CircBTBD7-420aa Regulates the Progression of Atherosclerosis

Figures

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  Figure 1. The Characteristics of circBTBD7 (A) The schematic diagram of circBTBD7 in chromosome 14. (B) Fluorescence in situ hybridization revealed the colocation of circBTBD7 and vascular smooth muscle cells. Scale bar: 100 μm. (C) Specific divergent primers detected circBTBD7 in complementary DNA in human coronary artery smooth muscle cells (HCASMCs). Sanger sequencing confirmed the back-splicing of circBTBD7. GAPDH served as a linear control. The black arrow indicates the junction site. (D) Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circBTBD7 and linear BTBD7 after RNase R digestion. (E) qRT-PCR analysis of the relative abundance of circBTBD7 and linear BTBD7 in HCASMCs treated with actinomycin D (10 μg/mL). (F) The relative subcellular localization of circBTBD7 was assessed by qRT-PCR. GAPDH and U6 were used as controls for cytoplasmic localization and nuclear localization. ∗∗∗P < 0.001. Data are mean ± SEM. Data were compared using unpaired Student’s t-test. bp = base pairs; cDNA = complementary DNA; chr = chromosome; DAPI = 4′,6-diamidino-2-phenylindole; gDNA = genomic DNA; HCASMC = human coronary artery smooth muscle cell; mRNA =…

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  Figure 2. CircBTBD7 Encodes an Undetected 420-aa Protein, circBTBD7-420aa (A) (Left) the putative ORF in circBTBD7 and the CDS in BTBD7 mRNA. (Right) circBTBD7-420aa is homologous to BTBD7, and the sequences of circBTBD7-420aa are shown. (B) Western blot confirmed the activity of theoretical ORF using empty vector, ORF-flag, and ORF mut-flag with anti-FLAG antibody. (C) The BTBD7 antibody recognized both BTBD7 and circBTBD7-420aa by Western blot. (D) The lysates from HCASMCs were separated by SDS-PAGE. Protein bands from 25 kDa to 80 kDa were cut for mass spectrometry analysis, and unique amino acid sequences (LLDPGLK) are shown. (E) Protein structure of circBTBD7-420aa predicted from Alphafold2. (F) (Left) The putative IRES activities of circBTBD7 were detected. IRES sequences were cloned to the pGL3-Basic vector. The pRL-TK vector acts as the negative control. (Right) The relative luciferase activity of Luc/Rluc was tested (n = 3). ∗∗P < 0.01; ∗∗∗P < 0.001. Data are mean ± SEM. Data were compared using analysis of variance. aa = amino acid; CDS = coding DNA sequence; FTMS = Fourier transform mass spectrometry; HCD = higher-energy collisional dissociation; IRES = internal ribosome entry…

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  Figure 3. The Inhibitory Effect of circBTBD7 on the Abnormal Proliferation and Migration of HCASMCs Is Mediated by circBTBD7-420aa (A) Quantitative real-time polymerase chain reaction and Western blot analysis demonstrated that circBTBD7 and circBTBD7-420aa were significantly down-regulated in ox-LDL–treated HCASMCs, respectively. (B) circBTBD7-420aa was located in both cytoplasm and nucleus detected by anti-FLAG antibody. Scale bars: 20 μm and 5 μm. (C) Cell proliferation was significantly inhibited by OE circBTBD7 and OE circBTBD7-420aa in the EdU assay. Scale bar: 50 μm. (D) The effect of circBTBD7 and circBTBD7-420aa on cell proliferation using the CCK-8 assay after stimulating HCASMCs. (E) HCASMC migration rate was assessed by Transwell assay. Scale bar: 200 μm. (F) HCASMC migration rate was assessed by wound-healing assay. Scale bar: 200 μm ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. Data are mean ± SEM. Comparisons in A used the unpaired Student’s t-test. Comparisons in C through F used analysis of variance. CCK-8 = Cell Counting Kit 8; EdU = 5-ethynyl-2′-deoxyuridine; NC = negative control; ns = not significant; OD = optical density; OE = over-expressed; oxLDL = oxidized low-density…

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  Figure 4. CircBTBD7-420aa Restrains Abnormal Proliferation and Migration of HCASMCs via SLC3A2 (A) (Left) IP assay was conducted using an IgG or anti-FLAG antibody in circBTBD7-420aa-flag-transfected HCASMCs. (Right) SLC3A2 was determined in circBTBD7-420aa binding partners. (B) Co-IP assay confirmed the interaction of circBTBD7-420aa and SLC3A2. (C) Immunofluorescence staining indicating that circBTBD7-420aa was colocalized with SLC3A2 in HCASMCs. Scale bar: 100 μm. (D) The 3-dimensional structure showed the binding position between circBTBD7-420aa and SLC3A2 using the swMATH website. (E) HCASMC migration rate was assessed by Transwell assay after down-regulation of SLC3A2. Scale bar: 200 μm. (F) The migration abilities inhibited by circBTBD7-420aa were restored by overexpression of SLC3A2 by Transwell assay. Scale bar: 200 μm. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. Data are mean ± SEM. Data were compared using analysis of variance. IgG = immunoglobulin G; IP = immunoprecipitation; M = marker; other abbreviations as in Figure 1, Figure 2, Figure 3.

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  Figure 5. CircBTBD7-420aa Restrains Abnormal Proliferation of HCASMCs via SLC3A2 (A) Cell proliferation was significantly inhibited through down-regulation of SLC3A2 in the EdU assay. Scale bar: 50 μm. (B) The proliferation abilities inhibited by circBTBD7-420aa were restored by overexpression of SLC3A2 in the EdU assay. Scale bar: 50 μm. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. Data are mean ± SEM. Data were compared using analysis of variance. Abbreviations as in Figure 1, Figure 2, Figure 3.

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  Figure 6. The Mechanism of circBTBD7-420aa Inhibiting SLC3A2 (A) HCASMCs were treated with CHX (20 μg/mL) for 0, 3, 6, or 9 hours, and circBTBD7-420aa could shorten the half-life of SLC3A2. (B) MG132, not CQ, inhibited the degradation of SLC3A2. (C) The possible ubiquitination sites predicted by the UbiBrowser database. (D) Western blot analysis of SLC3A2 ubiquitin levels in HEK-293T. (E) Western blot analysis of SLC3A2 ubiquitin levels after overexpression of circBTBD7-420aa in HEK-293T. (F) The specific ubiquitination site was determined by Western blot analysis. CHX = cycloheximide; Ub = ubiquitin; other abbreviations as in Figure 1, Figure 2, Figure 3.

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  Figure 7. CircBTBD7-420aa Inhibited the Development of Atherosclerosis (A) Hematoxylin and eosin and Masson staining of aortic samples from mice. Scale bar: 100 μm. (B) Immunohistochemistry analysis of aortic tissues was performed to evaluate the expression of SLC3A2. Scale bar: 100 μm. HE = hematoxylin and eosin; LV = lentivirus; NC = negative control.

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  Figure 8. Construction of Engineered Exosomes Loaded With circBTBD7-420aa Targeting SMCs (A) The vector was constructed using the pAAVS1-CMV-MCS cloning vector, including the circBTBD7-420aa-MP peptide plasmid (p547-plasmid) and eGFP-MP peptide plasmid (p549-plasmid) (a: promoter element and enhancer element; b: exosomal scaffold protein; c: ORFs). (B) The results of agarose gel electrophoresis showed that the size of the fragment was in line with the expectation (Lane M: Kilobase ladder; lane 1: p547-plasmid; lane 2: p547-plasmid digested by SmaI and NotI). (C) p547-plasmid was verified by Sanger sequencing. (D) Flow cytometry was used to detect exosomal external loaded polypeptides (HA-tag positive: osteopontin-targeting peptide; ISO positive: isotype control). (E) Western blot showed that V5 tag protein was expressed in both p547-293F cell and p547-Exo. (V5 tag protein binds to the target protein.) Exo = exosome; FITC-A = fluorescein Isothiocyanate-A; HA = hemagglutinin tag; ISO = isotype control; SMC = smooth muscle cell; SS-H = side-scatter-high; other abbreviations as in Figure 1, Figure 2, Figure 3.