NTS/IL-8通路对肝细胞肝癌侵袭迁移和EMT的影响

  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Hep3BNTR1hi，利用小干扰RNA构建NTR1低表达HCC细胞系HepG2NTR1-。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。 

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

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.