Differentially Expressed Circular Non-coding RNAs in Atherosclerotic Aortic Vessels and Their Potential Functions in Endothelial Injury

Figures

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  Figure 1. Atherosclerosis mice model was established successfully. (A–C) Serum content of total cholesterol, triglycerides, LDL-C of ApoE-/- mice increased significantly after feeding with high fat diet for 12 weeks, *P < 0.05, ***P < 0.001 vs. control, n = 6. (D) Serum content of HDL-C of ApoE-/- mice decreased significantly after feeding with high fat diet for 12 weeks, *P < 0.05 vs. control, n = 6. (E,F) H & E staining of aortic arches of control and AS mice showed atherosclerotic plaques and foam cell aggregation in AS vessels. (G,H) Masson staining of aortic arches of control and AS mice. (I) Expression of α-SMA detected by immunohistochemical staining of aortic arches of control and AS mice. (J) Expression of CD31 detected by immunohistochemical staining of aortic arches of control and AS mice; n = 3.

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  Figure 2. Expression spectrum of circRNAs was screened and analyzed by Arraystar circRNA Microarray Chip. (A) Data standardization. (B) The overall distribution of chip data. The horizontal and vertical axes represent circRNA expression of different samples respectively. The closer the data get to the center diagonal, the closer the circRNA expressed in two group; while the farther away the data get to the center diagonal, the greater the difference in circRNA expression level between two samples. (C) Cluster analysis of differentially expressed circRNA in each sample. Red represents high expressed circRNAs and green represents low expressed circRNAs. (D) Differentially expressed circRNA with statistically significant difference between control and AS group.

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  Figure 3. Validation of differentially expressed circRNAs in AS vessels. (A–E) Expression of mmu_circRNA_42617, mmu_circRNA_27503, mmu_circRNA_28589, mmu_circRNA_014193, mmu_circRNA_24705 in aortic arches of control and AS mice; (F–J) mmu_circRNA_35784, mmu_circRNA_35619, mmu_circRNA_39714, mmu_circRNA_36781,mmu_circRNA_37699 in aortic arches of control and AS mice; n = 3. ns, no significant difference.

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  Figure 4. Validation of differentially expressed circRNAs in MAECs induced by H2O2. (A) Effect of H2O2 on cell viability; ***P < 0.001 vs. Control, n = 4. (B,C) Effect of H2O2 on NO concentration of MAECs; 400×, n = 3. *P < 0.05, **P < 0.01 vs. Control. (D,E) Effect of H2O2 on ROS concentration of MAECs; 400×, n = 3. **P < 0.01, ***P < 0.001 vs. Control. (F,G) Expression of mmu_circRNA_36781 and mmu_circRNA_37699 in MAECs induced by 100 μM H2O2, n = 5–6.

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  Figure 5. Prediction and validation of complementary microRNAs of differentially expressed circRNAs. (A) The binding sites and 2D structure of circRNA KHDRBS1 with miR-30d-3p; (B) The binding sites and 2D structure of circRNA ABCA1 with miR-140-3p; (C–E) Expression of miR-30d-3p and miR-140-3p in MAECs induced by H2O2 and AS aorta, n = 3–6; (F–H) Expression of miR-140-3p in MAECs induced by H2O2 and AS aorta, n = 3–5.

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  Figure 6. Expression of predicted complementary mRNAs in AS aortas and H2O2-treated MAECs. (A) The binding sites and 2D structure of miR-140-3p with MAP2K6; (B) The binding sites and 2D structure of miR-30d-3p with TP53RK; (C) Expression of CD31 and MKK6 in control, AS near and AS far aortas, n = 3; (D) Expression of TP53RK in control, AS near and AS far aortas, n = 3; (E–G) Expression of CD31, MKK6 and TP53RK in H2O2-treated MAECs, n = 3–4.

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  Figure 7. Underlying target genes of differentially expressed circRNAs and their potential function in regulating endothelial injury and atherosclerosis.