ABSTRACT: To construct human recombinant lentiviral expression vector of microRNA-210 (miR-210) and to explore the over-expression of miR-210 on the capillary formation in human umbilical vein endothelial cells 12 (HUVE-12).
The recombinant lentiviral expression vector of pGCSIL-green fluorescent protein (GFP)-pre-miR-210 was constructed by molecular cloning and transfected to HUVE-12 (LV-miR-210-GFP group), only pGCSIL-GFP was transfected as control group (LV-GFP group). The miR-210 expression activity was evaluated by GFP reporter through fluorescence detection and real-time fluorescent quantitative PCR. The ephrinA3 protein expression was measured by flow cytometry. The concentration of vascular endothelial growth factor (VEGF) in culture supernatant was determined by ELISA. The cells were cultured in 96-well culture plate coated with Matrigel to assess the ability of capillary formation.
The recombinant plasmid pGCSIL-GFP-pre-miR-210 was confirmed by restriction endonuclease analysis and DNA sequencing. Fluorescence detection showed that the fluorescence intensity of GFP was highest between 48 and 72 hours after transfection. Real-time fluorescent quantitative PCR showed that the miR-210 expression of LV-miR-210-GFP group was 9.72 times higher than that in LV-GFP group (t = -11.10, P = 0.00). Flow cytometry analysis showed that the positive cell rate of enphrinA3 in LV-miR-210-GFP group (12.52% +/- 0.67%) was significantly lower than that in LV-GFP group (73.22% +/- 1.45%) (t = -66.12, P = 0.00). The concentration of VEGF in supernatant in LV-miR-210-GFP group was significantly higher than that in LV-GFP group [(305.29 +/- 16.52) pg/mL vs. (42.52 +/- 3.11) pg/mL, t = -27.06, P = 0.00]. In vitro capillary-like formation assay showed that the number of capillaries was significantly larger in LV-miR-210-GFP group than in LV-GFP group (17.33 +/- 6.33 vs. 6.33 +/- 2.33, t = -2.83, P = 0.04).
The recombinant lentiviral expression vector of miR-210 is constructed successfully and HUVE-12 over-expressing miR-210 can significantly increase the capillary formation, which facilitates further study on the molecular functions of miR-210 in angiogenesis.
Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 05/2012; 26(5):587-91.