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Circ_CLASP2 Regulates High Glucose-Induced Dysfunction of Human Endothelial Cells Through Targeting miR-140-5p/FBXW7 Axis.

ABSTRACT: Hyperglycemia exposure results in the dysfunction of endothelial cells (ECs) and the development of diabetic complications. Circular RNAs (circRNAs) have been demonstrated to play critical roles in EC dysfunction. The current study aimed to explore the role and mechanism of circRNA CLIP-associating protein 2 (circ_CLASP2, hsa_circ_0064772) on HG-induced dysfunction in human umbilical vein endothelial cells (HUVECs). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the levels of circ_CLASP2, miR-140-5p and F-box, and WD repeat domain-containing 7 (FBXW7). The stability of circ_CLASP2 was identified by the actinomycin D and ribonuclease (RNase) R assays. Cell colony formation, proliferation, and apoptosis were measured by a standard colony formation assay, colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay, and flow cytometry, respectively. Western blot analysis was performed to determine the expression of related proteins. Targeted correlations among circ_CLASP2, miR-140-5p, and FBXW7 were confirmed by dual-luciferase reporter assay. High glucose (HG) exposure downregulated the expression of circ_CLASP2 in HUVECs. Circ_CLASP2 overexpression or miR-140-5p knockdown promoted proliferation and inhibited apoptosis of HUVECs under HG conditions. Circ_CLASP2 directly interacted with miR-140-5p via pairing to miR-140-5p. The regulation of circ_CLASP2 overexpression on HG-induced HUVEC dysfunction was mediated by miR-140-5p. Moreover, FBXW7 was a direct target of miR-140-5p, and miR-140-5p regulated HG-induced HUVEC dysfunction via FBXW7. Furthermore, circ_CLASP2 mediated FBXW7 expression through sponging miR-140-5p. Our current study suggested that the overexpression of circ_CLASP2 protected HUVEC from HG-induced dysfunction at least partly through the regulation of the miR-140-5p/FBXW7 axis, highlighting a novel therapeutic approach for the treatment of diabetic-associated vascular injury.

SUBMITTER: Zhang Q

PROVIDER: S-EPMC7990784 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background Endothelial dysfunction and deregulated microRNAs (miRNAs) participate in the development of sepsis and are associated with septic organ failure and death. Here, we explored the role of miR-15b-5p on inflammatory pathways in lipopolysaccharide (LPS)-treated human endothelial cells, HUVEC and TeloHAEC. Methods The miR-15b-5p levels were evaluated in LPS-stimulated HUVEC and TeloHAEC cells by quantitative real-time PCR (qRT–PCR). Functional experiments using cell counting kit-8 (CCK-8), transfection with antagomir, and enzyme-linked immunosorbent assays (ELISA) were conducted, along with investigation of pyroptosis, apoptosis, autophagy, and mitochondrial reactive oxygen species (ROS) by cytofluorometric analysis and verified by fluorescence microscopy. Sirtuin 4 (SIRT4) levels were detected by ELISA and immunoblotting, while proprotein convertase subtilisin-kexin type 9 (PCSK9) expression was determined by flow cytometry (FACS) and immunofluorescence analyses. Dual-luciferase reporter evaluation was performed to confirm the miR-15b-5p–SIRT4 interaction. Results The results showed a correlation among miR-15b-5p, PCSK9, and SIRT4 levels in septic HUVEC and TeloHAEC. Inhibition of miR-15b-5p upregulated SIRT4 content, alleviated sepsis-related inflammatory pathways, attenuated mitochondrial stress, and prevented apoptosis, pyroptosis, and autophagic mechanisms. Finally, a PCSK9 inhibitor (i-PCSK9) was used to analyze the involvement of PCSK9 in septic endothelial injury. i-PCSK9 treatment increased SIRT4 protein levels, opposed the septic inflammatory cascade leading to pyroptosis and autophagy, and strengthened the protective role of miR-15b-5p inhibition. Increased luciferase signal validated the miR-15b-5p–SIRT4 binding. Conclusions Our in vitro findings suggested the miR-15b-5p–SIRT4 axis as a suitable target for LPS-induced inflammatory pathways occurring in sepsis, and provide additional knowledge on the beneficial effect of i-PCSK9 in preventing vascular damage by targeting SIRT4. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s11658-023-00482-5.

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_CLASP2 Regulates High Glucose-Induced Dysfunction of Human Endothelial Cells Through Targeting miR-140-5p/FBXW7 Axis.

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