靶向VEGF的miRNA对恶性黑色素细胞增殖和凋亡的影响

发布时间：2018-05-20 16:50

本文选题：黑色素瘤 + miRNA　；参考：《南方医科大学学报》2014年03期

【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(P0.01)。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(P0.05),与空白组比较细胞增殖抑制更加明显(P0.01);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(P0.01)。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 study the effects of exogenous miRNA targeting vascular endothelial growth factor (VEGF) gene on the proliferation and apoptosis of malignant melanoma cells. Methods according to the sequence of VEGF gene, four pairs of miRNA interference fragments were designed and synthesized, and the eukaryotic expression vectors were constructed, namely X-26-1-3OX-26-2n-1OX-26-3-2 and X-26-4-4, respectively. The integrity of the inserted sequence was identified by sequencing analysis. The four recombinant expression vectors were transfected into melanoma cell line SKmel-28 by liposome method. RT-PCR was used to detect the mRNA expression of VEGF gene in the transfected cells. Western blotting was used to detect the effect of miRNA on the expression of VEGF protein. MiRNA interference sequence. MTS and flow cytometry were used to detect the inhibitory effect of synthetic VEGF targeting miRNA on the growth of SKmel-28 and the effect on apoptosis of tumor cells. Results the sequence analysis showed that the sequence of the four inserted fragments was correct. After transfection of SKmel-28 cells, the expression of VEGF mRNA and protein in tumor cells was significantly inhibited, and there was significant difference compared with the control group (P 0.01). The strongest inhibitory effect of recombinant vector was X-26-2n-1MTS assay and flow cytometry analysis showed that after transfection of X-26-2n-1, the cell proliferation was significantly inhibited, and the inhibitory effect was time-dependent. Compared with the negative control group, the difference was significant (P 0.05), the inhibition of cell proliferation was more obvious than that of the blank group, and the early apoptosis rate, the late apoptosis rate and the total apoptosis rate of tumor cells were significantly higher than those of the control group. Conclusion exogenous miRNA targeting VEGF can significantly reduce the expression of SKmel-28 VEGF gene and protein, inhibit the proliferation and induce apoptosis of melanoma cells.
【作者单位】：广东省人民医院//广东省医学科学院皮肤科;广东省人民医院//广东省医学科学院普通外科;
【基金】：广东省科技计划项目(2010B080701022,2010B080701021)
【分类号】：R739.5

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