Hepatitis C virus NS4B protein induces epithelial-mesenchymal transition by upregulation of Snail

Abstract

Background: Chronic hepatitis C virus (HCV) infection is an important cause of hepatocellular carcinoma (HCC). Epithelial to mesenchymal transition (EMT) is a key process associated with tumor metastasis and poor prognosis. HCV infection, HCV core and NS5A protein could induce EMT process, but the role of NS4B on EMT remains poorly understood.

Methods: We overexpressed HCV NS4B protein in HepG2 cells or Huh7.5.1 cells infected by HCVcc, the E-cadherin expression, N-cadherin expression and the EMT-associated transcriptional factor Snail were determined. The migration and invasion capabilities of the transfected cells were evaluated using wound-healing assay. Additionally, we used Snail siRNA interference to confirm the relation of HCV NS4B and Snail on EMT promotion.

Results: HCV NS4B increased the expression of EMT related markers and promoted cell migration and invasion. Snail knock-down almost completely eliminated the function of NS4B protein in EMT changes and reversed cell migration capacity to lower level. HCV NS4B protein could reduce the expression of Scribble and Hippo signal pathway were subsequently inactivated, resulting in the activation of PI3K/AKT pathway, which may be the reason for the up-regulation of Snail.

Conclusions: This study demonstrates that HCV NS4B protein induces EMT progression via the upregulation of Snail in HCC, which may be a novel underlying mechanism for HCV-associated HCC development, invasion and metastasis.

Keywords: Cadherin; Epithelial to mesenchymal (EMT); HCV; Hippo pathway; Snail.

Fig. 1

HCVcc-infected cells presented the changes of EMT. The expression of E-cadherin, N-cadherin and Snail in Huh7.5.1 cells (Control group) and HCVcc infected Huh7.5.1 cells (HCVcc group). *P < 0.05, **P < 0.01

Fig. 2

HCV NS4B induced hepatocellular EMT. a HepG2 cells were transfected with pEGFPC1 plasmid (control group) and pEGFPC1-NS4B plasmid (NS4B group), 48 h later, the expression of E-cadherin and N-cadherin in control group and NS4B group. b HepG2 cells were transfected with pEGFPC1 plasmid (control group), 1, 3, 5 μg pEGFPC1-NS4B plasmid respectively, 48 h later, the expression of E-cadherin and N-cadherin in each group. c qRT-PCR analysis the mRNA expression of E-cadherin and N-cadherin in control group and NS4B group. d Immunofluorescence analysis of E-cadherin and N-cadherin in control group and NS4B group. *P < 0.05, **P < 0.01

Fig. 3

HCV NS4B promoted EMT due to upregulation of Snail. a HepG2 cells were transfected with pEGFPC1 plasmid (control group) and pEGFPC1-NS4B plasmid (NS4B group), 48 h later, the expression of Snail in control group and NS4B group. b HepG2 cells were transfected with pEGFPC1 plasmid (control group), 1, 3, 5 μg pEGFPC1-NS4B plasmid respectively, 48 h later, the expression of Snail in each group. c qRT-PCR analysis the mRNA expression of Snail in control group and NS4B group, The HepG2 cells were transfected with 3 μg pEGFPC1-NS4B plasmids in the qRT-PCR assay. d Western blotting analysis the expression of E-cadherin, N-cadherin, snail and HCV-NS4B in plasmids transfected cells, Snail siRNA for knockdown Snail. Co-transfected Snail siRNA and 3 μg pEGFPC1-NS4B plasmids into HepG2 cells, the expression level of Snail in HepG2 cells decreased after Snail siRNA transfection, and the phenomenons that downregulation of E-cadherin and upregulation of N-cadherin reversed while knockdown of snail. Con ~ for control group, n.s. ~ for no significant different, *P < 0.05, **P < 0.01

Fig. 4

HCV NS4B promoted hepatoma cell migration and invasion. Would healing assay were used to examine the migration and invasion capabilities of the HepG2 cells were transfected with HCV NS4B, cotransfected snail or control siRNA and NS4B. Microscopic observation of cell migration and recorded at 0, 12 and 24 h after scratching

Fig. 5

Western blotting analysis the key elements of Scribbe-Hippo-PI3K/AKT pathway in the HepG2 cells with HCV NS4B transfected. a HepG2 cells were transfected with pEGFPC1 plasmid (control group) and pEGFPC1-NS4B plasmid (NS4B group), 24 and 48 h later, the expression of Scribble, p-Yap and total Yap in control group and NS4B group. b HepG2 cells were transfected with 1, 3 μg pEGFPC1 plasmid and 1, 3 μg pEGFPC1-NS4B plasmid, 48 h later, the expression of p-akt, total-akt in each group

Fig. 6

The delineation of a potential molecular mechanism underlying EMT caused by HCV NS4B