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OBJECTIVE@#To explore the expression of microRNA-132 (miR-132) and its potential role in the development of atherosclerosis (AS).@*METHODS@#Thirty AS samples and 30 samples of normal peripheral vessels were collected from atherosclerotic patients undergoing peripheral angiostomy in our hospital for detecting the expression level of miR-132 using RT-qPCR. The expression of miR-132 in human umbilical vein endothelial cells (HUVEC) was up-regulated by liposome transfection, and intracellular reactive oxygen species (ROS), localization relationship between ROS and mitochondria, functional changes of mitochondrial reactive oxygen superoxide species (mtROS), mitochondrial membrane potential (MMP) and opening of mitochondrial permeability transition pore (mPTP) were analyzed by flow cytometry and laser confocal microscopy. The activity of mitochondrial redox respiratory chain complex (type I, II, III, IV and V) in HUVECs was detected using ELISA, and the expression levels of key iron death proteins were detected with Western blotting.@*RESULTS@#RT-qPCR results showed that miR-132 was significantly up-regulated in atherosclerotic plaques compared with normal vascular samples (P < 0.001). Compared with control HUVECs, HUVECs overexpressing miR-132 showed a significantly increased level of intracellular ROS (P < 0.001), and most of ROS was colocalized with mitochondria. HUVECs overexpressing miR-132 also showed significantly decreased MMP (P < 0.001) and obviously increased mtROS (P < 0.001) and opening of mPTP (P < 0.001), which led to mitochondrial REDOX respiratory chain stress disorder. The key iron death protein GPX4 was significantly down-regulated and the oxidized protein NOX4 was significantly increased in miR-132-overexpressing HUVECs (P < 0.001).@*CONCLUSION@#MiR-132 promotes atherosclerosis by inducing mitochondrial oxidative stress-mediated ferroptosis, which may serve as a promising therapeutic target for AS.
Subject(s)
Humans , Apoptosis , Atherosclerosis/genetics , Ferroptosis , Human Umbilical Vein Endothelial Cells/metabolism , Membrane Potential, Mitochondrial , MicroRNAs/metabolism , Mitochondria/metabolism , Oxidation-Reduction , Oxidative Stress , Reactive Oxygen Species/metabolismABSTRACT
AIM:To investigate the role of microRNA-132 (miR-132) on alveolar macrophage inflammation. METHODS: Rat alveolar macrophage cell line NR8383 was transfected with miR-132 mimic, mimic negative control ( NC) , miR-132 inhibitor, or inhibitor NC.The cells were divided into transfection group, transfection +lipopolysaccha-ride ( LPS) group, and transfection +LPS +acetylcholine ( ACh) group.The mRNA expression of acetylcholinesterase ( AChE) was detected by real-time PCR.The protein levels of AChE, signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3 (p-STAT3) in the cells, and nuclear factor-κB (NF-κB) in the cytoplasm and nu-cleus were analyzed by Western blot.The activity of AChE in the culture supernatant was measured by AChE activity assay kit.The nuclear translocation of NF-κB was detected by immunofluorescence assay.RESULTS: Up-regulation or down-regulation of miR-132 had no effect on the mRNA expression of AChE.However, up-regulation of miR-132 decreased the protein level of AChE compared with mimic NC group (P<0.05).Transfection with miR-132 inhibitor increased the pro-tein expression of AChE compared with inhibitor NC group ( P<0.05 ) .In the alveolar macrophages treated with LPS+ACh, the inhibition of nuclear translocation of NF-κB p65 in miR-132 mimic group was more effective than that in mimic NC group ( P<0.05) .The inhibitory effect in miR-132 inhibitor group was weaker than that in inhibitor NC group ( P<0.05 ) .The inhibitory effect of miR-132 mimic on the protein levels of STAT3 and p-STAT3 was stronger than that of mimic NC (P<0.05).CONCLUSION:miR-132 in LPS-stimulated alveolar macrophages reinforced ACh-mediated anti-inflam-matory reaction by targeting AChE to suppress ACh hydrolyzation, which was related to the suppression of NF-κB and STAT3 activation.
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AIM:To evaluate the regulatory effect of microRNA-132 ( miR - 132 ) in human umbilical vein endothelial cell ( HUVEC) . ●METHODS: ln vitro cultured human umbilical vein endothelia cells in hypoxic environment for 6h, then maintained under normal oxygen condition for 3h, 6h, 12h, 24h. miR-132 and peroxisome-proliferator-activated receptor-γ coactivator-1α ( PGC-1α) expression was detected by quantitative Real - time polymerase chain reaction and Western blot analysis. Human umbilical vein endothelial cells transfected miR-132 mimic and miR-132 inhibitor( anti-miR-132 ) were measured by quantitative Real-time polymerase chain reaction and Western blot. ●RESULTS: miR - 132 and PGC - 1α expression was significantly ( P ●CONCLUSION:miR-132 level is highly expressed in the HUVEC under hypoxia and may be an effect of regulation for PGC-1α.
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[ ABSTRACT] AIM:To investigate the effect of microRNA-132 ( miR-132 ) transfection on the lipopolysaccharide ( LPS)-induced inflammation in rat alveolar macrophages.METHODS:The rat alveolar macrophage NR8383 cultured with-out pyrogen in vitro were divided into blank control group, negative control group and transfected group.The cells in the 3 groups were transfected with phosphate buffer solution ( PBS) , Lipofectamine 2000 and synthesized miR-132 mimic respec-tively.The cell proliferation was detected by Cell Counting Kit-8 ( CCK-8) assay.Real-time PCR was used to detect the ex-pression of miR-132 in the cells.After NR8383 cells were stimulated with LPS for 6 h, the NF-κB DNA-binding activity was measured by electrophoretic mobility shift assay ( EMSA) .The expression of tumor necrosis factor-α( TNF-α) and interleu-kin-6 (IL-6) in NR8383 cells was assayed by Western blotting.RESULTS: After transfection, the expression of miR-132 was significantly higher than that in blank control group and negative control group.The growth of NR8383 cells in transfect-ed group was significantly inhibited compared with blank control group and negative control group ( P<0.05 ) .After the cells were stimulated with LPS, the productions of NF-κB, TNF-αand IL-6 in transfected NR8383 cells were decreased com-pared with blank control group and negative control group (P<0.05).CONCLUSION:Transfection of alveolar macropha-ges with miR-132 significantly suppresses the cell growth, and inhibits inflammatory responses induced by LPS.
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ObjectiveTo investigate the effects of sleep deprivation on expressions of Mir-132,mir-134 in the different regions of rat brain.MethodsAll the male SD rats were divided into control group ( normal sleep group),sleep deprivation (SD).The modified multiple platform method (MMPM) was used to establish sleep deprivation model.Mir-132,mir-134 level was detected by real time PCR.ResultsMir-132 were significantly increased in SD groups in hippocampus compared with the control groups ( 51.87 ± 8.13 vs 67.25 ± 7.59 ) (P ＜0.01 ).Mir-134 were significantly decreased in SD groups compared with the control groups( 1.82 ±0.15 vs 1.45± 0.12 )(P ＜ 0.01 ).There were no statistically significant differences in cortex and thalamus (P ＞ 0.05 ).Cortex mir-132 level in SD group and control group was 1.57 ±0.10,1.48 ±0.11 respectively,and it was 1.37 ±0.09,1.36 ±0.11 in thalamus;Cortex mir-134 level in SD group and control group was 98.26 ± 5.17,100.80 ±4.15respectively,and it was 97.56 ± 6.28,91.01 ± 4.07 in thalamus.ConclusionThe upregulation of mir-132 and downregulation of mir-134 implies that two miRNAs did opposite actions in the processes of sleep deprivation.This findings indicate that hippocampus mir-132,mir-134 levels in the SD rat may reflect associated depressive patho-physiological processes.