Study On The Molecular Mechanisms Of NTS/IL-8 Pathway Meadiated Regulation Of EMT In HCC

Objective: This study is aimed to elucidate the molecule mechanism involved in the neurotensin(NTS) /interleukin-8(IL-8) pathway and its effect on the invasion and epithelial-mesenchymal transition(EMT) in hepatocellular carcinoma(HCC).Methods: The expression of IL-8, vascular endothelial growth factor(VEGF),matrix metalloproteinase-9(MMP-9),inflammation markers(CD68,CD163 and CD31), as well as EMT–related proteins(E-cadherin,β-catenin) were examined in 100 paraffin-embedded HCC samples using immunohistochemistry staining method.The correlation between the expression of above proteins and patients’ respective clinical and pathological characteristics was analyzed. Hepatoma cell line Hep3 B cells were genetically modified to establish multiple NTS-sensitive HCC cell lines bearing various levels of NTR1 by gene transfection or si RNA interference. The secretion of IL-8 in varied NTS-sensitive Hep3 B cell lines after exogenous NTS stimulation was deteted using ELISA assay. The scratch repair test and transwell invasion assay were used to evaluate the migration and invasion potentials of HCC cells, and Western blot assay was applied to detect the expression of multiple EMT-related proteins in HCC cells with or without blocking IL-8 receptors. The expression and phosphorylation of a series of functional proteins along the MAPK, NF-κB, PI3 K and PKC signaling pathways were detected using Western Blot assay. Blocking the activation of the MAPK and NF-κB signaling pathway via SP600125, PD98059, SB203580 and BAY117082 was performed to elucidate the underlying molecular mechanisms of NTS induced IL-8 and IL-8 induced HCC invasion and EMT.Results: In HCC samples, the posive expression rate of NTS, NTR1 and IL-8 was 19%, 41% and 49%, respectively.The Spearman’s rank correlation shows that NTS is positively correlated with NTR1( r = 0.322,P = 0.001) as well as IL-8( r = 0.331,P = 0.001). Among the 19 NTS+ HCC samples, 14(73.68%)samples show postive expression of NTR1 and 16(84.21%)samples show positive expreesion of IL-8, which implied the activation of NTS/IL-8 pathway in certain HCC subgroup. NTS/IL-8+ samples displayed higher frequency of uncomplete tumor envelope and portal vein invasion in HCC patients(P=0.048,P<0.01), who usually experiencedworse prognosis with a shorter OS(P=0.019). The positive rates of MMP-9 and VEGF in the NTS/IL-8 pathway activated(NTS/IL-8+) HCC samples were significantly higher than the other samples(P=0.001,P=0.020). The numbers of M2 type tumor-associated macrophages(M2 TAMs) and micro-vessel density(MVD) were also higher as well(P<0.001,P<0.001). The expression of E-cadherin decreased,β-catenin was translocated to cytoplasm and the expression of N-cadherin enhanced in NTS/IL-8+ samples(P=0.026,P=0.040,P=0.003). Exogenous NTS stimulation and NTR1 over-expression could enhance the secretion of IL-8(P=0.005, P=0.03). Bloking IL-8 recepors decreased the wound closure rate(P<0.001), attaneuated the numbers of invasive cells(P=0.002) and reversed the EMT process, including upregulation of E-cadherin, downregulation of N-caherin and β-catenin. NTS stimulate the secretion of IL-8 is associated with the activation of the MAPK and NF-κB pathways, rather than the PKC pathway and the PI3 K pathway. The phosphorylation of P38, ERK, JNK, IκB and P65 was upregulated, and the nuclear expression of NF-κB family P65 and P50 was identified. SP600125, PD98059, SB203580 and BAY117082 were applied to block the MAPK and NF-κB signal pathways, which significantly inhibited the secretion of IL-8 and tumor invasion accompanied by reversing the expression of EMT-related proteins.Conclusion:The activation of the NTS/IL-8 pathway was detected in HCC tissues which significantly correlated with local inflammation and EMT of HCC. NTS stimulation induced the synthesis and secretion of IL-8 in HCC cells via activating the MAPK and canonical NF-κB pathways, and thus promoted tumor EMT and HCC invasion.