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Circ_USP36/miR-182-5p/KLF5 axis regulates the ox-LDL-induced injury in human umbilical vein smooth muscle cells

ABSTRACT: Atherogenesis is a chronic inflammatory process, closely related to high morbidity and mortality. Circular RNAs (circRNAs) were reported to function in atherosclerosis. However, the functional impact of circRNA ubiquitin-specific Protease 36 (circ_USP36) on atherosclerosis and the possible mechanism are still unclear. Serum specimens were collected from atherosclerosis patients and healthy volunteers. Human umbilical vein smooth muscle cells (HUVSMCs) exposed with 25 ?g/mL oxidized low-density lipoprotein (ox-LDL) were utilized to simulate atherosclerosis. Expression of circ_USP36, microRNA (miR)-182-5p and Kruppel-like factor 5 (KLF5) was determined via quantitative real-time polymerase chain reaction or western blot assay. Cell viability and apoptosis were evaluated by Cell Counting Kit-8 and flow cytometry. Cell metastasis, including migration and invasion, was assessed via Transwell assay. Biomarker protein was analyzed by western blot. The relationship among circ_USP36, miR-182-5p and KLF5 was confirmed by dual-luciferase reporter and RNA pull-down assays. Circ_USP36 and KLF5 were up-regulated, while miR-182-5p was down-regulated in atherosclerosis patients and ox-LDL-induced HUVSMCs. Circ_USP36 knockdown inhibited proliferation and metastasis of ox-LDL-induced HUVSMCs by up-regulating miR-182-5p. MiR-182-5p targeted KLF5, and ameliorated ox-LDL-mediated injury of HUVSMCs. Circ_USP36 knockdown down-regulated KLF5 expression by sponging miR-182-5p. Knockdown of circ_USP36 alleviated ox-LDL-mediated injury of HUVSMCs by modulating miR-182-5p/KLF5 axis, potentially providing a treatment target for atherosclerosis.

SUBMITTER: Zhao Q

PROVIDER: S-EPMC7791491 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:Atherosclerosis (AS) remains the leading cause of mortality throughout the world, and vascular endothelial cell dysfunction is one of the key events leading to this pathology. In recent years, there has been an increased interest in the role of circulating RNAs in various diseases; these noncoding RNAs can regulate gene products by acting as microRNA (miR) sponges. Furthermore, it has been shown that foam cells exhibit high expression levels of hsa_circ_0005699 (circ_0005699); however, to the best of our knowledge, no previous study has investigated the role of circ_0005699 in the regulation of vascular endothelial function. The present study employed human umbilical vein endothelial cells (HUVECs), which have been widely used to study vascular endothelial cell function. In addition, apolipoprotein E (ApoE)-deficient mice were used, which have been shown to rapidly develop AS and are widely used as a model of this disease. Cellular and biochemical techniques were performed, including gene transfection and short hairpin RNA-mediated gene silencing for cell transfection, luciferase reporter gene assay to confirm predicted genes, Cell Counting Kit-8 assay and flow cytometry to assess cell viability and apoptosis, and reverse transcription-quantitative PCR and western blotting for detection of mRNA and protein expression. In the present study, the expression levels of circ_0005699 were increased by oxidized low-density lipoprotein in a time- and dose-dependent manner in HUVECs; this was also associated with increased apoptosis of these cells. In addition, the expression levels of circ_0005699 were elevated, along with increased levels of inflammatory cytokines, in ApoE-deficient mice. An RNA pull-down assay indicated that circ_0005699 can bind miR-450b-5p to decrease its expression, whereas silencing of circ_0005699 resulted in increased expression of miR-450b-5p. In addition, the online bioinformatics tool starBase predicted NFKB1 as a target gene of miR-450b-5p, which was further confirmed by the luciferase reporter gene assay. Notably, knockdown of circ_0005699 resulted in the increased survival of HUVECs, which was associated with decreased protein expression levels of NFKB1 and inflammatory cytokines. By contrast, the effects of circ-0005699 silencing on survival were reversed by miR-450b-5p inhibition or NFKB1 overexpression. In conclusion, knockdown of circ_0005699 may ameliorate endothelial cell injury through regulation of the miR-450b-5P/NFKB1 signaling axis.

Project description:Background: Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of atherosclerosis. LncRNA OIP5 antisense RNA 1 (OIP5-AS1) has been found to be associated with the development of atherosclerosis. In this study, we further investigated the molecular basis of OIP5-AS1 in atherosclerosis pathogenesis. Methods: Oxidative low-density lipoprotein (ox-LDL) was used to treat human umbilical vein endothelial cells (HUVECs). The levels of OIP5-AS1, miR-135a-5p, and Krüppel-like factor 5 (KLF5) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Cell viability, migration, and apoptosis were evaluated using the Cell Counting Kit-8 (CCK-8), Transwell, and flow cytometry, respectively. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and malondialdehyde (MDA) were determined with enzyme-linked immunosorbent assay (ELISA). Targeted interactions among OIP5-AS1, miR-135a-5p, and KLF5 were confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Animal studies were performed to assess the role of OIP5-AS1 in atherosclerosis progression in vivo. Results: Our data showed the significant upregulation of OIP5-AS1 in atherosclerosis serum and ox-LDL-stimulated HUVECs. The silencing of OIP5-AS1 protected against ox-LDL-triggered cytotoxicity in HUVECs and diminished lipids secretion in ApoE-/- mice. Moreover, OIP5-AS1 functioned as a molecular sponge of miR-135a-5p, and miR-135a-5p was a functional mediator of OIP5-AS1 in regulating ox-LDL-induced HUVEC injury. KLF5 was a direct target of miR-135a-5p, and the increased expression of miR-135a-5p alleviated ox-LDL-induced cytotoxicity by downregulating KLF5. Furthermore, OIP5-AS1 influenced KLF5 expression through sponging miR-135a-5p. Conclusion: The current work identified that the silencing of OIP5-AS1 protected against ox-LDL-triggered cytotoxicity in HUVECs at least in part by influencing KLF5 expression via acting as a miR-135a-5p sponge.

Project description:Recent findings have revealed that aberrant miR-125a-5p expression is involved in the development of atherosclerosis. The present study aimed to investigate the precise mechanism of microRNA (miR)-125a-5p in atherosclerosis. Human vascular smooth muscle cells (HVSMCs) were treated with 20 µg/ml oxidized low-density lipoprotein (ox-LDL) for 24 h and were employed as in vitro models of atherosclerosis. Reverse transcription quantitative (RT-qPCR) assays were used to detect miR-125a-5p levels. Immunofluorescence analysis was conducted to assess α-smooth muscle actin (α-SMA) expression. Western blotting and RT-qPCR assays were performed to measure the expression levels of NACHT, LRR and PYD domains-containing protein 3 (NLRP3), apoptosis associated speck-like protein (ASC), caspase-1, active interleukin (IL)-1β and C-C motif chemokine 4-like (CCL4). Furthermore, the association between miR-125a-5p and CCL4 was assessed using a double luciferase analysis. In addition, VSMCs were transfected with miR-125a-5p mimics (30 nM), miR-125a-5p inhibitor (100 nM) or small interfering RNA against CCL4 (si-CCL4, 50 pM), respectively to further investigate the function of miR-125a-5p in ox-LDL-treated HVSMCs. The present study found that the expression levels of miR-125a-5p were significantly downregulated in HVSMCs, whereas the expression levels of α-SMA, NLRP3, ASC, caspase-1, IL-1β and CCL4 were markedly upregulated following ox-LDL treatment. Overexpression of miR-125a-5p in the absence of ox-LDL treatment decreased NLRP3, IL-1β and CCL4 expression, whereas inhibition of miR-125a-5p exhibited the opposite effects. The results of double luciferase analysis confirmed that CCL4 was a direct target of miR-125a-5p. Moreover, transfection of si-CCL4 into HVSMCs significantly decreased the ox-LDL-induced expression of NLRP3, ASC, caspase-1 and IL-1β proteins. Taken collectively, the results of the present study suggested that miR-125a-5p could negatively regulate the NLRP3 inflammasome by targeting CCL4 in ox-LDL-treated HVSMCs. The data provide new insight to the inhibition of atherosclerosis progression.

Project description:BackgroundKawasaki disease (KD) is an autoimmune disease with systemic vasculitis as the main pathological change, and is most common in children under 5. The role of long non-coding RNAs (lncRNAs) in human diseases has been highlighted. LncRNA Slco4a1 was reported to promote cell growth and act as an oncogenic regulator in cancer. However, the role of lncRNA Slco4a1 in KD remains unclear. This study aimed to investigate the role and mechanism of lncRNA Slco4a1 in KD.MethodsEnzyme linked immunosorbent assay (ELISA), qRT-PCR, Western blot, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining were conducted to explore the function of lncRNA Slco4a1. The interaction between POU5F1 and miR-335-5p was analyzed by the RIP assay and dual luciferase assay.ResultsLncRNA Slco4a1 was significantly upregulated in the serum of KD patients compared with healthy controls. LncRNA Slco4a1 was upregulated in human umbilical vein endothelial cells (HUVECs) stimulated with KD serum. LncRNA Slco4a1 overexpression could promote the expression of inflammatory factors and apoptosis in HUVECs. The number of inflammatory cells and the infiltration area of the coronary artery in KD rats were decreased after lncRNA Slco4a1 silencing. Furthermore, lncRNA Slco4a1 is a sponge of miR-335-5p and negatively regulated the expression of miR-335-5p. POU5F1 was the downstream target of miR-335-5p, and miR-335-5p overexpression could upregulate the expression of POU5F1. Additionally, miR-335-5p overexpression could inhibit the expression of inflammatory factors and apoptosis in HUVECs. We further investigated the effect of lncRNA Slco4a1 on the mitogen-activated protein kinase (MAPK) signaling pathway, and the results showed that lncRNA Slco4a1 could promote the activation of the MAPK signaling pathway.ConclusionsTogether, these results indicated that lncRNA Slco4a1 could regulate the progression of HUVECs in KD by targeting the miR-335-5p/POU5F1 axis, providing new insights for KD treatment.

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Circ_USP36/miR-182-5p/KLF5 axis regulates the ox-LDL-induced injury in human umbilical vein smooth muscle cells

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