Circulating microRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver disease

Abstract

MicroRNAs miR-122, miR-34a, miR-16 and miR-21 are commonly deregulated in liver fibrosis and hepatocellular carcinoma. This study examined whether circulating levels of these miRNAs correlate with hepatic histological disease severity in patients with chronic hepatitis C infection (CHC) or non-alcoholic fatty-liver disease (NAFLD) and can potentially serve as circulating markers for disease stage assessment. We first used an in vitro model of hepatitis C virus (HCV) infection to measure the extracellular levels of these four miRNAs. Whereas miR-21 extracellular levels were unchanged, extracellular levels of miR-122, miR-34a and to a lesser extent miR-16, steadily increased during the course of HCV infection, independently of viral replication and production. Similarly, in CHC patients, serum levels of miR-122, miR-34a and miR-16 were significantly higher than in control individuals, while miR-21 levels were unchanged. There was no correlation between the serum levels of any of these microRNAs and HCV viral loads. In contrast, miR-122 and miR-34a levels positively correlated with disease severity. Identical results were obtained in an independent cohort of CHC patients. We extended the study to patients with NAFLD. As observed in CHC patients, serum levels of miR-122, miR-34a and miR-16 were significantly higher in NAFLD patients than in controls, while miR-21 levels were unchanged. Again, miR-122 and miR-34a levels positively correlated with disease severity from simple steatosis to steatohepatitis. In both CHC and NAFLD patient groups, serum levels of miR-122 and miR-34a correlated with liver enzymes levels, fibrosis stage and inflammation activity. miR-122 levels also correlated with serum lipids in NAFLD patients.

Conclusion: Serum levels of miR-34a and miR-122 may represent novel, noninvasive biomarkers of diagnosis and histological disease severity in patients with CHC or NAFLD.

Figure 1. Differential expression of miRNAs in supernatant of HCV-infected cells.

(A) Detection of intracellular and extracellular HCV RNA. (B) miR-122, miR-34a, miR-16 and miR-21 levels in uninfected and HCV-infected Huh7.5 cells at 5, 10 and 15 days post-infection. Cycle threshold (C_T) values were converted to an absolute value based on the standard curve. The expression levels are presented as the mean ± standard error of the mean (SEM) (copy number/ml of supernatant or copy number/µg of total RNA) of three independent experiments.

Figure 2. Up-regulation of serum miR-122, miR-34a and miR-16 in chronic hepatitis C patients.

Serum levels of miR-122, miR-34a, miR-16 and miR-21 in (A) a first set of 18 CHC patients and (B) an independent set of 35 CHC patients. C_T values were converted to an absolute value based on the standard curves. Serum miRNA expression levels are expressed in copy number/ml. In the box-plot displays, the bold line indicates the median per group, the box represents 50% of the values and horizontal lines show minimum and maximum values of the calculated non-outlier values. For miR-34a, the dashed lines represent the levels corresponding to C_T values between 35 and 37 (1.2×10⁴ – 0.3×10⁴copies/ml).

Figure 3. Serum levels of miR-122, miR-34a, miR-16 and miR-21 and histological liver disease severity in CHC patients.

CHC group was subdivided into (A) CHC-early (F0-F1) and CHC-advanced groups (F3-F4) and (B) further subdivided according to individual fibrosis stage.

Figure 4. Serum levels of miR-122, miR-34a, miR-16 and miR-21 in NAFLD patients.

(A) Expression of miR-122, miR-34a, miR-16 and miR-21 in serum from healthy controls and NAFLD patients. (B) NAFLD patients were divided into two groups based on the NAFLD activity score (NAS): NAFLD-simple steatosis (NAFLD-SS) with NAS≤4 and non-alcoholic steatohepatitis (NASH) with NAS ≥5. (C) NAFLD-SS and NASH groups were further subdivided based on individual NAS scores.

Figure 5. Receiver operating characteristic (ROC) analysis of expression of miRNAs in CHC and NAFLD patients.

ROC curves with corresponding area under the ROC curve (AUC) for (A): mir-122 and miR-16 from CHC-early versus controls; (B) mir-122 and miR-16 from NAFLD-SS (NAS 1-4) versus controls; (C) miR-122 and miR-34a from CHC-early versus CHC-advanced; (D) miR-122 and miR-34a from NAFLD-SS(NAS 1-4) versus NASH (NAS 5–7).