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

Abstract

Background: Circular non-coding RNA (circRNA) has a variety of biological functions. However, the expression profile and potential effects of circRNA on atherosclerosis (AS) and vascular endothelial injury have not been fully elucidated. This study aims to identify the differentially expressed circRNAs in atherosclerotic aortic vessels and predict their potential functions in endothelial injury. Method: ApoE-/- mice were fed with high-fat diet for 12 weeks to induce AS. Atherosclerotic plaques were evaluated by H&E and Masson staining and immunohistochemistry; differentially expressed circRNAs were detected by Arraystar Circular RNA Microarray and verified by RT-PCR; the potential target mircoRNAs of circRNAs were predicted by miRanda, Tarbase, Targetscan and their expression changes were verified by RT-PCR; the potential target genes of mircoRNAs were predicted by Targetscan and verified by Western blot; the signaling pathways that they might annotate or regulate and their potential functions in vascular endothelial injury were predicted by gene enrichment analysis. Results: Fifty two circRNAs were up-regulated more than twice and 47 circRNAs were down-regulated more than 1.5 times in AS aortic vessels. Mmmu_circRNA_36781 and 37699 were up-regulated both in AS aortic vessels and H₂O₂-treated mouse aortic endothelial cells (MAECs). The expression of miR-30d-3p and miR-140-3p, the target microRNA of circRNA_37699 and circRNA_36781, were downregulated both in AS vessels and H₂O₂-treated MAECs. On the contrary, MKK6 and TP53RK, the potential target gene of miR-140-3p and miR-30d-3p, were upregulated both in AS aortic roots and H₂O₂-treated MAECs. Besides, gene enrichment analysis showed that MAPK and PI3K-AKT signaling pathway were the most potential signaling pathways regulated by the differentially expressed circRNAs in atherosclerosis. Conclusions: Mmu_circRNA_36781 (circRNA ABCA1) and 37699 (circRNA KHDRBS1) were significantly up-regulated in AS aortic vessels and H₂O₂-treated MAECs. They have potential regulatory effects on atherosclerosis and vascular endothelial injury by targeting miR-30d-3p-TP53RK and miR-140-3p-MKK6 axis and their downstream signaling pathways.

Keywords: atherosclerosis; ceRNA; circRNA; microRNA; vascular endothelium.

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.

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.

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.

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.

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.

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.

Figure 7

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