Circular RNA circCHFR Facilitates the Proliferation and Migration of Vascular Smooth Muscle via miR-370/FOXO1/Cyclin D1 Pathway

Abstract

Circular RNA (circRNA) is a novel subgroup of noncoding RNA in the human transcriptome playing a vital role in the atherosclerosis of cerebrovascular disease. However, the in-depth mechanism by which circRNA regulates the vascular smooth muscle proliferation and migration is still elusive. Here, a novel identified circRNA, circCHFR, was validated to be aberrantly overexpressed in the ox-LDL-induced vascular smooth muscle cell (VSMCs). Functionally, the circCHFR silencing by oligonucleotide transfection suppressed the proliferation and migration ability of VSMCs. Mechanically, bioinformatics tools and luciferase reporter assay state that circCHFR acts as a sponge of miR-370, and miR-370 targets the 3' UTR of FOXO1. Furthermore, the transcription factor FOXO1 could bind with the promoter region of CCND1 mRNA and promote Cyclin D1 expression. In summary, this finding states the vital role of the circCHFR/miR-370/FOXO1/Cyclin D1 axis and provides a profound understanding about the circRNA in smooth muscle cells and atherosclerosis.

Keywords: CCND1; FOXO1; atherosclerosis; circCHFR; circular RNA; vascular smooth muscle cells.

Figure 1

Microarray Analysis Unveils the Aberrantly Expressed circRNA in the ox-LDL-Induced VSMCs (A) Volcano plot presents the differently expressed circular RNAs (fold change > 2, p value < 0.05) in these VSMCs treated with ox-LDL and blank control. (B) The normalization of differently expressed circular RNAs. (C) Heatmap analysis visualized these circular RNA in the VSMCs. (D, E, and F) RT-PCR was performed to measure the expression of hsa_circ_0002984 (D), hsa_circ_0029589 (E), and hsa_circ_0010283 (F). *p < 0.05, **p < 0.01.

Figure 2

The Knockdown of circCHFR Suppresses the Proliferation and Migration of VSMCs (A) Three siRNA specifically targeting the circCHFR (si-circCHFR) were transfected into VSMCs to knock down circCHFR level. (B) Wound-healing assay for migration was performed using VSMCs transfected with two siRNAs targeting circCHFR. (C) CCK-8 assay stated the proliferative ability of VSMCs after circCHFR silencing compared with the blank transfection. (D) Transwell assay for the migration revealed the migrated VSMCs through the chamber membrane after circCHFR silencing. (E) Western blot analysis revealed the FOXO1 and Cyclin D1 protein in VSMCs with the circCHFR silencing transfection. *p < 0.05, **p < 0.01.

Figure 3

circCHFR Functions as the Sponge of miR-370 (A) Venn plot showed the potential miRNAs for circCHFR using multiple bioinformatics tools. (B) The analysis of circCHFR and miR-370 sequence indicated the several complementary binding sites within circCHFR and miR-370. Luciferase reporter assay confirmed the binding within circCHFR and miR-370. (C) RT-PCR illustrated the miR-370 level in the circRNA siRNA transfection of the VSMCs. (D) RT-PCR illustrated the miR-370 level in the VSMC-treated ox-LDL. (E) CCK-8 assay showed the proliferation of VSCs transfected with miR-370 mimics. *p < 0.05, **p < 0.01.

Figure 4

FOXO1 Acts as the Functional Target of miR-370 in VSMCs (A) FOXO1 protein functioned as the downstream target of miR-370 with complementary binding sites at the 3′ UTR. Luciferase reporter assay validated the relative fluorescence intensity when the FOXO1 3′ UTR wild-type or mutant was co-transfected with miR-370 mimics or control. (B) The transfection of miR-370 mimics could decrease the FOXO1 mRNA level in VSMCs. (C) Western blot showed the FOXO1 protein level in the ox-LDL-induced VSMCs. (D and E) The transfection of miR-370 mimics (D) or inhibitor (E) could decrease or enhance, respectively, the FOXO1 protein levels. *p < 0.05, **p < 0.01.

Figure 5

FOXO1 Enhances the Transcriptional Expression of CCND1 (A) The three putative binding elements that FOXO1 could target with the promoter region of CCND1 gene. (B) Chromatin immunoprecipitation (ChIP) assay illustrated the combining degree of FOXO1 with these element regions. (C) The wild-type and mutant element region sequence were constructed for the luciferase reporter assay. (D) Luciferase reporter assay revealed the molecular incorporation of FOXO1 and the candidate element region. (E) Western blot showed the FOXO1 level in VSMCs transfected with FOXO1 enhanced plasmid. (F) RT-PCR revealed the CCND1 mRNA. (G) Western blot showed the Cyclin D1 level in VSMCs. *p < 0.05, **p < 0.01.

Figure 6

Schematic Diagram Illustrated the Role of circCHFR on the VSMCs’ Proliferation and Migration through the miR-370/FOXO1/Cyclin D1 Pathway