靶向VEGF的miRNA对恶性黑色素细胞增殖和凋亡的影响  

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

作　　者：蒋源[1] 韩永智[1] 孙建[2] 

机构地区：[1]广东省人民医院//广东省医学科学院皮肤科,广东广州510080 [2]广东省人民医院//广东省医学科学院普通外科,广东广州510080

出　　处：《南方医科大学学报》2014年第3期358-363,共6页Journal of Southern Medical University

基　　金：广东省科技计划项目(2010B080701022;2010B080701021)

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。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＆lt;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＆lt;0.01） and increased the early, late and overall apoptosis rates of the cells （P＆lt;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.

关 键 词：黑色素瘤 血管内皮生长因子 细胞增殖 凋亡 

分 类 号：R739.5[医药卫生—肿瘤]