南方医科大学学报

南方医科大学学报 ›› 2014, Vol. 34 ›› Issue (03): 358-.

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靶向VEGF的miRNA对恶性黑色素细胞增殖和凋亡的影响

蒋源，韩永智，孙建   

  • 出版日期:2014-03-20 发布日期:2014-03-20

Targeting microRNA-mediated suppression of vascular endothelial growth factor gene
expression and proliferation in malignant melanoma cells in vitro

  • Online:2014-03-20 Published:2014-03-20

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摘要： 目的研究靶向血管内皮生长因子（VEGF）基因的外源性miRNA对恶性黑色素瘤细胞增殖及凋亡的影响。方法根据
VEGF基因序列设计合成4对miRNA干扰片段，构建质粒真核表达载体，分别为X-26-1-3、X-26-2n-1、X-26-3-2和X-26-4-4，测
序分析鉴定插入序列的完整性；脂质体法将该4 个重组表达载体转染黑色素瘤细胞株SKmel-28，RT-PCR 检测转染细胞中
VEGF基因的mRNA表达变化，Western blotting 检测miRNA对VEGF蛋白表达水平的调控效应，明确抑制效果最为显著的
miRNA干扰序列。MTS法和流式细胞仪分别检测人工合成的靶向VEGF的miRNA对SKmel-28生长的抑制作用，以及对肿瘤
细胞凋亡的影响。结果测序分析证实4个重组体插入片段的碱基序列均完全正确，成功转染SKmel-28细胞后，均能显著抑制
肿瘤细胞VEGF mRNA和蛋白的表达，与对照组相比有显著性差异（P<0.01）。抑制效应最强的重组载体是X-26-2n-1，MTS法
和流式细胞仪检测显示，SKmel-28细胞转染X-26-2n-1后，细胞增殖受到明显抑制，增殖抑制效应具有时间依赖性，与阴性对照
组比较差异具有显著性（P<0.05），与空白组比较细胞增殖抑制更加明显（P<0.01）；肿瘤细胞早期凋亡率、晚期凋亡率、总凋亡率
也显著高于对照组（P<0.01）。结论外源性靶向VEGF的miRNA能够显著降低黑色素瘤细胞SKmel-28 VEGF基因和蛋白的
表达水平，抑制肿瘤细胞增殖，诱导细胞凋亡。

Abstract: 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.