TGF-β上调Notch3诱导上皮间质转化在促进肺癌骨转移中的作用  

The role of TGF- β upregulated Notch3 by inducing EMT on bone metastasis in non-small cell lung cancer

作　　者：杨雪[1] 冯征[2] 张宁[1] 王亚芳[1] 赵正维[2] 周勇安[2] 

机构地区：[1]第四军医大学唐都医院肿瘤科,陕西西安710038 [2]第四军医大学唐都医院胸外科,陕西西安710038

出　　处：《现代肿瘤医学》2016年第18期2851-2857,共7页Journal of Modern Oncology

基　　金：陕西省自然科学基金(编号:2014JM2-8148)

摘　　要：目的:研究TGF-β上调Notch3表达诱导EMT在肺癌骨转移中的作用。方法:免疫荧光、蛋白印记和RT-PCR方法检测Notch3在TGF-β诱导下上皮、间质标记物以及上皮间质转化相关转录因子Twist、Snail和ZEB-1的表达变化;利用慢病毒包装Notch3正义表达载体和ZEB-1 siRNA载体分别上调Notch3和抑制ZEB-1的表达后,采用免疫荧光、蛋白印记方法检测抑制EMT转录因子ZEB-1的表达后,对上皮间质标记物的影响;进而通过体外迁移和侵袭试验观察抑制ZEB-1的表达后对Notch3高表达细胞系的迁移和侵袭能力的影响;最后体外ELISA法检测骨转移相关分子p THr P和IL-6的表达,探讨Notch3通过诱导EMT在促进肺癌骨转移中的作用。结果:Notch3 siRNA抑制其表达后能够逆转TGF-β诱导的上皮标记物和间质标记物的表达变化,这一现象是通过影响EMT转录因子ZEB-1实现的。进而利用慢病毒包装系统,构建了Notch3过表达细胞株和ZEB-1 siRNA细胞株,免疫荧光和蛋白印记试验发现,Notch3过表达载体能够抑制上皮标记物的表达,上调间质标记物的表达,促进EMT的发生,而抑制ZEB-1的表达后能够逆转这一现象。最后,体外侵袭试验和ELISA试验发现,Notch3高表达能够增加肺癌细胞的侵袭能力以及p THr P和IL-6的分泌,而抑制ZEB-1的表达后可明显降低其转移能力以及p THr P和IL-6的分泌。结论:Notch3参与了TGF-β诱导肺癌细胞上皮间质转化过程,并在NSCLC骨转移中发挥作用。Objective： To explore the role of TGF- β up- regulated Notch3- induce EMT on bone metastasis in NSCLC. Methods： Immunofluorescence,Western blot and RT- PCR assay were used to detect the protein expression mRNA level of epithelial,mesenchymal markers and EMT- related transcriptional factors twist,snail and ZEB- 1 expression inhibited by Notch3 siRNA exposed TGF- β stimuli. Lentivirus package Notch3 sense expression vector and siRNA targeting ZEB- 1 were transfected in NSCLC cell lines. Immunofluorescence and Western blot assay were used to detect the protein expression mRNA level of epithelial and mesenchymal markers in ZEB- 1 siRNA cell lines co-transfected Notch3 overexpression cell lines. In vitro transwell invasive assay was used to observe the effect of ZEB- 1siRNA on metastasis ability in Notch3 overexpression cell lines. ELISA assay was used to detect the bone metastasis relative molecular p THrp and IL- 6 expression. Results： TGF- β induced a strong morphological transformation,promoting epithelial- mesenchymal transition（ EMT）,inhibition of Notch3 by siRNA could reverse it. Western blot and RT- PCR assays revealed that force overexpression of Notch3 induced the expression and activity of ZEB- 1,subsequently suppression of E- cadherin and up- regulation of Fibronectin,which contributing to EMT and invasion. Western blot and immunofluorescence assays found that RNA interference（ RNAi）- mediated ZEB- 1 blocked Notch-induced EMT- like transformation,and subsequently reverse of E- cadherin and down- regulation Fibronectin. In vitro invasion assay showed that inhibition of ZEB- 1 can decrease Notch3- prompted invasion and p THr P,IL- 6 expression. Conclusion： Notch3 involved in TGF- β induced EMT- like transformation and bone metastasis in NSCLC.

关 键 词：NOTCH3 TGF-Β 上皮间质转化 骨转移 非小细胞肺癌 

分 类 号：R734.2[医药卫生—肿瘤]