Circular RNA circ_0029589 regulates proliferation, migration, invasion, and apoptosis in ox-LDL-stimulated VSMCs by regulating miR-424-5p/IGF2 axis.

Circular RNAs (circRNAs) have been identified to be critical mediators in the progression of atherosclerosis (AS). However, the exact roles and molecular mechanism of circ_0029589 in AS are far from understood. Vascular smooth muscle cells (VSMCs) stimulated by oxidized low-density lipoprotein (ox-LDL) were served as a cellular model of AS. The expression levels of circ_0029589, microRNA (miR)-424-5p, and insulin-like growth factor 2 (IGF2) were measured by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot analysis. Cell proliferation was determined by Cell Counting Kit-8 (CCK-8) assay and colony formation assay. Cell apoptosis, migration and invasion were examined by flow cytometry and transwell assay. The relationship between miR-424-5p and circ_0029589 or IGF2 was predicted by starbase and verified by dual-luciferase reporter assay. Circ_0029589 and IGF2 were upregulated and miR-424-5p was downregulated in VSMCs treated with ox-LDL. Silence of circ_0029589 inhibited proliferation, migration and invasion but induced apoptosis in ox-LDL-treated VSMCs. MiR-424-5p was a target of circ_0029589 and its knockdown reversed the effects of circ_0029589 interference on proliferation, migration, invasion, and apoptosis in ox-LDL-stimulated VSMCs. IGF2 was a target of miR-424-5p and miR-424-5p overexpression suppressed proliferation, migration and invasion while promoted apoptosis in ox-LDL-treated VSMCs by downregulating IGF2. Circ_0029589 positively modulated IGF2 expression by sponging miR-424-5p. Circ_0029589 silence might inhibit the progression of AS by regulating miR-424-5p/IGF2 axis, providing a novel mechanism for pathogenesis of AS.

Yu H ，Zhao L ，Zhao Y ，Fei J ，Zhang W ... -  《-》

Circ_0010283/miR-377-3p/Cyclin D1 Axis Is Associated With Proliferation, Apoptosis, Migration, and Inflammation of Oxidized Low-density Lipoprotein-Stimulated Vascular Smooth Muscle Cells.

Circular RNAs have been reported as vital regulators and promising therapeutic targets in multiple human diseases, including atherosclerosis (AS). However, the functional roles of circ_0010283 in AS remain unclear. The real-time quantitative polymerase chain reaction was used to determine the expression levels of circ_0010283, microRNA (miR)-377-3p, and cyclin D1 (CCND1) in serum samples. The vascular smooth muscle cells (VSMCs) were treated with oxidized low-density lipoprotein (ox-LDL) to establish the in vitro cell model of AS. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide and clonal colony-forming assays were performed to assess cell proliferation. The apoptosis was determined by flow cytometry assay. The migration of VSMCs was examined by wound healing and transwell assays. Western blot analysis was used to quantify protein expression. The association among circ_0010283, miR-377-3p, and CCND1 was confirmed by dual-luciferase reporter assay. We found that the serum level of circ_0010283 was upregulated in patients with AS and treatment with ox-LDL also increased the expression of circ_0010283 in VSMCs. Treatment with ox-LDL also increased proliferation, migration, and inflammation while inhibited apoptosis in VSMCs, which was overturned by silencing of circ_0010283. Moreover, miR-377-3p was a target of circ_0010283, and downregulation of miR-377-3p counteracted circ_0010283 silencing-induced effects on ox-LDL-stimulated VSMCs. The overexpression of miR-377-3p inhibited proliferation, migration, and inflammation while induced apoptosis of VSMCs by targeting CCND1. CCND1 was a target of miR-377-3p, and circ_0010283 acted as the miR-377-3p sponge to increase CCND1 expression. Circ_0010283 regulated proliferation, apoptosis, migration, and inflammation of ox-LDL-stimulated VSMCs through modulating miR-377-3p and CCND1.

Zhang P ，Wang W ，Li M 《-》

Circular RNA circ_0002984 Facilitates the Proliferation and Migration of Ox-LDL-Induced Vascular Smooth Muscle Cells via the Let-7a-5p/KLF5 Pathway.

Circular RNAs (circRNAs) play an important role in the progression of atherosclerosis (AS). This study aimed to explore the exact role and mechanism of circ_0002984 in oxidized low-density lipoprotein (ox-LDL)-mediated human vascular smooth muscle cells (HVSMCs). The model of smooth muscle cell phenotype switching was constructed by treating HVSMCs with ox-LDL. The levels of circ_0002984, let-7a-5p, and kruppel-like factor 5 (KLF5) were measured by quantitative real-time PCR or western blot assay. Cell proliferation, migration, and apoptosis were detected by Cell Counting Kit-8 (CCK-8), EdU staining, wound healing assay, transwell assay, and flow cytometry. The expression of cleaved-caspase-3 and KLF5 was examined by western blot. The relationship between let-7a-5p and circ_0002984 or KLF5 was verified by dual-luciferase reporter assay or RIP assay. The results showed that circ_0002984 and KLF5 were up-regulated, while let-7a-5p was down-regulated in AS patients and ox-LDL-disposed HVSMCs. Silence of circ_0002984 suppressed proliferation and migration, and promoted apoptosis in ox-LDL-stimulated HVSMCs. Moreover, circ_0002984 sponged let-7a-5p to regulate the proliferation, migration, and apoptosis in ox-LDL-resulted HVSMCs. In addition, KLF5 was a target of let-7a-5p and its overexpression reversed the effect of let-7a-5p on the proliferation, migration, and apoptosis in ox-LDL-treated HVSMCs. Also, circ_0002984 positively regulated KLF5 expression by absorbing let-7a-5p. The promotion effect of circ_0002984 on the proliferation and migration of ox-LDL-treated HVSMCs was reversed by KLF5 silencing. Taken together, depletion of circ_0002984 inhibited the proliferation and migration of ox-LDL-stimulated HVSMCs, which might be achieved by modulating the let-7a-5p/KLF5 axis.

Chen F ，Jiang R ，Yu X 《-》

Circ_GRN Promotes the Proliferation, Migration, and Inflammation of Vascular Smooth Muscle Cells in Atherosclerosis Through miR-214-3p/FOXO1 Axis.

Dysfunction of vascular smooth muscle cells (VSMCs) assumes a fundamental part in the pathogenesis of atherosclerosis (AS). Circular RNA granulin precursor (circ_GRN) was identified to promote the proliferation and invasion of human VSMCs (HVSMCs) in an in vitro AS model. However, the underlying mechanisms remain unclear. Levels of circ_GRN, microRNA (miR)-214-3p, and forkhead box protein O1 (FOXO1) were detected using quantitative real-time polymerase chain reaction and Western blot assays. The proliferation, migration, and inflammatory response of HVSMCs were evaluated by using flow cytometry, colony formation, Cell Counting Kit-8, Western blot, transwell assays, and enzyme-linked immunosorbent assay, respectively. The binding interaction between miR-214-3p and circ_GRN or FOXO1 was detected by dual-luciferase reporter assay. In this study, we found that circ_GRN was elevated in the serum of AS and oxidized low-density lipoprotein (ox-LDL)-induced HVSMCs. The in vitro AS model was established by exposing HVSMCs to ox-LDL, and we found circ_GRN knockdown reversed ox-LDL-evoked cell proliferation, migration, and inflammation. In a mechanical study, miR-214-3p directly bound to circ_GRN or FOXO1, and circ_GRN could regulate FOXO1 expression by competitively binding to miR-214-3p. Importantly, we demonstrated that miR-214-3p inhibition attenuated the protective effects of circ_GRN knockdown on ox-LDL-induced HVSMCs; besides that, miR-214-3p overexpression abolished ox-LDL-triggered HVSMC proliferation, migration, and inflammation, which were counteracted by FOXO1 upregulation. In conclusion, circ_GRN promoted the proliferation, migration, and inflammation of HVSMCs through miR-214-3p/FOXO1 axis in ox-LDL-induced AS model in vitro, suggesting the potential involvement in an AS process, which provided a potential candidate for future clinic intervention in AS.

Li X ，Li L ，Dong X ，Ding J ，Ma H ，Han W ... -  《-》

Circ_0002984 Enhances Growth, Invasion, and Migration in PDGF-bb-Induced Vascular Smooth Muscle Cells Through miR-379-5p/FRS2 Axis.

The accumulation of vascular smooth muscle cells (VSMCs) is considered to play important roles in atherosclerosis (AS) development and progression. Circ_0002984 was found to be increased in oxidized low-density lipoprotein (ox-LDL) human VSMCs (HVSMCs). However, the function and mechanism of circ_0002984 in VSMC dysfunction remain unknown. In this study, the expression of circ_0002984, microRNA (miR)-379-5p, and fibroblast growth factor receptor substrate 2 (FRS2) was detected using quantitative real-time polymerase chain reaction and western blot. Cell proliferation, cell cycle, migration, and invasion were detected using Cell Counting Kit-8, flow cytometry, and transwell assays. The binding interaction between miR-379-5p and circ_0002984 or FRS2 was confirmed by the dual-luciferase reporter assay. Collectively, this study found that circ_0002984 was elevated in platelet-derived growth factor type bb (PDGF-bb)-induced HVSMCs. Circ_0002984 knockdown abrogated PDGF-bb-induced proliferation, migration, and invasion in HVSMCs. Mechanistically, circ_0002984 was confirmed to target miR-379-5p, and miR-379-5p upregulation reversed the protective effects of circ_0002984 knockdown on PDGF-bb-induced HVSMCs. Besides, when FRS2 was a target of miR-379-5p, miR-379-5p restoration abolished PDGF-bb-evoked HVSMC dysfunction, which was attenuated by the overexpression of FRS2. Moreover, circ_0002984 could regulate FRS2 expression through sponging miR-379-5p in HVSMCs. Collectively, these results demonstrated that circ_0002984 promoted PDGF-bb-induced VSMC proliferation, migration, and invasion through the regulation of miR-379-5p/FRS2 axis, suggesting a new insight into the pathogenesis of AS and the potential application of circ_0002984 in AS treatment.

Zheng X ，Liu J ，Gong X ，Zhang X ，Ma S ... -  《-》