The Regulation Of MiR-328 On Human Umbilical Vein Endothelial Cell Induced By High Glucose And Its Signaling Mechanism In Endothelial-mesenchymal Transition

Objective: The purpose of this study was to investigate a novel rolel of miR-328 in endothelial–mesenchymal transition(EndMT) induced by high glucose in human umbilical vein endothelial cells(HUVECs) and its signaling mechanism.Methods: HUVECs were cultured in high glucose environment to induce EndMT; The recombinant lentiviruses were produced by miR-328 and antagomiR-328 transfection of HUVECs. The experiment was divided into nine groups:(1): HUVECs + normal glucose(5.5mmol / L),(2): HUVECs + mannitol group,(3): HUVECs + high glucose(30mmol / L),(4): HUVECs + miR-328,(5): HUVECs + miR-328 virus negative control,(6): HUVECs + high glucose + antagomiR-328,(7): HUVECs + high glucose + antagomiR-328 virus negative control,(8): HUVECs + high glucose + U0126(10μmol/L),(9): HUVECs + miR-328 + U0126(10μmol/L). Double immunofluorescent staining was used to determine expression of EndMT markers; Changes in miR-328 expression was examined by RT-PCR; The expression of typeⅠⅢ collagen, p-MEK1/2 and p-ERK1/2 were examined by western blot.Results: 1. Double immunofluorescent staining showed that the HUVECs showed positive staining for CD31 and α-SMA in high glucose group.2. Compared with the control group, the expression of miR-328 was up-regulated(p <0.05) in HUVECs treated by high glucose or miR-328. Compared with high glucose group or miR-328 group, miR-328 expression were less pronounced after treatment of HUVECs with U0126. 3. The expression of type ⅠⅢ collagen were increased in HUVECs treated by high glucose or miR-328 when compared with control group(p <0.05).while transfection with antagomiR- 328, the expression of type Ⅰ Ⅲ collagen were less pronounced than high glucose group(p <0.05).Compared with high glucose group or miR-328 group, type ⅠⅢ collagen expression were less pronounced after treatment with U0126.4. The expression of p-MEK1 / 2 and p-ERK1 / 2 were increased in HUVECs treated by high glucose or miR-328 in comparison to the control group(p <0.05); a lower expression of p-MEK1 / 2 and p-ERK1 / 2 were observed in antagomiR- 328 group or U0126 group than in high glucose group or miR-328 group.Conclusion: The phenomenon of EndMT in HUVECs was induced by high glucose, and the expression of miR-328 was increased at the same time; overexpression of miR-328 induced EndMT in HUVECs, while antagomiR-328 suppressed the phenomenon of EndMT; miR-328 induced EndMT were related with MEK1 / 2-ERK1 / 2 signaling pathway.