Epithelial-mesenchymal Transition Induced By HCVc Protein In Cholangiocarcinoma And Its Molecular Mechanism

Purpose: Cholangiocarcinoma (CC) is associated with chronic hepatitis C virus(HCV) infection. Hepatitis C virus core protein(HCVc) is an important protein product encoded by the HCV genome, it's function involved in interaction of cell protein, cell signal transduction, carcinogenesis and lipids metabolism. It perhaps participates carcinogenesis of cholangiocarcinoma by triggering NF-κB signaling pathways. Epithelial-mesenchymal transition(EMT) was defined by the formation of mesenchymal cells from epithelia in different embryonic territories. Recently, increasing evidences suggested that EMT was involved in cancer invasion and metastasis. In a view of significance of NF-κB signaling pathway in EMT, we studied the correlation between HCVc protein and CC EMT, intended to prove HCVc protein can induce CC EMT. The high positive expression of LOXL2 which is a key regulator of EMT was observed in CC tissues and the positive expression of LOXL2 was associated with CC tissues'invasion and metastasis, so we studied the effect of LOXL2 in the CC EMT induced by HCVc protein to preliminaryly reveal the mechanism of CC EMT.Experimental Design: In the first part of the experiment, we examined the expression of HCVc protein, epithelial markers:E-cadherin,β-catenin andα-catenin, mesenchymal markers:N-cadherin,vimentin and fibronectin by immunohistochemistry, and assessed their correlation and clinic pathological significance. In the second part of the experiment, we transfected a recombinant plasmid vector containing HCVc gene into QBC939 cell to observe the morphological change under microscope 5 days after transfection.The expression and localization of HCVc and LOXL2 proteins, epithelial and mesenchymal markers were determined by RT-PCR,Realtime PCR,immunocytochemistry and western blotting. In the third part, we co-transfected recombinant plasmid vector containing HCVc gene and LOXL2 shRNA to assesse the effect of LOXL2 in QBC939 EMT induced by HCVc protein.Results:1. In CC tissues, the positive expression rate was observed in 47.1% for HCVc protein, 50% for N-cadherin, 44.1% for Vimentin, 55.9% for Fibronectin and the decreased expression rate was E-cadherin for 55.9%,α-catenin for 70.6%,β-catenin for 55.9%, The positive expression of HCVc protein were associated with the decreased expression of E-cadherin,α-catenin and the positive expression of N-cadherin,Vimentin,Fibronectin(P<0.05), a positive-correlation between the expression of HCVc protein and metastasis of ganglia lymphatica and other organs were found(P<0.05). HCVc protein perhaps promote cholangiocarcinoma tissues'infiltration and metabasis by inducing it's EMT.2. The HCVc gene was obtained from PGST-HCVc195 plasmid. After it transfected into QBC939 cell, the high expression of HCVc mRNA and protein were observed by RT-PCR and Realtime PCR, immunocytochemistry and western blotting. 3. The QBC939 cell transfected with HCVc gene underwent a morphological change from a classic epithelial morphology to a spindle-like shape 5 days after transfection. In addition to this, the HCVc protein significantly decreased E-cadherin expression, but increased vimentin and fibronectin expression in QBC939 cell.4. The cell migration and invasion assays indicated that transfection of HCVc gene drastically enhanced the migratory and invasive potentials of QBC939 cell.5. After the QBC939 cell transfected with HCVc gene 48 hours, the expression of LOXL2 was significantly increased by HCVc protein.6. The HCVc gene was obtained from PGST-HCVc195 plasmid and LOXL2 shRNA gene was obtained from pGenesil-shLOXL2, after they co-transfected into QBC939 cell, the high expression of HCVc and the decreased expression of LOXL2 mRNA and protein were observed by RT-PCR,Realtime PCR,immunocytochemistry and western blotting.7. After co-transfection 48 hours, the decreased expression of E-cadherin and increased expression of vimentin and fibronectin induced by HCVc protein were inhibited by LOXL2 gene interference in QBC939 cell.Conclusions: The clinic pathological data indicated that loss of E-cadherin and gain of vimentin, fibronectin may lead to a more aggressive tumor behavior in CC tissues, it may has relation with HCVc protein. HCVc gene transfection data suggested that it can induce QBC939 cell EMT. The result of HCVc gene and LOXL2 shRNA co-transfection indicated that LOXL2 can inhibit EMT induced by HCVc protein in QBC939 cell, so we presumed that QBC939 undergoing EMT maybe triggered by the key EMT-inducing factor LOXL2. Furthermore, HCVc protein enhances migratory and invasive potentials of QBC939 cell.