Nan fang yi ke da xue xue bao = Journal of Southern Medical University · 2014
[Targeting microRNA-mediated suppression of vascular endothelial growth factor gene expression and proliferation in malignant melanoma cells in vitro].
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Jiang Yuan, Han Yongzhi, Sun Jian
Аннотация
OBJECTIVE: To explore the inhibitory effect of targeting miRNA on the expression of vascular endothelial growth factor (VEGF) and cell proliferation in malignant melanoma (MM) SKmel-28 cells.
METHODS: Recombination miRNA plasmid vectors targeting VEGF gene were transfected into SKmel-28 cells via Lipofectamine 2000. The integrity of the inserted fragments was detected using colony PCR and sequence analysis. The expression of VEGF mRNA and protein in SKmel-28 cells was detected by RT-PCR and Western blotting, respectively. MTS assay was used to determine the inhibitory effect of a selected targeting miRNA on SKmel-28 cell proliferation, and the apoptosis of SKmel-28 cells was detected using flow cytometry.
RESULTS: Transfection with the targeting miRNAs significantly down-regulated the expressions of VEGF mRNA and protein in SKmel-28 cells (P<0.01), and the miRNA construct X-26-2n-1 showed the highest inhibitory effect. The miRNA X-26-2n-1 significantly suppressed SKmel-28 cell proliferation in a time-dependent manner (P<0.01) and increased the early, late and overall apoptosis rates of the cells (P<0.01).
CONCLUSION: The targeting miRNA we constructed can effectively suppress the cell proliferation and induce apoptosis of SKmel-28 cells by down-regulating the expressions of VEGF gene.
METHODS: Recombination miRNA plasmid vectors targeting VEGF gene were transfected into SKmel-28 cells via Lipofectamine 2000. The integrity of the inserted fragments was detected using colony PCR and sequence analysis. The expression of VEGF mRNA and protein in SKmel-28 cells was detected by RT-PCR and Western blotting, respectively. MTS assay was used to determine the inhibitory effect of a selected targeting miRNA on SKmel-28 cell proliferation, and the apoptosis of SKmel-28 cells was detected using flow cytometry.
RESULTS: Transfection with the targeting miRNAs significantly down-regulated the expressions of VEGF mRNA and protein in SKmel-28 cells (P<0.01), and the miRNA construct X-26-2n-1 showed the highest inhibitory effect. The miRNA X-26-2n-1 significantly suppressed SKmel-28 cell proliferation in a time-dependent manner (P<0.01) and increased the early, late and overall apoptosis rates of the cells (P<0.01).
CONCLUSION: The targeting miRNA we constructed can effectively suppress the cell proliferation and induce apoptosis of SKmel-28 cells by down-regulating the expressions of VEGF gene.