Regulatory Role Of MicroRNAs In Endothelial Senescence And Vascular Function

Background and AimsAging is an independent risk factor for cardiovascular diseases, such as coronary artery, hypertension, stroke. With the increase of age, there are many crucial age-associated functional and structural changes, among which endothelial dysfunction has been demonstrated to be an early step in the development of atherosclerosis. However, it remains largely unknown how vascular cells, particularly endothelial cells, appeared senescent-like phenotype and how the senescence impairs the vascular functions and contributes to the age-related vascular diseases over time.MicroRNAs (MiRNAs), small noncoding RNAs, play an important role in regulating endothelial senescence. It is reported that miR-216a/miR-181b are upregulated in senescent endothelial cells. But the effect of miR-216a/miR-181b in the progress of endothelial senescence and the underlying mechanisms are still unclear.Methods1. Primary human umbilical vein endothelial cells (HUVECs) were cultured. PDL44 was identified as senescent HUVECs. To compare the expression of miR-216a/ miR-181b in PDL8 and PDL44 HUVECs, miR-216a/miR-181b were detected by real-time PCR.2. HUVECs were infected with has-miR-216a recombinant lentiviruses. To identify HUVECs senescent status, senescence-associated beta-galactosidase (SA-β-Gal) activity, cyclins (p53, p21), telomere length, telomerase activity were detected.3. The abilities of cell proliferation, migration and adhesion were assessed by MTS test, scraching test, and cell adhesion test, respectively.4. To investigate the mechanism of miR-216a/miR-181b in regulating endothial function, miR-216a/miR-181b mimics were transfected into PDL8 HUVECs to detect the expression change of target protein. The dual luciferase reporter assay was used to analyze whether miRNAs can interact with target protein directly.Results(一) Regulatory role of miR-216a in vascular endothelial senescence and function1. Compared with PDL8 HUVECs, the percentage of SA-β-gal-positive cells was increased in PDL44 HUVECs by 4.7-folds (P<0.001). The expression of p53 and p21 was increased in PDL44 HUVECs by 69%(P=0.003) and 61%(P=0.01), respectively. And telomere length was shorter in PDL44 HUVECs by 42%(P=0.01). Compared with PDL8 HUVECs, the expression of miR-216a was increased in PDL44 HUVECs by 59%(P=0.028).2. Compared with negative control, the stable miR-216a lentiviral transfection cells appeared significantly morphological changes and growth arrest when they were passaged to PDL20. The percentage of SA-β-gal-positive cells was increased in PDL20 HUVECs by 61%(P=0.001). The expression of p53 and p21 was significantly up-regulated and telomerase activity was decreased by 13%(P=0.031) in PDL20 HUVECs. However, telomere length did not significantly change between negative control and the stable miR-216a lentiviral transfection cells.3. Compared with negative control, the proliferation and migration abilities of stable miR-216a lentiviral transfection cells was decreased by 15%(P<0.001) and 16% (P<0.001), the adhesion abilities were increased by 95%(P=0.002) in PDL20 HUVECs. In contrast, inhibition of miR-216a can significantly increase endothelial proliferation and migration and decrease abhesion abilities.4. Bioinformation analysis found there were potential binding sites between SMAD7 and miR-216a. Overexpression miR-216a can inhibit SMAD7 expression by 50% (P=0.02). In dual-luciferase assay, miR-216a inhibited luciferase activites by 91%(P<0.001).(二) Regulatory role of miR-181b in vascular endothelial senescence and function1. Compared with PDL8 HUVECs, miR-181b was increased by 64%(P=0.046) in PDL44 HUVECs.2. In vitro analysis of PDL8 HUVECs, overexpression of miR-181b inhibited endothelial proliferation by 22%(P<0.001) and migration by 23%(P<0.001), and had no significant effect on endothelial tube formation (P>0.05).3. In PDL44 HUVECs, the mRNA and protein expression of IGF1R were decreased by 39%(P=0.004) and 45%(P=0.014), respectively. The luciferase assay showed that IGF1R was the putative target of miR-181b, but, overexpression of miR-181b in PDL8 HUVECs did not change the expression of IGF1R both on mRNA and protein level (P>0.05). However, IGF1R was surprisingly up-regulated by miR-181b under hypoxia condition (P=0.005).ConclusionIn our study, miR-216a induced a premature senescent-like phenptype. Moreover, miR-216a can enhance adhesion abilities and inhibited proliferation and migration of endothelial cells. MiR-216a may affect endothelial function by inhibiting SMAD7 expression. MiR-181b can inhibit proliferation and migration of endothelial cells, and the role was associated with up-regulation of the angiogenesis-related IGF1R gene under hypoxia condition, of which mechanisms need a further study.