Protective Effects of MicroRNA-126 on Human Cardiac Microvascular Endothelial Cells Against Hypoxia/Reoxygenation-Induced Injury and Inflammatory Response by Activating PI3K/Akt/eNOS Signaling Pathway

Abstract

Objective: This study explored the protective effects of the microRNA-126 (miR-126)-mediated PI3K/Akt/eNOS signaling pathway on human cardiac microvascular endothelial cells (HCMECs) against hypoxia/reoxygenation (H/R)-induced injury and the inflammatory response. Methods: Untreated HCMECs were selected for the control group. After H/R treatment and cell transfection, the HCMECs were assigned to the H/R, miR-126 mimic, mimic-negative control (NC), miR-126 inhibitor, inhibitor-NC, wortmannin (an inhibitor of PI3K) and miR-126 mimic + wortmannin groups. Super oxide dismutase (SOD), nitric oxide (NO), vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) were measured utilizing commercial kits. Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were performed to detect miR-126 expression and the mRNA and protein expression of inflammatory factors. Western blotting was used to determine the expression of key members in the PI3K/Akt/eNOS signaling pathway. ACCK-8 assay and flow cytometry were employed to examine cell proliferation and apoptosis, respectively. The angiogenic ability in each group was detected by the lumen formation test. Results: Compared to the control group, p/t-PI3K, p/t-Akt and p/t-eNOS expression, NO, VEGF and SOD levels, cell proliferation and in vitro lumen formation ability were decreased, while the ROS content, interleukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-α expression and cell apoptosis were significantly increased in the H/R, mimic-NC, miR-126 inhibitor, inhibitor-NC, wortmannin and miR-126 mimic + wortmannin groups. Additionally, in comparison with the H/R group, the miR-126 mimic group had elevated p/t-PI3K, p/t-Akt and p/t-eNOS expression, increased NO, VEGF and SOD contents, and strengthened cell proliferation and lumen formation abilities but also exhibited decreased ROS content, reduced IL-6, IL-10 and TNF-α expressions, and weakened cell apoptosis, while the miR-126 inhibitor and wortmannin group exhibited the opposite results. Furthermore, decreased p/t-PI3K, p/t-Akt and p/t-eNOS expressions, decreased NO, VEGF and SOD contents, cell proliferation and lumen formation abilities, as well as increased ROS content, increased IL-6, IL-10 and TNF-α expression, and increased cell apoptosis were observed in the miR-126 mimic + wortmannin group compared to themiR-126 mimic group. Conclusions: These findings indicated that miR-126 protects HCMECs from H/R-induced injury and inflammatory response by activating the PI3K/Akt/ eNOS signaling pathway.

Low-Intensity Pulsed Ultrasound Prevents the Oxidative Stress Induced Endothelial-Mesenchymal Transition in Human Aortic Endothelial Cells

“…It is known that hydrogen peroxide (H 2 O 2 ) can induce EndMT [12], which may contribute to vascular diseases and endothelial cell dysfunction. In addition, accumulation of reactive oxygen species (ROS) can directly damage endothelial cells and indirectly modulate endothelial cell function via altering expression and function of the related genes [13]. EndMT is a cellular process where endothelial cells undergo significant morphological changes with loss of their endothelial characteristics and gain of mesenchymal properties that ultimately leads to transformation into stromal fibroblasts [14].…”

miR-126 in Peripheral Blood Mononuclear Cells Negatively Correlates with Risk and Severity and is Associated with Inflammatory Cytokines as well as Intercellular Adhesion Molecule-1 in Patients with Coronary Artery Disease

“…The anti-inflammatory effect of miR-126 was highlighted by another study, which showed that miR-126 protects human cardiac microvascular endothelial cells (HCMECs) against the H/R-induced injury and inflammatory response via targeting the PI3K/Akt/eNOS signaling pathway. In addition, a decrease in VEGF in the miR-126 inhibitor group and an increase in VEGF in the miR-216 mimic group were found in their study, suggesting the miR-126 expression might be negatively correlated with the VEGF level in HCMECs [39]. However, a previous study revealed that the level of miR-126 is decreased under hypoxic conditions and subsequently reduces the expression of VEGF both in vitro and in vivo [40].…”

YAP Inhibits the Apoptosis and Migration of Human Rectal Cancer Cells via Suppression of JNK-Drp1-Mitochondrial Fission-HtrA2/Omi Pathways

“…The mitochondrial transmembrane potential was analyzed using a JC-1 Kit (Beyotime, China). Images were captured using a fluorescence microscope (OLYMPUS DX51; Olympus, Tokyo, Japan) and were analyzed with Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD) to obtain the mean densities of the regions of interest, which were normalized to those of the control group [38]. …”

“…Yap has 504 amino acids (aa), including an N-terminal TEAD-binding domain (TBD, 48-102 aa), a WW domains (WW, 171-263 aa) and a C-terminal transcriptional activation domain (TAD, 292-488 aa) [38]. Therefore, three types of YAP mutants were constructed via base deletion to generate mutant-Yap (102-504 aa), which lacks the TBD, mutant-Yap (263-504 aa), which lacks the TBD and WW, and mutant-Yap (1-263 aa) , which lacks the TAD according to a previous study [8].…”