Hepatitis C Virus Core Protein-induced Downregulation Of E-cadherin By Stabilizition Of Snail

Objective: Hepatocellular carcinoma (HCC) is one of the malignanciesseriously threatening to the health of humankind. Multiple risk factors canresult in the development of hepatocellular carcinoma，one of the mostimportant risk factors for developing HCC is chronic Hepatitis C virus(HCV). Up to170million people have infected with HCV worldwide and80%of them will develop into chronic hepatitis C, and then progress intoliver fibrosis or cirrosis, even HCC. One main reason for the high mortalityof HCC is attributed to the features of high malignant degree and earlymetastasis. So far, the majority of studies suggest that the proteins encodedby HCV, especially HCV Core, not only play an important role in thedevlepoment of HCV-related liver diseases, but also participate in theprocess of HCC metastasis with some unclarified mechanisms.Epithelial–mesenchymal transition (EMT) results in the coordinateddissolution of cell–cell adhesions, loss of apical-basolateral polarity, and thereorganization of the actin cytoskeleton to promote mesenchymal cellmigration and invasion. Previous researchs associated with EMT mostlyfocus on the embryonic development period, that is, most of these studiesaim to investigate the effects of EMT in normal embryonic developmentproscess such as the mesoderm and heart growth as well as neural crestformation. Recent studies demonstrated that EMT is closely related to the migration, invasion and metastasis in a variety of tumors including breast,pancreatic, colon cancer, hepatoma and melanoma. However the specificmetastatic mechanisms of EMT in the HCV-related HCC have still not beenclearly elucidated.Migration or invasion ability enhancement and apoptosis suppressionare the main cellular phenotypic changes of EMT. Canonical EMTbiomarkers not just contain the E-cadherin downregulation, overexpressionof vimentin, N-cadherin and fibronectin and the nuclear translocation ofβ-catenin, but include the upregulation of inhibitory transcription factorssuch as Snail, Slug, ZEB1, ZEB2and E47which control the E-cadherinexpression.E-cadherin is a kind of functional protein aossciated with theformation of cellular tight junction. Deficiency of E-cadherin will increasethe ability of invasion and metastasis, on the contrary, the recovery ofE-cadherin level can restore the epithelial phonetype and decrase themigration ability in tumor cells. In addtion, E-cadherin ectopic expressioncan also inhibit the transcription of specific expressing genes inmesenchymal cell. In consequence, loss expression of E-cadherin isrecongnized as a hallmark of EMT.Previous study has shown that Snail, by identifying conservativesequence5’-CAGGTG-3’, is capable of binding E-box elements within theCDH1promoter regions, repressing the activity of CDH1, and finallydownregulating the E-cadherin expression. Recent researchs on HCC havedemonstrated a negative correlation between E-cadherin’s low expressionand the expression quantity of Snail, that is, high Snail expression is relatedto high rates of metastasis and poor prognosis for HCC patients. Ourprevious studies identified that HCV core protein is able to decrease theexpression of E-cadherin. Of note, it remains unclear whether HCV-encoded viral core proteins have a direct role in stabilization of the expression of snailand then further regulate the level of E-cadherin to promote the migration,invasion and metastsis in HCC. As a consequence, the aim of this study is tofurther investigate the molecular mechanism of HCV core regulating theE-cadherin expression and reveal the specific role of HCV core in HCCmetastasis.Methods: We used wound healing, invasion assay, Real-time PCR,Western blot, immunofluorescence to investigate HCV Core induced-EMTin core-expression Huh7cells. Then the effects of Core protein onAKT/GSK-3β signaling patheway were assessed by western blot,immunofluorescence, siRNA technology. We constructed deletion mutationof CDH1promoter and site-directed mutation of E-box on the CDH1promoter region. And then we determined whether HCV Core could inhibitCDH1promoter activity and examined which E-box is the strongest activityof CDH1promoter by using the luciferase assay system. ChIP was furtherused to determine the levels of Core and Snail bounded to the CDH1promoter region in core-expressing cells. Besides, IP was used to determinewhether core binds to Snail. Moreover, we assessed the role of core proteinon tumor formation and metastasis in xenograte model and liver metastasismouse model. Then we determined the relationship of core, Snail anddownstream molecular E-cadherin in the tumor samples retrieved from liverof mice using immunohistochemistry.Results： In core-expression Huh7cells, HCV Core protein haddecreased expression of the epithelial marker E-cadherin and increased thelevel of the mesenchymal markers vimentin, Fibronectin. More importantly,similar molecular alterations occurred upon HCV replication system. Wefurther performed migration assay and wound healing test in core-expressinghuh7cells transfected with E-cadherin. Core-induced EMT was blocked by increasing the level of E-cadherin，which substantiated that with decreasingthe E-cadherin expression, Core promoted hepatoma cells invasion,migration and metastasis. The molecular events that Core protein activatesEMT-related pathway were further investigated. The results showed thatHCV Core did not activate TGF-β signaling in our experiment model.However, AKT/GSK3β signaling pathway was activated during HCVCore-induced EMT progress, which leads to inhibition of expression ofE-cadherin through stabilization of Snail.Indeed, the activity of the CDH1promoter showed a significantdecrease in Ad-core-infected Huh7cells, whereas, the GFP control had noeffect on CDH1promoter activity. Mutational analysis of CDH1promoterdemonstrated that each of three E-boxes located within the fragmentsupported core repressive CDH1promoter activity, and the first E-box(from-80to-75) displayed the strongest activity. Moreover, a combinedmutation with E-box1(from-80to-75) and E-box3(from-30to-25)caused moderate upregulation of CDH1promoter activity to a greaterextent than in cells transfected with reporter construction carry singlemutation alone. To further validate molecular mechanism through whichHCV Core regulates E-cadherin promoter activity, we performed chromatinimmunoprecipitation (ChIP) assay in core-expressing Huh7cells. Ourresults indicated that Core enhanced the binding of Snail to the proximalregion of the TSS of CDH1, Moreover, HCV Core specifically bounds toCDH1promoter region, indicating HCV Core bounding to the TSS ofCDH1region by recruitment of Snail. We wondered whether interactionbetween Core and Snail could directly suppress E-cadherin expression.Using immunoprecipitation (IP) assay, we found that core and Snail couldinteract virtually in Huh7cells. Collectively, these data indicated that coreand Snail formed a repressor complex to silence E-cadherin. Consistent with the increased level of Snail, HCV Core also enhancedthe expression of MMP-2, MMP-9and VEGF, conversely, the expressionof E-cadherin was dramatical decreased. These data suggested that HCVCore promoted tumor growth and aggressiveness in vivo. To further explorethe role of core in HCC metastasis in vivo, we mimicked the mechanism ofhuman HCC metastasis after the formation of primary HCC in liver of nudemouse. The results indicated that HCV Core promoted intrahepatic andlung metastasis in the liver implanted with core-expressing Huh7cells.While, sustainable knockdown of Snail restored core-mediateddown-regulation E-cadherin expression, decreased the expression ofMMP-2and inhibited Core-promoted tumor metastasis.Conclusion: Our study demonstrated that HCV core-induced Snailstablizition is mediated by the activation of the PI3K/AKT/GSK3β pathway,which results in the downregulation of E-cadherin and facilitating the EMTof hepatoma cells in vitro and in vivo. Both a novel molecular mechanism ofHCV-induced EMT which relays on the core-snail transcriptionalcorepressor complex and E-cadherin low expression and snail-dependentpromotion of core-associated hepatocarcinogenesis are illuminated inpresent investigation. These results not only provide new evidence to clarifythe specific cause and process of HCV-related hepatocellular carcinoma, butalso offer a new orientation for its clinical treatment.