Atherosclerosis (AS) is a common vascular disease, which can cause apoptosis of vascular endothelial cells. Notoginsenoside R1 (NGR1) is considered an anti-AS drug. MicroRNAs (miRNAs) are believed to play a vital role in cell apoptosis and angiogenesis. This study aimed to explore the mechanism of NGR1 for treating AS through miRNAs. Flow cytometry was used to detect the apoptosis rate. The levels of inflammatory cytokines interleukin (IL)-6 and IL-1b were detected using ELISA. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels were measured using corresponding assay kits. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to detect miR-221-3p expression. Dualluciferase reporter and RNA immunoprecipitation assays were carried out to examine the relationship between miR-221-3p and toll-like receptors 4 (TLR4). Also, western blot analysis was performed to determine the levels of TLR4 and nuclear factor kappa B (NF-kB) signaling pathway-related proteins. Oxidized low-density lipoprotein (ox-LDL) induced human umbilical vein endothelial cells (HUVECs) apoptosis, inflammation, and oxidative stress. NGR1 alleviated the negative effect of ox-LDL through promoting the expression of miR-221-3p in HUVECs. TLR4 was a target of miR-221-3p, and its overexpression could reverse the inhibition effects of miR-221-3p on apoptosis, inflammation, and oxidative stress. NGR1 improved miR-221-3p expression to inhibit the activation of the TLR4/NF-kB pathway in ox-LDL-treated HUVECs. NGR1 decreased ox-LDL-induced HUVECs apoptosis, inflammation, and oxidative stress through increasing miR-221-3p expression, thereby inhibiting the activation of the TLR4/NF-kB pathway. This study of the mechanism of NGR1 provided a more theoretical basis for the treatment of AS.
The hypoxia‐reoxygenation (H/R) model helps analyze myocardial infarction triggered by acute myocardial ischemia, which induces cardiomyocyte proliferation and apoptosis. The Gene Expression Omnibus database was used to obtain the GSE74205 and GSE3866 microarray data, including microRNA (miRNA) and messenger RNA profiles, to catalog potential key miRNAs and genes. The role of rno‐mir‐496 expression in cardiomyocyte proliferation within 10 days of birth was established. The microRNA Target Prediction Database (miRDB) database—via Gene Ontology annotation—predicted hook microtubule tethering protein 3 (Hook3), a key target gene of rno‐mir‐496, was closely related to cell proliferation. Upregulation of miR‐496 related to a significant reduction in apoptosis of H9c2 and human cardiomyocytes treatment with H/R. Moreover, transfection of H9c2 cells with miR‐496 mimics, which were pretreated with H/R for 12 hours, increased Ki67 levels, proliferating cell nuclear antigen and Bcl‐2 proteins; and decreased cleaved caspase‐3 and Bax protein levels, as determined by reverse transcription‐polymerase chain reaction and Western blot assays. A dual‐luciferase reporter system confirmed that miR‐496 targets the Hook3 suppressor. Hook3 overexpression stimulated apoptosis in H/R‐treated cells, thus reducing cell proliferation. Upregulated miR‐496 activated phosphatidylinositol‐3‐kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling, while Hook3 exhibited the inverse trend in H/R‐treated H9c2 cells. In summary, with Hook3 functionality's aid, miR‐496 upregulation defends cells from H/R‐induced apoptosis and stimulates cell proliferation. miR‐496 targets Hook3 to trigger the PI3K/Akt/mTOR signaling pathway for antiapoptotic and proliferative effects.
Our objective was to explore the effects of miR-92a and miR-126 on myocardial apoptosis in mouse ischemia-reperfusion model and further investigate the underlying mechanisms. Eighteen Kunming mice were selected and randomly divided into sham operation group and ischemia-reperfusion group with nine mice in each group. Cardiac muscle tissue was stained with Evans blue to confirm myocardial infarction and ischemia. Annexin V/PI double staining was used to detect the apoptotic rate of myocardial cells, and terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) was used to detect the number of apoptotic cells; Western blot was used to detect expression of Caspase 3 to evaluate the apoptosis of mouse myocardial cells; qRT-PCR was used to detect expression of miR-92a and miR-126 in mouse myocardium, and Western blot was used to detect expression of HSP70 in two groups. Evans blue staining results showed that there was a large area of ischemia in myocardium of ischemia-reperfusion mice with marked infarction, suggesting successful establishment of the model. In sham operation group, myocardial cells were mostly normal cells. Annexin V/PI double staining of flow cytometry result showed that the apoptotic rate was 5.9 % in sham operation group and 37.0 % in ischemia-reperfusion group, respectively. Apoptosis detection results showed that apoptotic index (AI) of myocardial cells in ischemia-reperfusion mice was significantly higher than in sham operation group. In addition, qRT-PCR results showed that miR-92a expression in ischemia-reperfusion group was significantly higher than in sham operation group (F = 32.302, P = 0.000), and miR-126 expression in ischemia-reperfusion group was significantly lower than in sham operation group (F = 41.125, P = 0.000). Moreover, HSP70 detected by Western blot showed that HSP expression in ischemia-reperfusion group was significantly lower than in sham operation group. The change of miR-92a was in accordance with AI of myocardial cells. However, the change of miR-126 is in contrary with AI of myocardial cells, which may be related to the HSP70 expression in myocardial cells.
Studies have shown that long non-coding RNAs (lncRNA) play critical roles in coronary atherosclerotic heart disease (CAD). However, the function of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in CAD is unclear. In this study, we aimed to investigate the functions of lncRNA NEAT1 in CAD. RT-PCR and western blot analysis were carried out to examine the expressions of related RNAs. Colony formation assay, cell proliferation assay, apoptosis assay, and dual-luciferase reporter assay were conducted to investigate the abilities of colony migration, cell proliferation, apoptosis, and targeting. The results showed that NEAT1 was up-regulated in CAD blood samples and in human coronary endothelial cells (HCAECs). Transfection of pcNEAT1 significantly inhibited the survival rate of HCAECs and induced apoptosis of HCAECs. MiR-140-3p was down-regulated in HCAECs. NEAT1 directly targeted miR-140-3p, and the expression of miR-140-3p was inversely correlated with the expression of NEAT1 in CAD patients. In addition, co-transfection of NEAT1 with miR-140-3p mimic reversed the effect of pcNEAT1 on cell viability and apoptosis. mitogen-activated protein kinase 1 (MAPK1) was proved to be a target gene of miR-140-3p, and the miR-140-3p mimic was shown to reduce the expression of MAPK1 in HCAECs. pcNEAT1 significantly increased the expression level of MAPK1, while shNEAT1 significantly reduced the expression level of MAPK1. Our results revealed that lncRNA NEAT1 increased cell viability and inhibited CAD cell apoptosis possibly by activating the miR-140-3p/MAPK1 pathway, and lncRNA NEAT1 might serve as a potential therapeutic target for CAD.
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