Oxygenized Low-Density Lipoprotein-Induced ASMC Dysregulation Depends on circ_0000345-Mediated Regulatory Mechanism

Figures

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  Fig.1. Expression of miR-647 and circ_0000345 was negatively correlated in the AS patient’s serum.. (A) Pearson’s correlation coefficient analysis of the correlation between miR-647 and circ_0000345 in the AS patient’s serum. (B and C) Relative levels of circ_0000345 and miR-647 in the AS patient’s serum. (D and E) Relative expression levels of circ_0000345 and miR-647 in ASMCs stimulated by different doses of ox-LDL. ＊P＜0.05.

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  Fig.2. The characteristics of circ_0000345 in ASMCs. (A) Schematic illustration showing the circularization of RSF1 exons 6 and 7 to form circ_0000345. (B) RT-qPCR analysis of circ_0000345 and RSF1 mRNA in ASMCs-derived total RNA pre-treated with or without RNase R. (C) Relative expression levels of circ_0000345 and RSF1 mRNA after reverse transcription with random primers or oligo (dT)18 primers. (D) RT-qPCR analysis of circ_0000345 in ASMCs-derived nuclear RNA and cytoplasmic RNA. ＊P＜0.05.

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  Fig.3. Effects of circ_0000345 overexpression on ox-LDL-induced ASMC proliferation, migration, invasion, and inflammatory response. (A) RT-qPCR verifying the transfection efficiency of the circ_0000345 overexpression plasmid. (B–I) ASMCs with or without ox-LDL stimulation and circ_0000345/pCD-ciR-transfected ASMCs with ox-LDL stimulation were used. (B) MTT assays showing the viability of ASMCs. (C) Western blotting analysis showing the relative levels of PCNA protein in ASMCs. (D) Flow cytometry assays exhibiting the apoptosis of ASMCs. (E) Western blotting analysis showing the relative levels of Cleaved caspase-3 in ASMCs. (F and G) Wound healing and transwell invasion assays exhibiting the migration and invasion of ASMCs. (H and I) The release of proinflammatory cytokines TNF-α and IL-6 in ASMCs. ＊P＜0.05.

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  Supplementary Fig.1. Effects of circ_0000345 silencing on cell proliferation, migration, invasion, and inflammatory response in ox-LDL-stimulated ASMCs. (A-I) ASMCs were transfected with si-NC or si-circ_0000345 and then treated with ox-LDL. (A) Relative expression of circ_0000345 in the above treated ASMCs. (B) The viability of the above treated ASMCs was determined by MTT assay. (C) Relative protein levels of PCNA in the above treated ASMCs were detected by western blotting. (D) The apoptosis of the above treated ASMCs was detected via flow cytometry assay. (E) PCNA protein levels in the above treated ASMCs were measured via western blotting. (F and G) Cell migration and invasion in the above treated ASMCs were evaluated by wound healing and transwell invasion assays. (H and I) ELISA was used to measure the release of TNF-α and IL-6 in the above treated ASMCs. ＊P＜0.05.

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  Supplementary Fig.2.. Effect of circ_0000345 overexpression on the expression levels of 6 miRNAs (miR-942, miR-147, miR-647, miR-636, miR-515-5p, and miR-153-3p) in ASMCs was assessed by RT-qPCR. ＊P＜0.05.

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  Fig.4. Circ_0000345 acted as a miR-647 sponge. (A) The WT-circ_0000345 and MUT-circ_0000345 sequences were respectively inserted into the pGL3 plasmid. (B) RT-qPCR showing the transfection efficiency of miR-647 mimic. (C and D) Dual-luciferase reporter and RIP assays were used to validate the relationship between circ_0000345 and miR-647. (E) RT-qPCR showing the interference efficiency of si-circ_0000345. (F) Impacts of circ_0000345 inhibition and overexpression on miR-647 expression. ＊P＜0.05.

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  Fig.5. miR-647 impaired circ_0000345 overexpression-mediated effects on cell proliferation, migration, invasion, and inflammatory response in ox-LDL-stimulated ASMCs. (A–H) ASMCs with or without ox-LDL stimulation and circ_0000345/pCD-ciR/circ_0000345+miR-NC/circ_0000345+miR-647-transfected ASMCs with ox-LDL stimulation were used. (A) MTT assays analysis of the viability of ASMCs. (B) Western blotting detection of the relative levels of PCNA protein in ASMCs. (C) Flow cytometry assays evaluation of the apoptosis of ASMCs. (D) Western blotting investigation of the relative levels of Cleaved caspase-3 protein in ASMCs. (E and F) Wound healing and transwell invasion assays evaluation of the migration and invasion of ASMCs. (G and H) ELISA measurement of proinflammatory cytokines TNF-α and IL-6 in ASMCs. ＊P＜0.05.

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  Fig.6. PAPD5 acted as a target for miR-647. (A) The pGL3 plasmid containing WT-PAPD5 3′UTR or MUT-PAPD5 3′UTR was constructed, respectively. (B and C) The targeting relationship between miR-647 and PAPD5 was demonstrated by dual-luciferase reporter and RIP assays. (D and E) Relative levels of PAPD5 mRNA and protein in the AS patient’s serum. (F) Relative levels of PAPD5 protein in ASMCs stimulated by different doses of ox-LDL. (G) RT-qPCR showing the interference efficiency of miR-647 inhibitor. (H) Pearson’s correlation coefficient analysis of the correlation between miR-647 and PAPD5 mRNA in the AS patient’s serum. (I) Effects of miR-647 inhibition and overexpression on PAPD5 protein levels in ASMCs. ＊P＜0.05.

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  Fig.7. PAPD5 silencing impaired miR-647 knockdown-mediated effects on ASMC proliferation, migration, invasion, and inflammatory response under ox-LDL stimulation. (A) Western blotting showing the interference efficiency of si-PAPD5. (B–I) ASMCs with or without ox-LDL stimulation and anti-miR-NC/anti-miR-647/anti-miR-647+si-NC/anti-miR-647+si-PAPD5-transfected ASMCs with ox-LDL stimulation were used. (B) The viability of ASMCs was evaluated. (B) Relative levels of PCNA protein in ASMCs were detected. (C) The apoptosis of ASMCs was detected. (D) Relative levels of Cleaved caspase-3 protein in ASMCs were analyzed. (E and F) The migration and invasion of ASMCs were determined. (G and H) The release of proinflammatory cytokines TNF-α and IL-6 in ASMCs was measured. ＊P＜0.05.

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  Fig.8. Circ_0000345 regulated PAPD5 expression via adsorbing miR-647. (A) The correlation between circ_0000345 and PAPD5 in the AS patient’s serum. (B and C) Relative levels of PAPD5 protein in ASMCs with or without ox-LDL stimulation and circ_0000345/pCD-ciR/circ_0000345+miR-NC/circ_0000345+miR-647-transfected ASMCs with ox-LDL stimulation were detected. ＊P＜0.05.