Increased microRNA-155 expression in the serum and peripheral monocytes in chronic HCV infection

Abstract

Background: Hepatitis C Virus (HCV), a single stranded RNA virus, affects millions of people worldwide and leads to chronic infection characterized by chronic inflammation in the liver and in peripheral immune cells. Chronic liver inflammation leads to progressive liver damage. MicroRNAs (miRNA) regulate inflammation (miR-155, -146a and -125b) as well as hepatocyte function (miR-122).

Methods: Here we hypothesized that microRNAs are dysregulated in chronic HCV infection. We examined miRNAs in the circulation and in peripheral monocytes of patients with chronic HCV infection to evaluate if specific miRNA expression correlated with HCV infection.

Results: We found that monocytes from chronic HCV infected treatment-naïve (cHCV) but not treatment responder patients showed increased expression of miR-155, a positive regulator of TNFα, and had increased TNFα production compared to monocytes of normal controls. After LPS stimulation, miR-155 levels were higher in monocytes from cHCV patients compared to controls. MiR-125b, which has negative regulatory effects on inflammation, was decreased in cHCV monocytes compared to controls. Stimulation of normal monocytes with TLR4 and TLR8 ligands or HCV core, NS3 and NS5 recombinant proteins induced a robust increase in both miR-155 expression and TNFα production identifying potential mechanisms for in vivo induction of miR-155. Furthermore, we found increased serum miR-155 levels in HCV patients compared to controls. Serum miR-125b and miR-146a levels were also increased in HCV patients. Serum levels of miR-122 were elevated in cHCV patients and correlated with increased ALT and AST levels and serum miR-155 levels.

Conclusion: In conclusion, our novel data demonstrate that miR-155, a positive regulator of inflammation, is upregulated both in monocytes and in the serum of patients with chronic HCV infection. Our study suggests that HCV core, NS3, and NS5 proteins or TLR4 and TLR8 ligands can mediate increased miR-155 and TNFα production in chronic HCV infection. The positive correlation between serum miR-155 and miR-122 increase in cHCV may be an indicator of inflammation-induced hepatocyte damage.

Figure 1

Induction of miR-155 in peripheral monocytes of chronic HCV treatment naïve patients.A. Total RNA was isolated from the monocytes of healthy volunteers (n = 6), chronic HCV treatment naïve patients (n = 12) and sustained responders (n = 5) with miRNeasy kit (Qiagen). B. Monocytes of healthy controls and chronic HCV treatment naïve patients were stimulated with 100 ng/ml LPS for 6-8 h. TaqMan miRNA assay (Applied Biosystems) was used for detection of miR-155. RNU48 was used as a normalization control. The non-parametric Mann–Whitney test was employed for statistical analysis.

Figure 2

Decreased miR-125b expression in peripheral monocytes of chronic HCV treatment naïve patients.A-B. Total RNA was isolated from monocytes of healthy volunteers (n = 6), chronic HCV treatment naïve patients (n = 12) and sustained responders (n = 5) with miRNeasy kit (Qiagen). MiR-125b and miR-146a levels were detected by TaqMan miRNA assay (Applied Biosystems), and RNU48 was used as a normalization control. The non-parametric Mann–Whitney test was employed for statistical analysis.

Figure 3

Increased TNFα and TLR8 expression in peripheral monocytes of chronic HCV treatment naïve patients.A-C. Total RNA was isolated from monocytes of healthy volunteers treated or not (n = 6-8), and from chronic HCV treatment naïve patients stimulated or not (n = 9-10) with 100 ng/ml LPS for 6-8 h. Real time PCR was carried out using gene-specific primers for TNFα (A), TLR4 (B) and TLR8 (C) expression. 18 S was used as a normalization control and the non-parametric Mann–Whitney test was employed for statistical analysis.

Figure 4

Viral NS3, NS5 and core proteins and TLR4 and TLR8 ligands induce miR-155 and TNFα in monocytes of healthy controls.A-B. Monocytes of healthy controls (n = 5) were stimulated or not with 100 ng/ml LPS (TLR4 ligand), 5ug/ml CLO75 (TLR8 ligand), 5ug/ml viral core protein, NS3 and NS5 proteins (non-structural viral proteins), and E2 (viral envelop protein) for 6-8 h. The experiment was performed in duplicates. A. Total RNA was isolated and subjected to TaqMan miRNA assay for miR-155. B&C. TNFα mRNA expression was examined by real time PCR (B) and protein levels were determined by ELISA in cell-free supernatants (C). Two-tailed t-test was used for statistical analysis.

Figure 5

Increased serum miR-155, miR-125b and miR-146a levels in chronic HCV-infected patients.A-C. 100ul of serum was used for total RNA isolation from both healthy controls (n = 12-18) and chronic HCV patients (n = 30-36) with miRNeasy kit (Qiagen). TaqMan miRNA assays (Applied Biosystems) were used to detect miRNA levels and C.elegans (Cel)-miR-39 was used to normalize the ct values. The non-parametric Mann–Whitney test was used for statistical analysis.

Figure 6

Increased circulating miR-122 levels correlate with serum ALT, AST and miR-155 levels in chronic HCV-infected patients.A. 100ul of serum was used for total RNA isolation from both healthy controls (n = 12-13) and chronic HCV patients (n = 33) with miRNeasy kit (Qiagen). Serum miR-122 levels were detected with TaqMan miRNA assay as described in the methods. The non-parametric Mann–Whitney test was used for statistical analysis. B. Serum ALT or AST levels of healthy and chronic HCV-infected individuals. C-E. Correlation between serum miR-122 and ALT (C) and AST (D) and serum miR-155 (E) was determined with the Spearman method.