Transfection of adenovirus-mediated mircoRNA-126 gene into infant hemangioma endothelial cells in vitro.

ABSTRACT

Objective: To investigate the effects of microRNA-126 (miR-126) overexpression on hemangioma endothelial cells (HemECs). Methods: An adenoviral vector containing the miR-126 gene was constructed. HemECs were passaged and expanded and adenovirus-mediated green fluorescent protein (GFP) gene was transfected in vitro. The infection efficiency of adenovirus vector to HemECs was tested by Ad-GFP infection procedure. GFP expression efficiency was observed using a fluorescence microscope and flow cytometry was used to determine the best virus multiplicity of infection (MOI). The experiment was divided into the blank group, AD-GFP group, and AD-miR-126 group. The miR-126 group was transfected into HemECs in vitro with adenovirus-mediated miR-126 gene under optimal MOI conditions. RT-PCR was applied to detect expression of miR-126 gene in cells. The influence of recombinant adenovirus on cell activity was evaluated by CCK-8 assay. Flow cytometry was utilized to detect cell cycle and apoptosis. Results: HemECs could be effectively infected by adenovirus containing GFP gene in vitro, the transfection efficiency had the dose-effect relationship with multiplicities of infection (MOI). When MOI was 400, the infection efficiency was more than 90%. miR-126 expression in HemECs was significantly enhanced in miR-126 group (P<0.05). Compared to the control group, cell proliferation was significantly enhanced (P<0.05) and induced S-phase arrest significantly (P<0.05) when miR-126 was upregulated. In addition, compared with the control group, the early apoptotic rate was significantly decreased by upregulating miR-126 (P<0.05). Conclusion: miR-126 overexpression can successfully promote proliferation and inhibit apoptosis of HemECs. This work will provide the theoretical and experimental basis for further transplantation study in vivo.