BVES Inhibition Triggers EMT In Human Hepatocellular Carcinoma And Is Regulated By Proto-oncogenic Protein Netrin-1

OBJECTIVE: The leading cause of HCC-related mortality is cancer metastasiswhich includes cell adhesion to the extracellular matrix (ECM), ECM degradation,cellmigration,invasion and macrometastasis. BVES is a three-pass transmembrane protein,it has been reported to play a role in maintaining epithelial integrity and regulatingcell movement,which are the hot topics in cancer research. Recently, BVES has beenstudied to play an important role in the development of cancer, however, theunderlying mechanisms and upstream factors remain to be explored, the importanceof BVES in HCC, especially in the EMT process of HCC have never been reported. Inthis study, we detected the role of BVES in the EMT of HCC, studied the relationshipbetween BVES and proto-oncogenic protein Netrin-1，and discussed the mechanismsunderlying upon our studys on Netrin-1previously.METHODS: The expression of BVES was fist detected in HCC tissues and theircorresponding paracancerous tissues by qRT-PCR、 Western blot and IHC methods.RT-PCR was used to measure the expression of BVES in HCC cells with differentmetastatic potential. The siRNAs targeting the mRNAof BVES（BVES siRNA） andcontrol siRNA were constructed. After transfected siRNA into Huh7cells， theinterference efficiency and the expression of mesenchymal marker Vimentin andepithelial marker E-cadherin were detected by qRT-PCR and Western blot. The mRNA of Twist1、Snail1、MMP2/MMP9and IL-6were detected by qRT-PCR. F-actincytoskeletal was detected using TRITC-phalloidin in Huh7cells. The migratory andinvasive ability of Huh7cells after BVES inhibition were analyzed by cell migrationand invasion assay. The expression of BVES was further studied in HCC cellsoverexpressed of Netrin-1and after addition of recombinant human Netrin-1protein.Western blot was used to detect the impact of Netrin-1protein on PI3K/Akt signallingpathway. PI3K/Akt inhibitor LY294002was carry out to study the mechanism ofNetrin-1role in the regulation of BVES expression.RESULTS:14of the21HCC samples(66.7%) showed decreased BVESexpression compared with their pericarcinous tissues，BVES was only detected in thecytoplasm and membrane by IHC. BVES decreased in a metastaticpotential-dependent manner：the expression of BVES decreased as the metastaticpotential of HCC cell increased. After BVES inhibited by siRNA, the expression ofE-cadherin decreased68.20±0.72%（p <0.05), opposed to an3.71±0.93timesincrease of Vimentin（p <0.05); meanwhile，Huh7cell underwent reorganization ofthe F-actin cytoskeleton. To study the effects of BVES inhibition on hepatocellularcarcinoma cell migration, we detected the cells in Transwell after BVES knockdown，both migration and invasion ability were increased after BVES inhibition(p <0.01, n=5). MTT assay was carried out to have excluded the possibility that the enhancedmigration and invasion ability of BVES inhibition were caused by cell proliferation asBVES inhibition did not affect the proliferation of Huh7cells in24h.We also foundan increase in Twist1、Snail1、MMP2/MMP9and IL-6mRNA. Interestingly, we foundBVES expression was reduced in the Huh7cell transfected with Netrin-1, and BVESwas reduced accompanyed by the increase of the phosphorylation of Akt afteraddition of recombinant human Netrin-1protein. Further analysis showed thatPI3K/Akt inhibitor LY294002restored the downregulation of BVES caused byNetrin-1. CONCLUSIONS: We conclude that BVES is concerned with the metastasis ofHCC, BVES inhibition triggers EMT in human HCC cells. We propose that BVEScan be downregulated by Netrin-1, molecule mechanism study suggests PI3K/Aktsignalling pathway plays an important role in this process. Further study the roleBVES plays in Netrin-1induced EMT of HCC, may provide potential targets for theintervention of liver cancer metastasis.