Down-regulation of SIRT1 by microRNA34a miR-34a down-regulates the mechanism by which SIRT1 inhibits the function of endothelial progenitor cells

Objective To investigate the mechanism of microRNA34a (miR-34a) down-regulating silencing of information regulator 1 (SIRT1) in dysfunction of endothelial progenitor cells during oxidative stress, and provide a new explanation for the dysregulation of miRNA expression during dysfunction of endothelial progenitor cells, so as to find new treatment target for atherosclerosis. Methods In this experiment, endothelial progenitor cells were treated with different concentrations of hydrogen peroxide (H2O2), and the survival rate of the cells was detected by CCK-8. The miR-34a mimics, miR-34a inhibitors and their respective control groups were synthesized by chemical synthesis and transfected into endothelial progenitor cells. After the endothelial progenitor cells were stimulated with 500 μM H2O2, the expression level of miR-34a was verified by RT-PCR. The apoptosis of miR-34a induced by H2O2 was detected by flow cytometry. Furthermore, qRT-PCR and Western blotting were used to detect expression levels of mRNA and protein in SIRT1. Results The cell viability decreased with the increase of H2O2 concentration by CCK-8, which was concentration-dependent. Compared with control groups, the survival rate of cells added with 100μM, 200 μM, 500 μM and 800 μM was respectively (94±2)%, (85 ± 2) %, (78 ± 2)%, (32 ±1)%. The difference was statistically significant (F=16.3, P<0.001). After endothelial progenitor cells were stimulated by H2O2, the expression level of miR-34a mimics was higher than that of the control group (t=16, P=0.003), and the expression level of miR-34a inhibitors was lower than that of the control group (t=53, P<0.001). The apoptosis rate of miR-34a mimics group was significantly increased (t=30.4, P<0.001), and the apoptosis rate of miR-34a inhibitors group was significantly decreased (t=8.7, P<0.001). The qRT-PCR results showed that the relative expression level of the target gene SIRT 1 in the miR-34a mimics group was significantly lower than that of control group (t=87.7, P<0.001), while the miR-34a inhibitors group was significantly higher than that of control group (t=125, P<0.001). Western Blot results showed that the expression of SIRT 1 protein was significantly decreased in miR-34a mimics group (t=4.1, P=0.014), while that was significantly increased in miR-34a inhibitors group(t=7.1, P=0.002), compared with control group. Conclusions Under oxidative stress, the miR-34a mimics and inhibitor can modulate the expression of miR-34a, thereby altering the expression of the protein in SIRT1. Endothelial progenitor cell dysfunction may be associated with increased levels of miR-34a and decreased expression of its target protein SIRT1. Key words: Endothelial progenitor cells; Oxidative stress; miR-34a; Silent information regulator 1