Inhibitory effect of miR-146a on high glucose-induced apoptosis of retinal microvascular endothelial cells and its mechanism

Objective

To investigate the effect of microRNA-146a (miR-146a) on apoptosis of human retinal microvascular endothelial cells (HRMECs) induced by high glucose and its possible molecular mechanism.

Methods

HRMECs were cultured in vitro with 5.5 mmol/L D-glucose in the normal control group and 25 mmol/L D-glucose in the high glucose group for 48 hours, respectively.Normally cultured HRMECs were transfected by miR-146a mimics in the high glucose+ miR-146a mimics group or corresponding mimics control in the high glucose+ mimics control group by lipofection and cultured with 25 mmol/L D-glucose for 48 hours, respectively.Real-time fluorescent quantitative polymerase chain reaction (PCR) was performed to detect the expression level of miR-146a.MTT assay and flow cytometry were used to detect the activity and apoptosis of HRMECs, and Western blot was employed to detect the expression levels of apoptosis-associated protein B-cell lymphoma factor-2 (Bcl-2), Bcl-2 related X protein (Bax) and nuclear factor-κB (NF-κB) signaling-related proteins NF-κB p65 and p-NF-κB p65.

Results

The relative expression levels of miR-146a were 1.00±0.10, 0.22±0.02, 0.21±0.02 and 0.88±0.09, and the cell viability was (100.00±10.06)%, (68.41±6.67)%, (67.91±6.74)% and (90.46±8.97)%, and the apoptosis rates were (3.11±1.02)%, (27.28±3.56)%, (27.44±4.03)% and (7.29±2.11)% in the normal control group, high glucose group, high glucose+ mimics control group and high glucose+ miR-146a mimics group, respectively.The relative expression levels of miR-146a and the cell viability were significantly lower, and the cell apoptosis rate was significantly higher in the high glucose group than those in the normal control group, with statistical significant differences (all at P<0.05). The relative expression levels of miR-146a and the cell viability were significantly higher, and the cell apoptosis rate was significantly lower in the high glucose+ miR-146a mimics group than those in the high glucose group and the high glucose+ mimics control group, and the differences were statistically significant (all at P<0.05). The relative expression levels of Bax and p-NF-κB p65 protein were significantly higher, the relative expression level of Bcl-2 protein was significantly lower in the high glucose group than those in the normal control group, showing statistically significant differences (all at P<0.05). The relative expression levels of Bax and p-NF-κB p65 protein were significantly lower, and the relative expression level of Bcl-2 protein was significantly higher in the high glucose+ miR-146a mimics group than those in the high glucose group and the high glucose+ mimics control group, and the differences were statistically significant (all at P>0.05). There was no significant difference in the relative expression of NF-κB p65 protein among the groups (F=0.106, P=0.955).

Conclusions

Overexpression of miR-146a may inhibit the apoptosis of HRMECs induced by high glucose, and its mechanism may be related to the inhibition of NF-κB signaling pathway activation.

Diabetic retinopathy; MiR-146a; NF-κB signaling pathway; Retinal vascular endothelial cells