes larger, and a small part falls off from the bottom of the bottle. Immediately add 6-8 mL of fresh complete culture medium to terminate the digestion. Gently blow the cells off completely with a pipette, recover the cells and centrifuge at low speed at room temperature, centrifuge at 800 rpm for 5 minutes, discard the old culture medium, resuspend the cells with 1 mL of freezing solution (90% serum + 10% DMSO), place them in a cryovial, immediately insert into a cell program cooling box, freeze at -80°C overnight, and transfer to a liquid nitrogen tank the next day.

[0034] (3) Cell recovery Ensure 37°C warm water or a water bath is readily available. Remove the frozen cells from the liquid nitrogen tank and thaw them in a 37°C water bath. Once completely thawed, quickly transfer them to a 15mL centrifuge tube containing 1mL of complete culture medium. Centrifuge at 800 rpm for 5 minutes at room temperature to pellet the cells. Carefully discard the DMSO-containing medium and resuspend the cells in 1mL of fresh complete culture medium. After gently pipetting to evenly distribute the cells, transfer the cells to an appropriately sized cell culture flask (prepared with the appropriate amount of fresh complete culture medium). Finally, place the culture flask in a constant temperature cell incubator (37°C, 5% CO2).

[0035] Example 7 Ultracentrifugation extraction F.nucleatum Exosomes of dTHP-1 cells in infected / uninfected groups Fn -Ex and Ex, and then perform high-throughput sequencing to analyze the differentially expressed miRNAs in exosomes and perform functional prediction analysis based on GO and KEGG: (1) RNA extraction and library construction Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion) according to the manufacturer's instructions. The total RNA quantity and integrity were assessed using a Nanodrop 2000 (Thermo Fisher Scientific Inc., USA) and an Agilent 2100 Bioanalyzer (Agilent Technology, USA), respectively. Small RNA libraries were constructed using the NEBNext Small RNA Library Prep Set for Illumina (Cat. No. NEB#E7330S, NEB, USA) from 1 μg of total RNA per sample.

[0036] The protocol involved ligating sequencing adapters to both ends of the RNA, reverse-transcribing the adapter-attached RNA into cDNA, and then amplifying it through PCR. Subsequently, 140–160 bp of PCR products were isolated and purified by agarose gel electrophoresis to create a small RNA library. After the library quality was verified using an Agilent 2100 Bioanalyzer, it was sequenced using the Illumina Novaseq 6000 platform, generating 150 bp paired-end reads. Small RNA sequencing and analysis were performed by Ouyi Biotechnology Co., Ltd. (Shanghai, China).

[0037] (2) miRNA sequencing analysis method The raw image data files obtained by high-throughput sequencing are converted into raw sequencing sequences through base calling analysis, called RawData or RawReads. Low-quality reads are further filtered, and reads containing 5' primers and poly (A) tails, reads without 3' adapters and tag sequences, and reads with a length of less than 15nt or greater than 41nt are screened and filtered to obtain clean reads.

[0038] First, clean reads were aligned to the reference genome for length distribution. Sequences were then aligned to the Rfam v10.1 database (http: / / www.sanger.ac.uk / software / Rfam) using Bowtie software. rRNA, scRNA, Cis-reg, snRNA, and tRNA sequences were annotated and filtered. Subsequently, Bowtie software was used to align and annotate the sequences with cDNA sequences, the Repbase database, and the miRBase database (http: / / www.mirbase.org / ), removing degraded transcripts and repetitive sequences. Known miRNAs were identified and annotated, and expression patterns of known miRNAs across samples were analyzed. Differentially expressed miRNAs were filtered using a q-value < 0.05 and a FC > 2 or FC < 0.5 as default thresholds. Biological replicates were used to calculate q-values using the DEG algorithm in the R package.

[0039] (3) Statistical methods The miRNA expression level was calculated using the TPM (transcript per million) metric. The TPM formula is (number of reads aligned to each miRNA) / (number of reads aligned to the total sample) × 10 6 TPM stands for paired reads per million (paired reads per million) as a miRNA expression indicator, where the total number of paired reads is used to normalize the expression value. DESeq software was used to screen for differentially expressed miRNAs in the sample alignment groups. TPM was used to calculate the log2 expression of each sample, and differential expression between the two groups was screened for miRNAs that met the p ≤ 0.05 and ≥ 2-fold differential expression range.

[0040] (4) Differentially expressed miRNAs in exosomes The barplot of differential miRNA statistics after screening based on the screening criteria of P value < 0.05 and Fold Change (FC) ≥ 2 is shown in the figure below. Figure 5 As shown in A, FnThere were 19 significantly differentially expressed miRNAs in the -Ex group VS the Ex group, of which 8 were upregulated, namely hsa-let-7d-5p, hsa-miR-1-3p, hsa-miR-103a-3p, hsa-miR-503-5p and hsa-miR-664a-5p; and 11 were downregulated, namely hsa-miR-140-3p, hsa-miR-183-5p, hsa-miR-186-5p, hsa-miR-532-5p and hsa-miR-2110. The volcano plot of differential miRNAs is shown in Figure 2. Figure 5 As shown in B, the expression pattern cluster analysis is as follows Figure 6 As shown in the figure, hsa-miR-664a-5p was significantly upregulated with FC=7 and p value of 0.01, and its expression was stable within the group.

[0041] The sequence of hsa-miR-664a-5p is as follows: 5'-ACUGGCUAGGGAAAAUGAUUGGAU-3'; When preparing the nucleotide sequence table, replace "U" with "T" to prepare the nucleotide sequence table, specifically: 5'-ACTGGCTAGGGAAAATGATTGGAT-3' (SEQ ID No. 1).

[0042] (5) KEGG functional analysis of differentially expressed miRNAs in exosomes like Figure 7 As shown, KEGG functional analysis of differentially expressed miRNA target genes in miRNA-seq high-throughput sequencing revealed that target genes were significantly enriched in the MAPK signaling pathway, one of the important pathways for VSMC phenotypic transformation. Therefore, hsa-miR-664a-5p promotes VSMC proliferation, migration, and phenotypic transformation through the MAPK signaling pathway.

[0043] Example 8 qRT-PCR detection of infected / uninfected groups F.nucleatum The expression levels of hsa-miR-664a-5p in dTHP-1 cells and VSMC cells treated with different exosomes in the same group (1) Total RNA extraction ① Take out the cell culture plate and place it on ice. Aspirate the culture medium and wash it three times with pre-cooled sterile PBS buffer. ②Every 10cm 2Add 1-2 mL of RNAiso Plus (Takara, Code No. 9108 / 9109) to the growing cultured cells, transfer the cell lysate to a centrifuge tube, and repeatedly pipette until there is no obvious precipitation in the lysate. ③ Add chloroform (1 / 5 the volume of RNAiso Plus) to each tube, cover the centrifuge tube tightly, mix until the solution emulsifies and turns milky white, then let it stand at room temperature for 5 minutes; ④ Centrifuge at 12,000 × g for 15 minutes in a pre-cooled centrifuge at 4°C. The solution in the EP tube will separate into an upper aqueous phase, a middle denatured protein phase, and a lower organic phase. Carefully aspirate an appropriate amount of the upper supernatant. Add 0.5-1 times the volume of RNAiso Plus to the tube with isopropanol, mix thoroughly by inverting, and let stand at room temperature for 10 minutes.

[0044] ⑤ Centrifuge at 12,000 × g for 10 min at 4°C, discard the supernatant, add an equal volume of 75% ethanol to that of RNAiso Plus, and centrifuge at 7,500 × g for 5 min at 4°C. After discarding the supernatant, air-dry the precipitate for 5-10 min, and dissolve it in 30-50 μL of DEPC water.

[0045] (2) Tagging A and reverse transcription ① Prepare the following reaction system in a 0.2 mL RNase-free tube: Prepare the reaction system on ice according to Table 1; Table 1 Poly(A) / cDNA synthesis reaction system

[0046] ② Place the EP tube in a PCR instrument and incubate at 37°C for 1 hour according to the instructions, then heat at 85°C for 5 minutes to inactivate the enzyme and terminate the reaction; ③ Add 90 μL ddH2O to a final volume of 100 μL; ④ The obtained reaction product can be temporarily stored in a -20℃ refrigerator.

[0047] (4) qPCR quantification ① Preparation of fluorescent quantitative PCR reaction system (20 μL system): For real-time fluorescent quantitative PCR reaction using the TB Green dye method, prepare the reaction system according to Table 2 and add the sample to a 96-well PCR plate on ice in the dark. Table 2 Fluorescence quantitative PCR reaction system

[0048] ②qRT-PCR reaction: Set the qRT-PCR reaction parameters according to Table 3 and perform the experiment on a fluorescence quantitative PCR instrument Q6; Table 3 qRT-PCR machine parameters

[0049] The results of qRT-PCR detection of hsa-miR-664a-5p expression in cells are as follows Figure 8 As shown, Figure 8 A is F.nucleatum The test results of dTHP-1 cells in the infected group and the uninfected group (treatment method of Example 4) are as follows: F.nucleatum The dTHP-1 infection group significantly upregulated the expression of hsa-miR-664a-5p; Figure 8 B is the exosomes (Ex) produced by dTHP-1 and F.nucleatum Exosomes produced after infection with dTHP-1 ( Fn -Ex) were co-cultured with VSMC for 24 h. Compared with the Control group, the Ex group and Fn -Ex group, the gene expression levels of hsa-miR-664a-5p were increased, but Fn -The ascending effect of the Ex group was more significant.

[0050] Example 9 Detection of the effect of hsa-miR-664a-5p on the biological function of VSMC cells: (1) CCK8 detection of the effect of hsa-miR-664a-5p on VSMC cell proliferation: When the cell density of human aortic vascular smooth muscle cells (T / G HAVSMC) reached 80%-90%, 5000 cells were seeded per well in 96-well plates. The next day, serum-free medium was replaced to synchronize the cells. Double-stranded microRNA hsa-miR-664a-5p mimics and hsa-miR-664a-5p inhibitor were transfected according to the manufacturer's instructions to overexpress or knockdown hsa-miR-664a-5p, respectively. The medium was changed 4-6 hours after transfection. The next day, two exosomes (Ex and Fn -Ex) was added to medium containing Ex serum at a concentration of 40 μg / mL. Equal amounts were added to different groups and cultured in a constant-temperature cell incubator (37°C, 5% CO2) for 24 hours. On the third day, 10 μL of CCK8 detection reagent was added to each well. After incubation at 37°C in the dark for 1 hour, the OD value at a wavelength of 450 nm was measured using a microplate reader and the data were recorded for statistical analysis.

[0051] The nucleotide sequence of the RNA hsa-miR-664a-5p mimics is: 5'-ACUGGCUAGGGAAAAUGAUUGGAU-3', namely hsa-miR-664a-5p; The nucleotide sequence of hsa-miR-664a-5p inhibitor is: 5'-AUCCAAUCAUUUUCCCUAGCCAGU-3'; When preparing the nucleotide sequence table, replace "U" with "T" to prepare the nucleotide sequence table, specifically: 5'-ATCCAATCATTTTCCCTAGCCAGT-3' (SEQ ID No. 2).

[0052] The results of cell proliferation ability test were as follows Figure 9 As shown, Fn -Ex can significantly promote VSMC cell proliferation, and hsa-miR-664a-5p mimics also play a role in promoting VSMC proliferation; hsa-miR-664a-5p inhibitor weakens the effect. Fn -The role of Ex.

[0053] (2) Transwell migration assay and wound healing assay to detect the effect of hsa-miR-664a-5p on VSMC cell migration ability ① Transwell migration assay When the cell density of human aortic vascular smooth muscle cells (T / G HAVSMC) reached 80%-90%, after starvation for 1 day, 5000 cells were seeded into Transwell chambers. The next day, hsa-miR-664a-5pmimics and inhibitor were transfected according to the instructions. The corresponding transfection solution was added to the upper and lower chambers, and the solution was changed 4-6 hours after transfection. The next day, two exosomes (Ex and Fn -Ex) was added to medium containing Ex serum-depleted serum at a concentration of 40 μg / mL. Equal amounts were added to different groups and cultured in a constant temperature cell incubator (37°C, 5% CO2) for 24 hours. The Transwell chambers were removed, the medium aspirated, and the cells were rinsed three times with sterile PBS buffer. 4% paraformaldehyde was added to a clean 24-well plate, and the bottom of the chambers were immersed in the solution for fixation for 60 minutes. Subsequently, the 4% paraformaldehyde was discarded, the cells were gently rinsed three times with sterile PBS buffer, and the plates were placed in a 0.1% crystal violet solution and incubated in the dark for 30 minutes. Finally, the chambers were removed and rinsed three times with sterile PBS buffer to remove excess crystal violet. The cells inside the chambers were wiped clean with a cotton swab, allowed to dry briefly, and then observed under a microscope. Statistical analysis was performed using Image J.

[0054] ② Scratch test When the cell density of human aortic vascular smooth muscle cells (T / G HAVSMC) reached 80%-90%, 5×10 5Cells were plated at a density of 100 μL in a 6-well plate. The cells were starved for 1 day to achieve synchronization. On the third day, hsa-miR-664a-5p mimics and inhibitors were transfected according to the manufacturer's instructions. The medium was changed 4-6 hours after transfection. On the fourth day, a 200 μL yellow pipette tip was used to scratch the bottom of the 6-well plate vertically. The plates were washed three times with sterile PBS buffer to remove detached cells. Two types of exosomes (Ex and Fn -Ex) was added to medium containing Ex-deficient serum at 40 μg / mL. Equal amounts were added to different groups and images were taken under an inverted microscope (0 h). Cells were then cultured in a constant temperature incubator (37°C, 5% CO2). Images were taken of the same sites at the same time point the following day (24 h). Statistical analysis was performed using Image J.

[0055] The results of the test on the effect of hsa-miR-664a-5p on VSMC cell migration ability are as follows Figure 10 and Figure 11 As shown in Figure 2, hsa-miR-664a-5p mimics significantly promoted VSMC migration; exosomes can promote VSMC migration, infection F.nucleatum The promoting effect of exosomes in the 1:1 group was more significant, but this promoting effect could be weakened by the hsa-miR-664a-5p inhibitor.

[0056] (3) RT-qPCR detection of the effect of hsa-miR-664a-5p on VSMC cell phenotypic transformation-related genes and inflammatory factors ① The method and steps for total RNA extraction are the same as step (1) in Example 8; ②Reverse transcription; a. Removal of genomic DNA: Prepare the reaction system on ice as shown in Table 4; Table 4 Reaction system for genomic DNA removal

[0057] Place the EP tube in a PCR instrument and set it to 42°C for 2 minutes according to the instructions. The resulting reaction can be temporarily stored in a -20°C refrigerator. b. Reverse transcription reaction: Prepare the reverse transcription reaction system on ice as shown in Table 5; Table 5 Reverse transcription reaction system

[0058] c. Add the prepared reverse transcription reagent system (10 μL) to the resulting reaction mixture and set the PCR instrument parameters according to the instructions: 37°C for 15 min; 85°C for 5 s; ③The qPCR quantification method and steps are the same as step (4) in Example 8; qRT-PCR test results are as follows Figure 12 As shown in Figure 3, hsa-miR-664a-5p mimics significantly reduced the expression of VSMC cell contraction markers α-SMA and SM22α, and upregulated the expression of macrophage marker CD68 ( Figure 12 A). Fn -Ex inhibited the contractile phenotype of VSMC, but hsa-miR-664a-5p inhibitor upregulated VSMC contractile markers ( Figure 12 B). Overexpression of hsa-miR-664a-5p promotes the expression of inflammatory factors (MCP-1, MMP-2 and IL-1β) ( Figure 12 C); Ex and Fn -Ex can promote the expression of MCP-1, MMP-2 and IL-1β, Fn -Ex group had a more significant promoting effect, but hsa-miR-664a-5p inhibitor could reverse this result ( Figure 12 D).

[0059] Although the specific embodiments of the present invention have been described in detail in conjunction with the embodiments, this should not be construed as limiting the scope of protection of this patent. Within the scope described by the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. Use of macrophage exosome molecules induced by Fusobacterium nucleatum in the preparation of a preparation for inducing VSMC cells to transform into a macrophage-like phenotype, wherein the nucleotide sequence of the macrophage exosome molecules induced by Fusobacterium nucleatum is 5'-ACUGGCUAGGGAAAAUGAUUGGAU-3'.

2. The use according to claim 1, characterized in that: The Fusobacterium nucleatum is ATCC25586.

3. The use according to claim 1, characterized in that: Exosome-producing macrophages were induced from human monocytes.

4. The use according to claim 1, characterized in that: The preparation is a preparation for regulating the progress of atherosclerosis.

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