Animals
All animal experiments in this study were approved by the Ethics Committee of Yunnan Labreal Biotech Ltd. Co., and operations followed the standard operating procedures of the Laboratory Animal Center of Kunming Medical University (No. PZ20230109). A total of 40 eight-week-old male apolipoprotein (Apo E−/−) mice (22.0 ± 0.4 g) were provided by the Animal Center of Kunming Medical University. Firstly, DM mice were established via daily injecting with 50 mg/kg streptozotocin (STZ; Sigma-Aldrich, MO, USA) for five days [22]. The control group mice were accepted with an equal volume of citric acid (CA) for five consecutive days. The DM mice were confirmed with blood glucose > 16.7 mM then were randomly distributed into seven groups including DM + HFD group (n = 5), DM + HFD + pcDNA3.1 group (n = 5), DM + HFD + CEACAM1 group (n = 5), DM + HFD + NC inhibitor group (n = 5), DM + HFD + miR-449a inhibitor group (n = 5), DM + HFD + miR-449a inhibitor + Si-NC group (n = 5), DM + HFD + miR-449a inhibitor + Si-CECAM1 group (n = 5). Subsequently, the DM mice were fed a high-fat diet (HFD), including 0.25% cholesterol and 20% lard oil, for four weeks to establish the AS mouse model. The control mice were fed with standard mice chow. All mice were grown in living conditions with 18–22°C, 50% humidity, and a 12 h light/dark cycle. The formation of atherosclerotic plaque was confirmed with transabdominal aortic ultrasound, and the DM mice with an atherosclerotic plaque served as DM-induced AS mice. After four weeks, the DM + HFD mice were injected with lentivirus expressing CEACAM1 (2 × 107 TU/each mouse) or lentivirus expressing NC (2 × 107 TU/each mouse) by tail vein injection or miR-449a inhibitor and its negative control, or si-CECAM1 and its negative control. The control and DM + HFD group mice were injected with equal saline and then fed with standard mice chow for another sixteen weeks. At the end of the twentieth week, all mice were euthanized with excessive inhalation of isoflurane, and then blood samples and the thoracic aortic tissues were harvested from subsequent experiments. The body weight of each mouse was calculated, and the biochemical measurements, including oxidized low-density lipoprotein (Ox-LDL), T-CHO (total cholesterol), total triglycerides (TG), and blood glucose (BG), were measured. A flow chart of animals interfering in this study is shown in Supplementary Fig. 1.
Immunofluorescence (IF)
After the euthanasia, the aorta roots were separated from the mice and immediately fixed with 4% paraformaldehyde (PFA). The sections were paraffin-embedded and subjected to pathological and immunological staining. To investigate the levels of CEACAM1 on the surface of the aorta using the IF staining. The paraffin-embedded sections were sliced into the 4-µm thickness and dehydrated with xylene and gradient ethanol. The slices were incubated with primary CEACAM1 monoclonal Antibody (MA5-24338; Thermo Fisher Scientific, Waltham, MA, USA) at 25 µg/mL overnight at 4°C. After that, the sections were incubated with Goat Anti-Rat IgG NorthernLights™ NL557-conjugated Antibody (1:200, NL013; R&D Systems, Minneapolis, MN, USA) for 2 h at room temperature in the dark. The nuclear was stained with 4’, 6‑diamidino‑2‑phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO, USA) for 5 min. After washing the sections three times, the positive cells were observed and photographed using a confocal fluorescence microscope (UltraView Vox; PerkinElmer, MA, USA).
Histological staining and immunochemistry (IHC)
The aortic sinus lesions were detected using an Oil Red O staining kit (Beyotime, Shanghai, China) and Sirius Red Sigma-Aldrich (365548-25G; St. Louis, MO, USA, respectively. Oil Red O staining was used to examine the lipidosis. Briefly, the 4-µm sections were stained with Oil Red O staining solution for 20 min at room temperature according to the manufacturer’s instruction, then rinsed with phosphate buffer saline (PBS) three times, then lipid deposition was observed and imaged under an inverse microscope (Leica, Wetzlar, Germany). Additionally, Sirius Red staining was used to detect the interstitial collagen content of the atherosclerotic plaques. The sections were stained with Sirius red staining solution and incubated at room temperature for 30 min. The collagen of the plaques was observed and photographed under an inverse microscope (Leica, Wetzlar, Germany). In addition, the numbers of VSMCs and macrophages were examined using IHC staining with SMCs marker α smooth muscle actin (a-SMA, 1:1000; ab124964, Abcam, Cambridge, MA, USA) and macrophage marker (MOMA2, 1:500; ab33451, Abcam, Cambridge, MA, USA). Then, the plaque vulnerable index was then calculated according to the ratio between the Oil red O positive area plus the MOMA-2 positive area and the Sirius Red area plus a-SMA area. Moreover, the levels of VECs marker CD34 (1:2500; ab82289, Abcam, Cambridge, MA, USA) and vascular endothelial growth factor (VEGF, 1:100; MA5-13182, Thermo Fisher Scientific, Waltham, MA, USA) were detected with IHC staining. In brief, 4-µm sections were incubated with primary antibodies overnight at 4°C and stained with Goat anti-rabbit IgG HRP-conjugated secondary antibody (1:1000; ab150077, Abcam, Cambridge, MA, USA) at room temperature for 1 h. Finally, the sections were counterstained with hematoxylin for 1 min and blued with 1% ammonia water. The positive rate was measured by the product of the percentage of immunopositive area (0%, 0; 1–25%, 1; 26–50%, 2; 51–75%, 3; 76–100%, 4) and staining intensity (0, negative; 1, weak positive; 2, medium positive; 3, strong positive). The images were measured using Image J software.
Enzyme-linked immunosorbent assay (ELISA)
The levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8) were measured using the ELISA kits (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s suggestion. The expression of adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and macrophage cationic peptide 1 (MCP-1) was also examined using ELISA kits (Abcam, Cambridge, MA, USA).
Apoptosis analysis in vivo
TUNEL staining was used to determine the apoptotic rate of the VECs in vivo. In brief, the atherosclerotic plaque sections were stained with anti-CD34 antibody overnight at 4°C. Subsequently, the sections were stained with TUNEL staining solution (Beyotime, Shanghai, China) following the manufacturer’s protocol. After that, the sections were stained with Goat anti-rabbit IgG HRP-conjugated secondary antibody as previously described. Finally, the apoptotic VECs were calculated according to the total of the CD34 and TUNEL positive cells relative to the total number of the CD34 positive cells in the plaque.
Cell culture and transfection
Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HUVECs were cultured in the EGM-2 medium (Lonza, Basel, Switzerland). Then, HUVECs were incubated in a humid incubator with 37°C and 5% CO2. HUVECs were seeded into six-well plates at 4 × 105 cells/well density and cultured at 37°C with 5% CO2 until cells reached 80–90% confluence. HUVECs were stimulated with a high glucose (33 mM) medium for five days in the absence or presence of miRNA inhibitors and si-RNAs. Then, HUVECs were randomly divided into five groups, including the HG group, HG + NC inhibitor group, HG + miR-449a inhibitor group, HG + miR-449a inhibitor + Si-NC group, HG + miR-449a inhibitor + Si-CEACAM1 group. Exception of the control group, miRNA inhibitor, and interfering RNA subsequently transfected into HUVECs using Lipofectamine 3000 regents (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instruction.
Apoptosis analysis in vitro
The apoptosis was detected using the Annexin V-FITC/PI detection kit (Beyotime, Shanghai, China) according to the manufacturer’s suggestion. Briefly, the transfected-HUVECs were collected and resuspended in 500 µL PBS at a density of 1 × 105 cells. Then, cells were centrifuged at 1000 g for 5 min and resuspended with 195 µL Annexin V-FITC binding solution. After that, 5 µL Annexin V-FITC and 10 µL Propidium iodide (PI) staining solution were added to cells and then incubated for 20 min at room temperature in the dark. Finally, apoptotic cells were detected using flow cytometry (FCM).
RNA extraction and quantitative PCR (qPCR)
Total RNA and microRNA were extracted from tissues and cells using Trizol reagent (Takara, Dalian, China) and RNAiso for Small RNA (Takara, Dalian, China) following the manufacturer’s suggestion, respectively. RNA was reversed transcription into cDNA using PrimeScript™ RT reagent Kit (Takara, Dalian, China) according to the manufacturer’s protocol. Then, cDNA was performed qPCR analysis with TB Green® Fast qPCR Mix (Takara, Dalian, China) on an ABI Prism 7300 system (Applied Biosystems, Waltham, MA, USA). The amplification conditions include 95°C for 15 s, 95°C for 5 s, and 60°C for 30 for 42 cycles. The relative expression of genes was determined using 2−ΔΔCt methods, which calculated ΔCt = Ct (target gene) – Ct (reference gene) and ΔΔCt = ΔCt (experimental group) – ΔCt (control group). GAPDH and U6 were used to normalize gene expression. The primers were listed as follows, miR-449a, F, 5’-GTGTGATGAGCTGGCAGTGTA-3’, R, 5’-AGCAGTTGCATGTTAGCCGAT-3’. CEACAM1, F, 5’-GCTGGGACGTATTGGTGTGA-3’, R, 5’-GTCATTGGAGTGGTCCTGCC-3’. MMP-9, F, 5’-TCTATGGTCCTCGCCCTGAA-3’, R, 5’-TTGTATCCGGCAAACTGGCT-3’. U6, F, 5’-GGCTCAGAATCACCCCATGT-3’, R, 5’-CCTGGACGTGCAGATGACTT-3’. GAPDH, F, 5’-GGTCACCAGGGCTGCTTTTA-3’, R, 5’-CCCGTTCTCAGCCATGTAGT-3’.
Dual-luciferase reporter gene assay
The sequences of 3’UTR of CEACAM1 contained binding sites (CACUGCC) with miR-449a were amplified and inserted into luciferase reporter plasmids to construct the wild-type recombinational luciferase reporter genes (CEACAM1-WT). In addition, the binding site sequences were mutated into CUGACGC, and the mutant-type recombinational luciferase reporter genes (CEACAM1-MUT) were constructed. Then, the recombinational wild/mutant type luciferase reporters were con-transfected with miR-499a mimic or its negative control into HUVECs and incubated for 48 h. After that, the luciferase activity was measured using a dual-luciferase activity reporter system (Promega, Madison, WI, USA) according to the manufacturer’s protocol.
Western blot assay
Protein was isolated from tissues and cells using Cell lysis buffers (Takara, Dalian, China) following the suggestion of the manufacturer. Subsequently, the protein was separated by 10% SDS-PAGE gel, transferred with PVDF membranes, and blocked with 5% non-fat milk. After that, the membranes were incubated with rabbit anti-MMP9 antibody (1:1000; ab38898, Abcam, Cambridge, MA, USA), rabbit anti-TIMP1 antibody (1:1000; ab216432, Abcam, Cambridge, MA, USA), rabbit anti-CEACAM1 antibody (1:1000; #14,771, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-GAPDH antibody (1:2000; ab8245, Abcam, Cambridge, MA, USA) overnight at 4°C. The membranes were further incubated with HRP-labeled goat anti-rabbit IgG (1:2000; ab7090, Abcam, Cambridge, MA, USA) at room temperature for 1 h. Finally, bands were visualized using an enhanced chemiluminescence reaction solution (Bio-Rad, Hercules, CA, USA). GAPDH is used as an internal reference gene to normalize protein levels.
Migration and invasion assay
The cell migration and invasion were examined using the wound healing and Transwell assays. After HUVECs transfection or stimulation, cells were harvested and seeded into the six-well plates at the density of 1 × 106 cells/well, then incubated overnight at 37°C. Subsequently, the monolayer cells were scratched using a 200 µL pipette. The migrated areas of the migrated cells were observed and photographed at 0 h and 24 h. In addition, the transfected and stimulated HUVECs (2 × 105 cells/well) were collected and seeded onto the transwell insert, which was pro-coated with Matrigel (BD Biosciences, San Jose, CA, USA). 500 µL EGM-2 medium was added into the lower well and incubated for 24 h. Finally, the invaded cells on the opposite filters were fixed with methanol and stained with 1% crystal violet (Beyotime, Shanghai, China) according to the manufacturer’s protocol. And the invaded cells were observed and counted under an inverted microscope (Leica, Wetzlar, Germany).
Tube formation assay
The tube formation was performed using Matrigel (BD Biosciences, San Jose, CA, USA). After cell transfection and stimulation, 1 × 104 HUVECs were seeded onto the Matrigel pre-coated 96-well plates and incubated in the EGM-2 medium for 12 h. The tube formation of HUVECs was visualized, and the number of nodes was calculated according to at least three cells formed at a single point quantitated as a node.
Statistical analysis
All values in this study were expressed as mean ± stand deviation (SD). Comparison between multiple groups was performed using one/two-way analysis of variance (ANOVA), respectively. All statistical analyses were performed using GraphPad Prism 9 (GraphPad, Inc).
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