Effect of lentiviral vector-mediated VASH1 gene on chemosensitivity of human glioma U-87MG cells

FU Kai; JIANG Pu-cha; GONG Rui; WANG Wei

doi:10.13798/j.issn.1009-153X.2016.01.012

Abstract：Objective To construct the lentiviral vector over-expressing VASH1 and study the relationship between the expression of VASH1 and the chemosensitivity of human glioma U-87MG cells. Methods The lentiviral vector carrying human VASH1 gene (pGCL-GFP-VASH1), which was constructed and sequenced, was transinfected into 293 T cells. The transinfection efficiency was evaluated and then the lentiviral vector was transfected into the human glioma U-87MG cells. Transfection efficiency was determined by Fluorescence microscopy. The expression of VASH1 mRNA and protein in U87MG cells were detected respectively by RT-PCR and Western blot. The changes in the sensitivity of transfected U-87MG cells to the chemotherapeutic drugs including cispatin and temozolomide were determined by cell counting kit-8 (CCK-8). The apoptosis of U-87MG cells transfected with pGCL-GFP-VASH1 was determined by flow cytometry (FCM). Results The lentiviral vector-mediated VASH1 gene was successfully constructed and transfected into the U-87 cells. Immunofluorescence assay demonstrated that the transfection efficiency was above 70%. RT-PCR and Western blot analyses demonstrated that pGCL-GFP-VASH1 significantly increased the expressions of VASH1 mRNA and protein by 21.57% and 27.29% respectively in transfected U-87MG cells 96 hours after the transfection compared to the control group(P<0.01);CCK-8 results showed that when exposed to cisplatin or temozolomide, the survival rate of pGCL-GFP-VASH1-transfected U-87 cells were significantly decreased to 23.96 and 17.24% respectively, which were significantly lower than those in the control groups. FCM results showed that the apoptosis rate of U-87MG cells transfected with pGCL-GFP-VASH1 was significantly higher than that in the control groups (P<0.01). Conclusion The lentiviral vector-mediated VASH1, which can significantly increase the expression of VASH1 in U-87 MG cells, may enhance chemosensitivity and apoptosis of human glioma U-87MG cells.