A variant in the MICA gene is associated with liver fibrosis progression in chronic hepatitis C through TGF-β1 dependent mechanisms

Abstract

Hepatocarcinogenesis is tightly linked to liver fibrosis. Recently, two GWAS variants, MICA rs2596542 and DEPDC5 rs1012068 were identified as being associated with the development of HCV-induced hepatocellular carcinoma (HCC) in Japanese patients. The role of these variants on hepatic inflammation and fibrosis that are closely associated with HCC development is not known, nor are the biological mechanisms underlying their impact on the liver. Here, we demonstrate in 1689 patients with chronic hepatitis C (CHC) (1,501 with CHC and 188 with HCV-related HCC), that the MICA (T) allele, despite not being associated with HCC susceptibility, is associated with increased fibrosis stage (OR: 1.47, 95% CI: 1.05-2.06, p = 0.02) and fibrosis progression rate (hazards ratio: 1.41, 95% CI: 1.04-1.90, p = 0.02). The DEPDC5 variant was not associated with any of these phenotypes. MICA expression was down-regulated in advanced fibrosis stages. Further, (T) allele carriage was associated with lower MICA expression in liver and serum. Transforming growth factor-β1 (TGF-β1) expression suppresses MICA expression in hepatic stellate cells. Our findings suggest a novel mechanism linking susceptibility to advanced fibrosis and subsequently indirectly to HCC, to the level of MICA expression through TGF-β1-dependent mechanisms.

Figure 1

Association of MICA rs2596542 and DEPDC5 rs1012068 with inflammation, fibrosis stage. Association of MICA rs2596542 with fibrosis stage (a) and inflammation (b) and DEPDC5 rs1012068 with fibrosis stage (c) and inflammation (d) in the CHC cohort (n = 1501). P-values are univariate and provided for the additive model of inheritance. MICA rs2596542 (T) allele is the risk allele, the same risk allele in the GWAS by Kumar et al.. DEPDC5 rs1012068 (G) allele is the risk allele, the same risk allele in the GWAS by Miki et al..

Figure 2

Hepatic and serum MICA expression. Relative hepatic MICA mRNA expression in 94 patients with chronic hepatitis C virus infection and 28 controls (a); relative MICA mRNA expression in Huh7 cells infected with the JFH1 strain hepatitis C virus as compared to mock infected control cells (b); correlation between MICA rs2596542 genotype and hepatic MICA mRNA (c) and soluble MICA levels (n = 214) (d). The x axis shows the genotypes at rs2596542 and the y axis shows MICA expression level relative to GAPDH by quantitative real-time PCR or the concentration of soluble MICA in pg/ml by ELISA, respectively. The level of hepatic MICA mRNA correlates positively with serum soluble MICA levels in a sub-cohort of CHC with available RNA and serum (n = 58) (e); hepatic MICA mRNA according to hepatic fibrosis (f). The x axis shows hepatic fibrosis dichotomized as absent/mild (METAVIR stage F0–F1) or moderate/severe (METAVIR stage F2–F4), and the y axis shows hepatic MICA expression. The number of independent samples tested in each group is shown below the figure and P value was calculated using the two-tailed Student’s t-test or ANOVA test with Tukey test for correction for multiple comparisons.

Figure 3

Transforming growth factor beta influences MICA expression. Relative TGF-β1 mRNA expression in Huh7 cells infected with the JFH1 strain of the hepatitis C virus compared to mock infected control cells (a); relative hepatic TGF-β1 mRNA expression in 94 patients with chronic hepatitis C virus infection and 28 controls according to fibrosis stage (b); serum concentration of TGF-β1 in pg/ml levels according to hepatic fibrosis (n = 214) (c); MICA mRNA expression and protein levels at the hepatic stellate cell surface modulation by TGF-β1. MICA mRNA expression was assessed in control, TGF-β1 (2 ng/ml) with or without LY2109761 (a TGFβR-I/II inhibitor) (100 nM, was added 90 minutes before TGF-β1), or LY2109761 (100 nM) treated cells for 24 hours by real-time PCR (d), or 48 hours for flow cytometry (e); the level of soluble MICA was assessed in the supernatant of control or TGF-β1 (2 ng/ml) treated LX2 cells for 24 and 48 hours by ELISA (f). Results are expressed, as mean ± sem (n = 3) and P value was calculated using the two-tailed Student’s t-test or ANOVA test with Tukey test for correction for multiple comparisons. *P < 0.05,**P < 0.001, ***P < 0.0001.

Figure 4

Proposed model for the effect of MICA rs2596542 in modulating liver fibrosis risk. Patients carrying the MICA risk genotype have low hepatic and serum MICA expression. TGF-β1 further suppresses MICA expression on hepatic stellate cells, leading to increased liver fibrosis.