The mir-21 Inhibition Enhanced HUVEC Cellular Viability during Hypoxia-Reoxygenation Injury by Regulating PDCD4

Abstract

The purpose of this study was to explore the clinical value of altered plasma mir-21 expression level as a biomarker for the severity of coronary artery disease (CAD) and its molecular impact on HUVEC cellular injuries. Angiographically validated 56 patients with single-vessel CAD disease, 92 patients with double-vessel CAD, 139 complex coronary artery stenosis patients, and 56 healthy individuals (n = 343) were enrolled in this study. The expressions of plasma mir-21 were evidently and progressively higher while PDCD4 levels were significantly and steadily lower in single-, dual-, and multivessel occluded CAD patients than in healthy participants (P < 0.001). The relative expressions of mir-21 in hypoxia-reoxygenation- (HR-) exposed HUVECs were markedly upregulated, but PDCD4 concentrations were obviously downregulated as compared with normal control cells (P < 0.001). Moreover, altered circulatory mir-21 expression levels were able to significantly differentiate single- (AUC 0.893), double- (AUC 0.914), and multivessel stenosis CAD (AUC 0.933) patients from healthy subjects. Besides, the plasma mir-21 expressions in elderly (66-85 years) groups were remarkably higher than those in younger aged (25-45 years) subjects. Caspase-3 and ROS expression levels were remarkably elevated, but cellular viability noticeably declined in HR-induced HUVECs than in normoxic cells (P < 0.001). In contrast, mir-21 inhibition markedly reduced caspase-3 activity and ROS concentrations while significantly ameliorating HUVEC cellular viability in HR conditions. PDCD4 expressions in HR-exposed HUVECs were prominently decreased whereas mir-21 inhibition significantly enhanced PDCD4 levels (P < 0.001). Upregulated plasma mir-21 can be a valuable clinical biomarker for the detection of the severity of coronary artery stenosis patients. Elevated circulatory mir-21 concentrations have a positive correlation with aging. Inhibitory mir-21 evidently increased HUVEC cellular viability through upregulation of targeting PDCD4 and recommended a newer possible therapeutic molecule for the management of CAD patients.

Figure 1

The expression patterns of mir-21 and PDCD4. (a) Circulating mir-21 concentrations in healthy participants versus single-, double-, and multivessel lesion CAD subjects. (b) Expression levels of mir-21 among hypoxic reoxygenation (HR) and normal control HUVECs. (c) The PDCD4 expression patterns between healthy volunteers and disease groups. (d) Comparison of PDCD4 expressions in HR-exposed and normoxic-cultured HUVECs. ^∗∗P < 0.001.

Figure 2

ROC curve analysis was used for categorizing the severity of CAD patients: (a) healthy controls and single-vessel lesion CAD (AUC 0.893); (b) dual-vessel stenosis CAD with healthy subjects (AUC 0.914); (c) healthy volunteers and multivessel blocked CAD patients (AUC 0.933).

Figure 3

Expression patterns of mir-21 in HUVECs and luciferase analysis to determine the target: (a) the mir-21 expression levels in healthy and HR groups; (b) reporter gene assay luciferase expression levels in normoxic, HR, HR with transfected mir-21, and NC groups in HUVECs standardized by Renilla reniformis. ^∗∗P < 0.001.

Figure 4

Effects of mir-21 inhibition on HUVECs: (a) the relative expression levels of PDCD4 in normoxic and HR cells; (b) intracellular ROS generations in normal HUVEC versus HR-injured cells; (c) caspase-3 expression between normal culture and HR-incubated cells; (d) cellular viability in HR and normal, HR and HR transfected with mir-21 inhibition, and HR and NC groups in HUVECs. ^∗∗P < 0.001.

Figure 5

The relative expression of circulatory mir-21 in different aged subjects and both male and female genders: (a) the expression patterns of plasma mir-21 among healthy groups; (b) plasma mir-21 concentrations in single-vessel CAD subjects; (c) the expression of mir-21 levels in double-vessel CAD patients; (d) multivessel CAD patients' mir-21 levels; (e) healthy male and female groups' mir-21 expressions; (f) the levels of plasma mir-21 in male versus female single-vessel CAD groups; (g) the concentrations of circulatory mir-21 among male and female double-vessel stenosis CAD subjects; (h) the mir-21 levels between male and female multivessel blocked CAD patients.