Construction of recombinant lentivirus containing human mir-7-3 like sequence and its inhibitory effects on human glioma proliferation / 中华神经医学杂志

ABSTRACT

Objective To construct a lentiviral vector containing mir-7-3 gene and study the function of mir-7-3 gene and its role in glioma gene therapy.Methods The plasmid Lenti-GFP-mir-7-3 and packaging plasmids pRsv-REV,pMDlg-pRRE and pMD2G were co-transfected into the human embryonic kidney epithelial cell line 293T cells,and then,recombinant lentiviral FIV-CMV-GFP-mir-7-3 carried mir-7-3 gene and GFP gene was obtained; fluorescence expression of 293T cells was observed under fluorescence microscope.The supematant was collected,concentrated and identified,and then,it was used to transfect into the U251 glioma cells (positive transfection group); and blank control group (cells transfected with empty vector) and negative control group (parental cells) were also employed.Real time-PCR was used to examine the relative contents of mir-7-3 in U251 cells; Westem blotting was employed to detect the epidermal growth factor (EGFR) and serine/threonine kinase (AKT2) protein expressions; cell cycle was analyzed by flow cytometry and cell proliferative activities were measured by MTT method.Results Recombinant lentiviral FIV-CMV-GFP-mir-7-3 carried mir-7-3 gene and GFP gene was successfully constructed in 293T cells; electrophores showed that the sequence of RT-PCR product was consistent with the data ofmir-7-3 by DNA sequence analysis,indicating that the mir-7-3 gene was successfully cloned; strong green fluorwscence was observed by fluorwscent microscopy.The supernatant of lentivirus-transfected 293T cells was effectively infected into U251 cells and the relative content of mir-7-3 could be observed in the transfected U251 cells.As compared with those in the parental cells and the cells transfected with empty vector,the protein expressions of EGFR and AKT2 in the transfected group decreased significantly,reaching 38％ and 42％,respectively (P＜0.05).As compared with those in the parental cells and the cells transfected with empty vector,the cells at G0/G1 phase increased,the S phase fiaction was lower and the survival rates dramatically dropped in the mir-7-3 transfected cells 3,4,5 and 6 d after implanation.Conclusions The lentiviral vector containing mir-7-3 gene was constructed successfully.Mir-7-3 could specifically suppress EGFR and AKT2 expressions,induce gene silencing,inhibit cell growth,indicating that this way should be a new strategy in glioma gene therapy.