HBx Induces Epithelial-Mensenchymal Transition By Upregulating Snaill In Hepatocellular Carcinoma Cells

BACKGROUND AND OBJECTSThe chronic infection of hepatitis B virus (HBV) is an independent risk factor of hepatocellular carcinoma (HCC). The hepatitis B virus X protein(HBx) encoded by HBV genome is an important transcriptional regulator which is closely involved in transactivating of oncogenes and plays an important role in malignant transformation of hepatic cells. HBx is also a key mediator of HCC invasion and metastasis in some way of disrupting the adherens junctions of cell to cell, promoting degradation of intercellular substance, inducing epithelial to mesenchymal transition (EMT) of tumor cells.The functional loss of E-cadherin is the hallmark of EMT. Previous studies have shown that HCC with lower E-cadherin expression that showed poor differentiation and more likely introhepatic metastasis and portal vein invasion. The expression of E-cadherin is also closely related to the prognosis of HCC. Transcriptional silence of CDH1 which encoding E-cadherin is the most important mechanism of down-regulation of E-cadherin.Recently, zinc-finger transcription factor Snail,which plays an important role in the formation of mesoderm,has been described to directly repress transcription of the CDH1 by binding to the E-boxes (CACCTG sequence) on its promoter. Because of the strong capacity of repress CDH1 transcription and induction of EMT, Snail is also involved in the progression of various epithelial carcinomas , trigerring invasion, metastasis, and dedifferentiation. Clinical research has showed that Snail is related to the invasion, metastasis and prognosis of HCC. But no data showed that if there was somehow relationship between HBx and Snail. It is still not known that what kind of role snail played in the progression of HCC with HBV infection.In this study, we supposed that HBx could up-regulate the expression of Snail1 protein by activating the transcriptional activity of SNAI1 gene. Moreover, the Snail1 up-regulation can induce EMT of HCC, trigerrin the invasion and metastasis of hepatocellular carcinoma. The emphasis is that Snail1 is the key mediator in the progression of HBx promoting the invasion and metastasis of HCC.MATERIALS AND METHODS1. HBx, Snail1, E-cadherin, N-cadhe and Vimentin were detected in 74 cases of HCC via immunochemistry staining. The correlations of the proteins and the the correlations between expression of proteins and clinical pathological properties were analyzed. We expected to identified that if the expression of Snail1 protein was correlated to the pathological staging of HCC and HBx protein expression.2. We cloned SNAI1 gene coding domain into pAdeasy-1 to form recombinant adenovirus Ad-SNAI1. Transfecting Ad-SNAI1 into HepG2 cells was performed to induce exogenous overexpression of Snail1. Then to investigate EMT markers and the invasive and migratory properties of HCC.3. HepG2 cells were transfected with recombinant adenovirus Ad-HBx. Snail1, EMT markers and the cells'property of invasion and migrasion were measured. So we can see whether HBx could induce endogenous overexpression of SNAI1,and furthermore, induce EMT and promote invasion and migration by this way.4. Silencing the transcription of SNAI1 was performed to investigate whether loss of Snail1 could inverse EMT of HCC induced by HBx. Therefore, we cloned the proper shRNA of SNAI1 to pGPU6/GFP/Neo vector. To improve the efficiency of target gene silencing, we subcloned the hu6 promoter and shRNA from the vector to adenovirus.Then Ad-shSNAI1 and Ad-HBx were cotransfected to HepG2 cells. And then, Snail1, EMT markers and the cells'property of invasion and migrasion were measured.5. A series of 5'-flanking SNAI1 promoter were amplificated from human liver DNA, and then subcloned to pGL3-Basic vector. Transfected the pGL3-Basic-SNAI1 promoters into SMMC-7721 cells together with or without Ad-HBx.The activities of promoters was mesuered and compared. pRL-TK vector was cotransfected as the inner control.RESULTS1. There are 58 in 74 cases that with positive HBx expression, 47cases for Snail1. The expression of HBx and SNAI1 were correlated significantly in 45 cases which both positive expression for HBx and Snail1 (p=0.002). SNAI1 positive expression was correlated with the absence of E-cadherin, up-regulation of N-cadherin and Vimentin. Clinical data Analysis showed: HBx positive expression was correlated with the clinical features including TNM staging, tumor size, portal invasion, peri-liver invasion and HBV-DNA in peripheral blood. So we considered Snail1 may be testified as a marker for the invasive and migratory properties of HCC, and the up-regulation of SNAI1 was concerned with HBx.2. Successfully subcloned the SNAI1 coding domain to adenovirus vector pAdeasy-1. The recombinant adenovirus named Ad-SNAI1. We got high expression level of the target gene with Ad-SNAI1 transfeced into HepG-2 cells. The expression of E-cadhrein was down-regulated accomplished with N-cadhrein and Vimentin up-regulated and improved invasion and migration of the hepatocarcinoma cells.3. Transfected with Ad-HBx, the SNAI1 mRNA and Snail were all overexpressed in HepG2 cell line. E-cadhrein was greatly down-regulated together with N-cadhrein and Vimentin up-regulation. The hepatocarcinoma cells also showed improver invasion and migration.4. siRNA sequence of SNAI1 was designed and tested. Then the hU6-shSNAI1 segament was subcloned to adenovirus vector to form Ad-shSNAI1. Ad- shSNAI1 was cotransfected with HBx to HepG2 cell line. The epithelial phenotype was maintained with slight upregulation E-cadherin and downregulation of mensenchymal markers. Decreased invasion and migration of HepG2 cell lines was also observed. It proved that EMT and promoted invasion and migration induced by HBx could be reversed thoroughly by Snail1 silencing.5. Successfully subcloned the 5'-flanking SNAI1 promoters to pGL3Basic vector. HBx could improve the transcriptional activity of SNAI1 promoter. The segment (-869~-514) ( count from the transcriptional initiation site) played an important role in the procedure of the transcriptional regulation of SNAI1 by HBx.CONCLUSIONS1. The expression of SNAI1 was closely correlated with HBx. Both of them had related to the invasion and metastasis of HCC. 2. Ectogenetic Snail1 could induce EMT of HCC cells. Up-regulation of the Snail1 by HBx could induce EMT in HCC cells.3. Sanil1 silencing could efficiently revesed EMT of HCC cells induced by HBx, the cells maintained epithelial phenotype and the invasive & migratory abilities of HCC were decreased. It showed that Snail1 was the key mediator in the procedure of HBx induced EMT in HCC.4. HBx may upregulate the transcriptional activity of SNAI1 gene by activating PI3K-Akt pathway. The segment (-869~-514) ( count from the transcriptional initiation site) played an important role in the procedure of the transcriptional regulation of SNAI1 by HBx.