miR-638抑制尤文肉瘤细胞的增殖和血管拟态形成的影响

miR-638 Inhibits the Proliferation and Tubal Formation of Ewing Sarcoma Cells Partially through Regulating the VEGFA

该研究旨在探讨miR-638在尤文肉瘤细胞中的表达及其对尤文肉瘤细胞增殖和血管拟态形成能力的影响,进一步探讨其可能机制。应用q RT-PCR检测miR-638在尤文肉瘤细胞系(A673、SK-ES-1和RD-ES)中的表达;转染人工合成miR-638 mimic到RD-ES和SK-ES-1细胞内,运用CCK-8法和小管形成实验分别检测miR-638对A673和SK-ES-1细胞增殖及血管拟态形成能力的影响;应用q RT-PCR和Western blot探讨对血管内皮生长因子(vascular endothelial growth factor,VEGFA)在A673细胞内是否为miR-638的靶基因,回复实验验证VEGFA是否参与miR-638对A673细胞增殖和血管拟态形成的作用。结果表明,与人间充质干细胞(mesenchymal stem cells,MSC)相比,尤文肉瘤细胞内miR-638呈低表达趋势(P<0.05);转染miR-638 mimic后,RD-ES及SK-ES-1细胞内miR-638升高明显,表明miR-638 mimic转染成功;转染miR-638组尤文肉瘤细胞增殖能力下降(P<0.05),新生拟态血管分支数较对照组减少(P<0.05);转染miR-638组A673和SK-ES-1细胞内VEGFA m RNA及蛋白质水平均明显下降,提示miR-638能够调控尤文肉瘤细胞内VEGFA的表达;回复实验结果显示,同时转染了miR-638 mimic和pc DNA-empty组细胞内VEGFA的蛋白质水平最低,生长速度最慢,小管形成的分支数最少。miR-638在尤文肉瘤细胞中呈低表达,过表达miR-638能够抑制尤文肉瘤细胞增殖及血管拟态形成,该作用可能通过靶向调控VEGFA作用而实现的。

This work was to investigate the level of miR-638 in Ewing sarcoma cells（EWS） and its effects on the proliferation and tube formation of EWS, so as to further identify the putative mechanisms involved. The level of miR-638 was detected in EWS comparing with the mesenchymal stem cells（MSC） by q RT-PCR assays. We restored the level of miR-638 in RD-ES and SK-ES-1 cells through transfection with artificial miR-638 mimic. The CCK-8 and tubule formation experiments were used to explore the effects of miR-638 on the proliferation and tubal formation of EWS cells, respectively. Then q RT-PCR and Western blot assays were performed to explore the effects of miR-638 on the level of VEGFA. Rescue assays were performed to identify whether VEGFA was involved in miR-638-mediated suppressive effects on EWS. The levels of miR-638 in three Ewing sarcoma cell lines were significantly lower than those in MSC（P0.05）; Compared with the control groups, the proliferation rates and the number of branches in the formation were significantly decreased（P0.05）. q RT-PCR and Western blot analysis showed that over-expression of miR-638 could significantly inhibit the levels of m RNA and protein of VEGFA. The results of rescue showed that the level of miR-638 mimic and pc DNA-empty target gene VEGFA was the lowest, the growth rate was the slowest, and the number of branches was the least. miR-638 was suppressed in EWS cells, and its over-expression inhibited cell proliferation and tubal formation of EWS, which might be mediated targeting VEGFA partially.