RESUMEN
BACKGROUND: Human aortic vascular smooth muscle cells (HA-VSMCs) play vital roles in the pathogenesis of vascular diseases. Circular RNAs (circRNAs) have been reported to regulate the biological functions of HA-VSMCs. In this study, the functions of circRNA pecanex homolog (circPCNX) in platelet-derived growth factor-BB (PDGF-BB)-induced HA-VSMCs were investigated. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to determine the expression of circPCNX, DNA methyltransferase 1 (DNMT1), and microRNA-1278 (miR-1278). 5'-Ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry analysis, wound healing assay, and transwell assay were used to examine cell proliferation, cell cycle, and migration. Western blot assay was utilized to measure protein levels. RNA immunoprecipitation (RIP) assay, RNA pull down assay, and dual-luciferase reporter assay were adopted to analyze the relationships among circPCNX, miR-1278, and DNMT1. RESULTS: CircPCNX was upregulated in PDGF-BB-treated HA-VSMCs in a dose- or time-dependent manner. CircPCNX knockdown alleviated PDGF-BB-induced cell proliferation, cell cycle progression, and migration in HA-VSMCs. CircPCNX knockdown could reverse PDGF-BB-induced HA-VSMC progression by regulating DNMT1. Moreover, circPCNX was identified to regulate DNMT1 expression by sponging miR-1278. Inhibition of miR-1278 reversed circPCNX knockdown-mediated effects on cell proliferation and migration in PDGF-BB-induced HA-VSMCs. MiR-1278 overexpression suppressed PDGF-BB-stimulated HA-VSMC proliferation and migration by targeting DNMT1. CONCLUSION: CircPCNX promoted PDGF-BB-induced HA-VSMC proliferation and migration by elevating DNMT1 expression through sponging miR-1278.
Asunto(s)
Becaplermina , MicroARNs , Músculo Liso Vascular , Humanos , Becaplermina/farmacología , Movimiento Celular , Proliferación Celular , Células Cultivadas , MicroARNs/genética , MicroARNs/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Transducción de SeñalRESUMEN
Circular RNAs (circRNAs) are associated with the pathogenesis of human diseases, including atherosclerosis. Here, we undertook to investigate the biological role and mechanism of circRNA E3 ubiquitin-protein ligase (circ-CHFR) in atherosclerosis. The expression levels of circ-CHFR, miR-214-3p, and pregnancy-associated plasma protein A (PAPPA) were measured by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot in human aorta vascular smooth muscle cells (HA-VSMCs) exposed to oxidized low-density lipoprotein (ox-LDL). Cell proliferation, migration, and invasion capabilities were assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT), and transwell assays, respectively. The relationship between miR-214-3p and circ-CHFR or PAPPA was confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Our data showed that circ-CHFR was upregulated in HA-VSMCs after stimulation with ox-LDL. Downregulation of circ-CHFR inhibited the proliferation, migration, and invasion of HA-VSMCs exposed to ox-LDL. Mechanistically, circ-CHFR acted as a miR-214-3p sponge, and miR-214-3p was a molecular mediator of circ-CHFR regulation in ox-LDL-stimulated HA-VSMCs. PAPPA was a miR-214-3p target, and circ-CHFR regulated the expression of PAPPA by sponging miR-214-3p. Moreover, overexpression of miR-214-3p repressed the proliferation, migration, and invasion of ox-LDL-induced HA-VSMCs by decreasing PAPPA expression. Our findings suggest that the circ-CHFR/miR-214-3p/PAPPA axis regulates ox-LDL-induced proliferation, migration, and invasion in HA-VSMCs.
Asunto(s)
Aterosclerosis , Movimiento Celular , Proliferación Celular , MicroARNs , Músculo Liso Vascular , Proteína Plasmática A Asociada al Embarazo , ARN Circular , Aorta/metabolismo , Aorta/patología , Aterosclerosis/genética , Aterosclerosis/patología , Proteínas de Ciclo Celular , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Humanos , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacología , MicroARNs/genética , MicroARNs/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteína Plasmática A Asociada al Embarazo/metabolismo , ARN Circular/genética , Transducción de Señal , Ubiquitina-Proteína LigasasRESUMEN
BACKGROUND AND AIMS: Human vascular smooth muscle cells (HA-VSMCs) are an important cell type involved in atherosclerosis. Low density lipoprotein (LDL) is a lipoprotein particle that carries cholesterol into peripheral tissue cells, and oxidized modified LDL (ox-LDL) is a well-known inducer of the atherosclerosis-related phenotype switch in VSMCs, leading to the occurrence of atherosclerosis. Accumulating studies have revealed that long non-coding RNAs (lncRNAs) mediate the effect of ox-LDL on the atherosclerosis-related biological activities of HA-VSMCs, including proliferation, migration, and apoptosis. However, the mechanism of small nucleolar RNA host gene 12 (SNHG12) in ox-LDL-induced phenotype switch of VSMCs remains unclear. Thus, this research dug in whether SNHG12 mediated the influence of ox-LDL on HA-VSMCs and the potential mechanism. METHODS: Fundamental experiments and functional assays were performed to measure the function of SNHG12 on HA-VSMCs. Then, mechanism assays and rescue assays were performed to study the regulatory mechanism of SNHG12 in HA-VSMCs. RESULTS: SNHG12 reversed the influence of ox-LDL treatment in enhancing cell proliferative and migratory abilities and weakening apoptotic ability in HA-VSMCs. SNHG12 was a competitive endogenous RNA (ceRNA) competing with sprouty RTK signaling antagonist 2 (SPRY2) to bind to miR-1301-3p, thus up-regulating SPRY2 expression in ox-LDL-treated HA-VSMCs. Besides, SNHG12 recruited serine and arginine rich splicing factor 1 (SRSF1) to stabilize negative regulator of ubiquitin like proteins 1 (NUB1) expression. CONCLUSIONS: This study illustrated that SNHG12 inhibited cell proliferation, migration and facilitated cell apoptosis in ox-LDL-induced HA-VSMCs by up-regulating SPRY2 and NUB1.
Asunto(s)
MicroARNs , Músculo Liso Vascular , ARN Largo no Codificante , Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Movimiento Celular , Proliferación Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lipoproteínas LDL , Proteínas de la Membrana , Miocitos del Músculo Liso , Regulación hacia ArribaRESUMEN
The proliferation, migration and dedifferentiation of vascular smooth muscle cells (VSMCs) exert crucial roles in atherosclerosis (AS) progression. The aim of our study was to explore the influences of circular RNA 0004872 (circ_0004872) in platelet-derived growth factor-BB (PDGF-BB)-induced AS cell model and investigate the underlying mechanisms. Real-time quantitative polymerase chain reaction (RT-qPCR) was implemented for the expression detection of circ_0004872, mitogen-activated protein kinase 1 (MAPK1) messenger RNA (mRNA), microRNA-513a-5p (miR-513a-5p) and thioredoxin interacting protein (TXNIP). Cell proliferation was analyzed via Cell Counting Kit 8 (CCK8) assay. Cell migration was assessed via wound healing assay and transwell migration assay. Western blot assay was used to measure the expression of alpha smooth muscle actin (α-SMA), osteopontin (OPN), calponin and TXNIP. Dual-luciferase reporter assay and RNA-pull down assay were used for confirmation of interaction between miR-513a-5p and circ_0004872 or TXNIP. Circ_0004872 expression was elevated in PDGF-BB-induced human aortic vascular smooth muscle cells (HA-VSMCs) and carotid plaque tissues. Circ_0004872 silencing alleviated PDGF-BB-induced proliferation, migration and dedifferentiation in HA-VSMCs. MiR-513a-5p bound to circ_0004872, and circ_0004872 knockdown-induced effects in PDGF-BB-treated HA-VSMCs were largely attenuated by the silencing of miR-513a-5p. MiR-513a-5p bound to the 3' untranslated region (3'UTR) of TXNIP, and miR-513a-5p overexpression-mediated effects were counteracted by the transfection of pcDNA-TXNIP in PDGF-BB-induced HA-VSMCs. TXNIP was modulated by circ_0004872/miR-513a-5p signaling cascade in HA-VSMCs. Circ_0004872 accelerated PDGF-BB-induced proliferation, migration and dedifferentiation in HA-VSMCs through enhancing TXNIP level via sponging miR-513a-5p.
Asunto(s)
Proteínas Portadoras , MicroARNs , Músculo Liso Vascular , Becaplermina/metabolismo , Becaplermina/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Movimiento Celular , Proliferación Celular , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismoRESUMEN
AIMS: Atherosclerosis (AS) is a common cardiovascular disease with complicated pathogenesis. Long non-coding RNAs (lncRNAs) have been reported to be associated with AS progression. We aimed to explore the role and underlying mechanism of HOXA transcript at the distal tip (HOTTIP) in AS. MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of HOTTIP, miR-490-3p and high mobility group B 1 (HMGB1) in AS patients' sera and oxidized low-density lipoprotein (ox-LDL) induced human aortic vascular smooth muscle cells (HA-VSMCs). Cell Counting Kit-8 (CCK-8) assay and transwell assay were conducted to evaluate the proliferation and migration of HA-VSMCs, respectively. Western blot assay was carried out to determine the levels of proliferating cell nuclear antigen (PCNA), matrix metalloprotein 2 (MMP2), MMP9 and HMGB1. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to verify the targeting association between HOTTIP and miR-490-3p, as well as miR-490-3p and HMGB1. KEY FINDINGS: HOTTIP and HMGB1 were upregulated and miR-490-3p was downregulated in the sera of AS patients and ox-LDL-stimulated HA-VSMCs. HOTTIP knockdown suppressed ox-LDL induced proliferation and migration in HA-VSMCs. MiR-490-3p was identified as a target of HOTTIP and HOTTIP overexpression abolished the inhibition on cell proliferation and migration mediated by miR-490-3p in ox-LDL-induced HA-VSMCs. Moreover, miR-490-3p inhibition promoted cell proliferation and migration by directly targeting HMGB1 in ox-LDL-induced HA-VSMCs. Besides, HOTTIP knockdown repressed the activation of PI3K-AKT signaling pathway. SIGNIFICANCE: HOTTIP knockdown suppressed cell proliferation and migration by regulating miR-490-3p/HMGB1 axis and PI3K-AKT pathway in ox-LDL-induced HA-VSMCs.