PAI-1 participation in hepatocyte epithelial-mesenchymal transition induced by HCV NS5A or Core proteins.

Introduction and objectives

Chronic HCV infection induces the development of liver fibrosis mediated by hepatocyte epithelial-mesenchymal transition (EMT). Plasminogen activator inhibitor (PAI-1) has been associated with fibrosis. Objective: To evaluate the participation of PAI-1 in the EMT of hepatocytes induced by HCV proteins.

Materials and patients

Huh7 cells were transfected with 1µg of the plasmid to express HCV NS5A or Core proteins, cells were co-cultured with hepatic stellate cells (LX2), at 48 and 72 hours of co-culture, the expression of LX2 activation biomarkers were determined by western blot and RT-qPCR. Likewise, transcriptional expression of 84 genes associated with fibrosis in Huh7 was determined by RT-qPCR array. Bioinformatic analyses were performed with Enrichr and STRING. serpine1 (PAI-1) was selected as one of the differentially expressed genes, Huh7 cells were transfected with NS5A or Core, and after 24 hours gene silencing was performed with siPAI-1 (5nM) and pharmacological treatment with TM5275 (25 μM) to inhibit the PAI-1 function. After 24 and 48 hours of treatment, the expression of the viral proteins was validated by chemiluminescence and western blot. Likewise, the PAI-1 inhibition was validated and the translational expression of EMT markers (TGFβ1, Snail, E-cadherin, and Vimentin) was evaluated in Huh7 cells by western blot and densitometric analysis.

Results

NS5A and Core expression in Huh7 cells co-cultured with LX2 cells, induced transcriptional overexpression of TGFβ1, Col1α1, and Timp1, suggesting LX2 activation. We observed 28 genes differentially expressed in Huh7 (NS5A+) and 46 genes were differentially expressed in Huh7 (Core+) during co-culture with LX2 cells. Bioinformatics analyses were performed, and serpine1 (PAI-1) was identified as a differentially expressed gene. On the other hand, at the translational level, NS5A induced the overexpression of TGFβ1 and Snail (4-fold) and subexpression of E-cadherin (0.6-fold). Likewise at the translational level, Core induced the overexpression of Snail (2.5-fold) and subexpression of E-cadherin (0.4-fold), compared to the control, suggesting the EMT of Huh7. A gene silencing of 60% of PAI-1 was obtained in all groups. this silencing induces a reduction of 50% of vimentin expression at the translational level in all groups. On the other hand, TM5275 decreased the expression of TGFβ1 by 60% both in the control group and in the NS5A transfected cells. Likewise, TM5275 increased the expression of E-cadherin at the translational level by 60% both in the control group and in the Core transfected cells.

Conclusions

HCV proteins regulate the expression of molecular markers and signaling pathways in hepatocytes associated with the development of EMT, such as PAI-1. At the same time, PAI-1 inhibition negatively regulates this EMT, which is important to understand the pathophysiology of HCV damage.

Ethical statement: not applicable

Declaration of interests: None.

Funding: Financial support was provided by the Department of Biochemistry and Molecular Medicine and CIIViM, School of Medicine, Universidad Autónoma de Nuevo León (UANL), Monterrey, Nuevo León 64460, México.