| Objective:The purpose of this research was to construct a lentiviral vector containing mir-7-3gene and green fluorwscent protein(GFP) gene, and to detect the expression of mir-7-3gene in U251cells. To investigate the biological effects on gliomas by lentivirus-miRNA interference technology.Methods:The fragments containing all the mir-7-3gene were amplified by RT-PCR and were cloned into the lentivirus vectors labeled with GFP,which was transfected together with the packaging plasmids into293T cells by CaCl2, the supernatant was collected, concentrated,identifled, and to transfect U251cells of gliomas. fluorwscent microscopy was used to observe the fluorwscence,and real time-RT-PCR was used to examine the relative contents of mir-7-3in U251cells. U251cells line was transinfected by mir-7-3, Lt-non (negative control shRNA)and phosphate-buffered saline (PBS), respectively, then we analysed the mRNA expression level of endogenous EGFR and AKT2in tumor cells by RT-PCR and Western blot and used MTT and flow cytometry to detect cell proliferation and cycle.Results:Electrophores is showed that the sequence of the RT-PCR product was consistent with the data of mir-7-3by DNA aequence analysis, indicated that the mir-7-3gene was successfully cloned, strong green fluorwscence was observed by fluorwscent microscopy. The supernatant of lentivirus-transfected293T cells effectively infected U251cells and the relative content of mir-7-3in the transfected U251cells.In vitro, mir-7-3reduced expression level of endogenous AKT2and EGFR in tumor cell obviously, and suppressed the activity cell proliferation and cell cycle.Conclusion:RNAi technology inhibit the expression level of endogenous AKT2and EGFR in tumor cells and changed the phenotype of malignant glioma U251cell line. We find The inhibitory effect of transfection with mir-7-3on the growth of glioma cells in vitro.mir-7-3gene is expected to become a glioma means of gene therapy. |
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