Study On The Molecular Mechanisms Of Neurotensin Mediated Regulation Of Epithelial-mesenchymal Transition In HCC

Objective: This study aim to elucidate the underlying molecule mechanism of neurotensin(NTS)-induced epithelial-mesenchymal transition(EMT) and hepatocellular carcinoma(HCC) invasion.Methods: The level of NTS,Ki67 and EMT related proteins, including E-cadherin, N-cadherin, β-catenin and Vimentin in 100 cases of paraffin-embedded HCC samples were examined using immunohistochemistry staining method. The clinical and pathological characteristics were compared between high NTS patients and low NTS ones. Two hepatoma cells(Hep3B and Hep G2) with different levels of NTR1 was established by gene transfection and si RNA interference. The Brd U proliferation assay, MTT assay, Annexin V apoptosis assay, scratch repair experiments, Transwell invasion assay were used to compare the functional alterations of hepatoma cells upon different NTS stimulation. The expression of EMT markers was detected by Real-time PCR and Western blot. PCR Array and Western Blot assay were conducted to elucidate the underlying molecular mechanisms of NTS induced EMT. Blocking NTR1 and GSK3β activation via SR48692 and TSW119 were adopted to study if tatget therapy against NTS signaling pathway can reverse EMT and inhibit HCC invasion.Results: In 100 cases HCC samples, NTS+ samples accounted for 19.00% of total. NTS expression in tissues was significantly correlated with imcomplate envelope and portal vein invasion of HCC patients. NTS+ patients displayed poor prognosis, whose OS is significantly shorter than those of NTS- patients. In NTS+ samples, expression of E-cadherin decreased significantly, the β-catenin translocating to the cytoplasm and increasing expression of N-cadherin accompanied by. Exogenous NTS stimulating and NTR1 over-expression have no effect on proliferation and apoptosis of HCC cell lines, but can improve the wound closure rate and invasion cells number. Exogenous NTS stimulating and NTR1 over-expression also can repress E-cadherin and upregulated N-caherin, β-catenin and snail in HCC cell lines, which accompanied by increasing Wnt1, Wnt3, Wnt5, Axin, p-GSK3β, β-catenin expression. SR48692 and TSW119 can dowonregulated N-caherin, β-catenin and Wnt associated markers, and decreasd the invasion of hepatoma cells as well.Conclusion: High expression NTS correlated with aggressive phenotype and poor prognosis of HCC. NTS signal induces EMT of HCC cells via activation canonical Wnt/β-catenin pathway and thus promote HCC invasion and metatasis.