NTS/IL-8通路对肝细胞肝癌侵袭 迁移和EMT的影响

Effects of NTS-induced IL-8 on invasion and epithelial-mesenchymal transition in hepatocellular carcinoma

目的:探讨神经降压素(neurotensin,NTS)对肝细胞肝癌(hepatocellular carcinoma,HCC)细胞合成和分泌白细胞介素8(interleukin-8,IL-8)的影响及NTS/IL-8通路活化对HCC侵袭、迁移和上皮间质转化(epithelial-mesenchymal transition,EMT)的作用。方法:通过慢病毒基因转染构建神经降压素受体1(neurotensin receptor1,NTR1)高表达的HCC细胞系Hep3B^(NTR1hi),利用小干扰RNA构建NTR1低表达HCC细胞系HepG2^(NTR1-)。通过实时荧光定量PCR(RT-qPCR)和酶联免疫吸附(ELISA)实验检测外源性NTS刺激前后HCC细胞IL-8分泌量的变化;利用划痕修复实验和Transwell实验观察阻断IL-8受体后HCC细胞侵袭迁移能力的变化;采用Western blot比较阻断IL-8受体后HCC细胞EMT相关蛋白的表达变化。结果:外源性NTS刺激和高表达NTR1可促进Hep3B和HepG2细胞合成和分泌IL-8(P均<0.01),阻断或降低NTR1表达后IL-8的表达显著降低(P<0.05,P<0.01)。外源性NTS刺激和高表达NTR1可提高HCC细胞的侵袭和迁移能力(P均<0.05),阻断IL-8受体,即阻断NTS/IL-8信号下传,HCC细胞的划痕修复率和侵袭细胞数均降低(P均<0.001),同时伴有E-cadherin表达增加,N-cadherin、β-catenin表达降低。结论:外源性NTS刺激和高表达NTR1可以刺激HCC细胞合成和分泌IL-8,阻断NTS/IL-8信号下传会降低肝癌细胞EMT和侵袭迁移能力。

Objective：To determine the abnormal activation of the neurotensin （NTS）/interleukin-8 （IL-8） pathway and its effect on in-vasion and epithelial–mesenchymal transition （EMT） in hepatocellular carcinoma （HCC）. Methods： Hepatoma cell lines Hep3BNTR1hi and HepG2NTR1-were genetically modified by gene transfection or siRNA interference to establish various NTS-sensitive HCC cell lines bearing multiple levels of NTS receptor 1 （NTR1）. ELISA assay and real time RT-PCR were used to detect the difference of IL-8 protein secretion and RNA synthesis in varied NTS-sensitive Hep3B and HepG2 cell lines after exogenous NTS stimulation. The migration and in-vasion potentials of HCC cells were evaluated by scratch repair test and Transwell invasion assay. Western blot assay was used to de-tect the expression of EMT-related proteins in HCC cells with or without blocking IL-8 receptors. Results：Exogenous NTS stimulation and NTR1 overexpression enhanced the IL-8 RNA synthesis and protein secretion （P all〈0.01）. Exogenous NTS stimulation and NTR1 overexpression also increased the invasion of HCC cells （P all 〈0.05）. Blocking IL-8 receptors reduced the wound closure rate, de-creased the numbers of invasive cells, and reversed the EMT progress （P all〈0.001）, including the up-regulation of E-cadherin and down-regulation of N-cadherin andβ-catenin. Conclusion：In HCC cells, NTS stimulation and high expression of NTR1 induced the syn-thesis and secretion of IL-8, thereby promoting tumor EMT and HCC invasion.