microRNA抑制黑色素瘤VEGF基因表达及对细胞增殖和侵袭能力的影响

发布时间：2018-06-19 13:12

本文选题：黑色素瘤 + microRNA　；参考：《中山大学学报(医学科学版)》2014年02期

【摘要】：【目的】研究外源性靶向microRNA对恶性黑色素瘤细胞血管内皮生长因子(VEGF)基因表达的抑制作用,以及对癌细胞增殖和侵袭迁移能力的影响。【方法】根据VEGF基因序列人工设计合成4对microRNA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;脂质体法将该四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 Blot检测microRNA对VEGF蛋白表达水平的调控效应,明确抑制效果最为显著的microRNA干扰序列。MTS和EDU法分别检测靶向VEGF的microRNA对SKmel-28生长的抑制作用,流式细胞仪观察转染对肿瘤细胞凋亡的影响,Transwell检测细胞侵袭和迁移能力变化。【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,与对照组相比有统计学差异(P0.05)。抑制效应最强的重组载体是X-26-2n-1;MTS和EDU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,与对照组相比有统计学差异(P0.05),增殖抑制效应具有时间依赖性,细胞侵袭和迁移能力也显著减弱(P0.01);流式细胞仪检测显示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microRNA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,诱导肿瘤细胞凋亡,抑制肿瘤细胞的体外增殖和侵袭迁移能力。
[Abstract]:[objective] to study the inhibitory effect of exogenous targeted microRNA on the expression of vascular endothelial growth factor (VEGF) gene in malignant melanoma cells. [methods] according to the sequence of VEGF gene, 4 pairs of microRNA interference fragments were synthesized, and the eukaryotic expression vectors were constructed. The eukaryotic expression vectors were: 1: X-26-1-3X -26 -2n-1X -26-3-2 and X-26-4-4. The integrality of the inserted sequence was identified by sequencing. 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 in the transfected cells. Western Blot was used to detect the effect of microRNA on the expression of VEGF protein. The inhibitory effects of microRNA interference sequence. MTS and EDU on SKmel-28 growth were determined by microRNA targeting VEGF. The effect of transfection on apoptosis of tumor cells was observed by flow cytometry. Transwell was used to detect the changes of cell invasion and migration ability. [results] sequencing analysis confirmed that the base sequences of the four inserted fragments of the recombinant were all correct, and the SKmel-28 cells were successfully transfected. The expression of VEGF mRNA and protein in tumor cells was significantly inhibited compared with the control group (P 0.05). The expression of X-26-2n-1mMTS and EDU showed that the proliferation of SKM-28 cells transfected with X-26-2n-1 was significantly inhibited, with a significant difference compared with the control group (P 0.05), and the inhibitory effect was time-dependent. The ability of cell invasion and migration was also significantly decreased (P 0.01). Flow cytometry showed that tumor cells had early apoptosis rate and late apoptosis rate. The total apoptosis rate was also significantly higher than that of the control group (P 0.01). [conclusion] exogenous microRNA targeting VEGF can significantly reduce the expression of VEGF gene and protein in melanoma cells and induce apoptosis of tumor cells. Inhibition of tumor cell proliferation, invasion and migration in vitro.
【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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