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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: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.

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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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