摘要
目的:探讨Twist转录因子通过ERK通路对体外鼻咽癌CNE2细胞血管生成的影响。方法:利用Lipofectamine 2000将Twist基因干扰序列转染入体外培养的鼻咽癌CNE2细胞,提取细胞条件培养基(conditionedmedium,CM),用CM作用体外人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)后,CCK-8检测细胞活力,Transwell小室实验检测细胞侵袭能力,小管形成实验检验HUVECs体外血管生成能力,real-time PCR检测Twist mRNA在CNE2细胞中的表达,Western blot法检测CNE2细胞中ERK、p-ERK、p38、p-p38、JNK、p-JNK和VEGF的表达。另外,使用ERK、p38和JNK通路抑制剂作用CNE2细胞后,再检测通路抑制剂对体外HUVECs小管形成的影响。结果:CNE2细胞转染Twist干扰序列后可显著抑制Twist mRNA及蛋白表达水平;从沉默Twist基因的CNE2细胞提取的CM促进体外HUVECs活力、侵袭和小管形成的能力均明显被削弱;抑制CNE2细胞中Twist的表达可上调p-ERK并下调VEGF的表达(P <0.01),而对p-p38和p-JNK的表达无明显影响;ERK通路抑制剂PD98059可部分削弱Twist干扰序列对体外HUVECs小管形成的抑制作用。结论:沉默Twist基因可显著抑制体外鼻咽癌细胞的促血管生成作用,该作用可能部分通过上调ERK信号通路实现。
AIM:To investigate the effect of Twist transcription factor on angiogenesis of nasopharyngeal can-cer cells by regulating ERK signaling pathway in vitro.METHODS:Twist siRNA was transfected into the CNE2 cells with Lipofectamine 2000,and the CNE2 cell-conditioned medium(CM)was prepared,which was then used to treat human umbilical vein endothelial cells(HUVECs).The CCK-8 assay,Boyden chamber and capillary tube formation assay were used to evaluate the angiogenic activity of HUVECs.The mRNA expression of Twist in nasopharyngeal cancer cells was detected by real-time PCR.The protein levels of Twist,ERK,p-ERK,p38,p-p38,JNK,p-JNK and VEGF were detected by Western blot.The ERK,p38 and JNK signaling pathway inhibitors were employed to treat HUVECs,and their effect on the capillary tube formation of HUVECs was analyzed.RESULTS:The expression of Twist at mRNA and protein levels in the CNE2 cells was significantly inhibited after transfection with Twist siRNA(P<0.01).The abilities of proliferation,invasion and capillary tube formation of HUVECs were markedly suppressed after knockdown of Twist expression(P<0.01).The protein level of p-ERK was significantly elevated in the CNE2-siTwist cells,while VEGF expression was decreased.Knockdown of Twist did no change the levels of p-p38 and p-JNK.The ERK signaling pathway inhibitor partly abrogated the anti-angiogenic effect of Twist knockdown.CONCLUSION:Knockdown of Twist inhibits the angiogenesis of nasopharyngeal cancer cells in vitro partly through activation of ERK signaling pathway.
作者
孙旭鸯
陈湧
SUN Xu-yang;CHEN Yong(Department of Otolaryngology,Lishui Central Hospital,Lishui 323000,China;Department of Gastrointestinal Surgery,The Second People Hospital of Yueyang,Yueyang 414000,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2019年第2期298-304,共7页
Chinese Journal of Pathophysiology
基金
湖南省卫计委科研计划项目(No.B2017232)
关键词
鼻咽癌
TWIST转录因子
ERK信号通路
血管生成
Nasopharyngeal carcinoma
Twist transcription factor
ERK signaling pathway
Angiogenesis
