Serum levels of microRNAs can specifically predict liver injury of chronic hepatitis B

Abstract

Aim: To investigate whether circulating microRNAs (miRNAs) can serve as molecular markers to predict liver injury resulted from chronic hepatitis B (CHB).

Methods: The profiles of serum miRNA expression were first generated with serum samples collected from 10 patients with CHB and 10 healthy donors (Ctrls) by microarray analysis. The levels of several miRNAs were further quantitated by real-time reverse transcription polymerase chain reaction with serum samples from another 24 CHB patients and 24 Ctrls. Serum samples of 20 patients with nonalcohlic steatohepatitis (NASH) were also included for comparison. The comparison in the levels of miRNAs between groups (CHB, NASH and Ctrl) was analyzed with Mann-Whitney U-test. The correlation between miRNAs and clinical pathoparameters was analyzed using Spearman correlation analysis or canonical correlation analysis. The receiver-operator characteristic (ROC) curves were also generated to determine the specificity and sensitivity of each individual miRNA in distinguishing patients with CHB from Ctrls.

Results: miRNA profile analysis showed that 34 miRNAs were differentially expressed between CHB and Ctrl subjects, in which 12 were up-regulated and 22 down-regulated in CHB subject (fold change > 2.0 and P < 0.01). The median levels of miR-122, -572, -575 and -638 were significantly higher (P < 1.00 × 10(-5)) while miR-744 significantly lower (P < 1.00 × 10(-6)) in CHB compared with the Ctrl. The levels of miR-122, -572 and -638 were also higher (P < 1.00 × 10(-3)) while the level of miR-744 lower in CHB (P < 0.05) than in NASH, although the difference between them was not as significant as that between CHB and Ctrl. ROC curve analysis revealed that the levels of miR-122, -572, -575, -638 and -744 in serum were sensitive and specific enough to distinguish CHB, NASH and Ctrl. Multivariate analysis further showed that the levels of these miRNAs were correlated with the liver function parameters. Most significantly, it was the scatter plot of principal component with the levels of these miRNAs, but not the parameters of liver function, which clearly distinguished CHB, NASH and Ctrl subjects.

Conclusion: Serum levels of miR-122, -572, -575, -638 and -744 are deregulated in patients with CHB or NASH. The levels of these miRNAs may serve as potential biomarkers for liver injury caused by CHB and NASH.

Keywords: Chronic hepatitis B; Liver injury; Nonalcohlic steatohepatitis; Serum microRNAs.

Figure 1

Serum levels of microRNAs in chronic hepatitis B, nonalcohlic steatohepatitis and healthy donors. The levels of serum miR-122 (A), miR-638 (B), miR-572 (C), miR-575 (D), miR-744 (E) and miR-30c (F) in patients with chronic hepatitis B (CHB) (n = 24), with nonalcohlic steatohepatitis (NASH) (n = 20) and healthy donors (Ctrl) (n = 24) were measured by quantitative reverse transcription polymerase chain reaction. The line at each group represents the median value of indicated miRNA. The values are normalized to miR-24 and shown in log₁₀ scale at y-axis. P values on the top are NASH vs Ctrl, on the left are CHB vs Ctrl and on the right are NASH vs Ctrl.

Figure 2

Receiver-operator characteristic curve analyses. Receiver-operator characteristic curves of the miR-122, -638, -572, -575 and -744 were established to discriminate chronic hepatitis B (CHB) from healthy donor (Ctrl) (A), nonalcohlic steatohepatitis (NASH) from Ctrl (B) and CHB from NASH (C).

Figure 3

Aberrant levels of serum miR-122, -572, -575, -638 and -744 correlate with the liver pathological parameters. A: Canonical correlation analysis. The correlation between microRNA (miRNA) variables and liver function parameter variables were calculated by canonical correlation analysis. All the data were log₁₀ transformed. Correlation coefficient r = 0.74 and P < 1.00 × 10^-4. miRNAs: miR-122, -572, -575, -638 and -744; Liver function parameters: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), alkaline phosphatase (ALP), total bilirubin (TBIL) and bile acid; B: Comparison of serum miRNAs, ALT and AST in chronic hepatitis B (CHB) and nonalcohlic steatohepatitis (NASH). The comparison among the levels of ALT, AST, miR-122, -572, -575, -638 and -744 in serum samples collected from patients with NASH and CHB (indicated on x-axis). The relative change of ALT, AST and miRNA expression levels were expressed ratio in log₂ compared with healthy control (Ctrl) (indicated on y-axis). The values of ALT, AST and miRNA fold change are the average of samples from CHB (n = 24), NASH (n = 20) and Ctrls (n = 24), and the SD is shown as an error bar; C, D: Scatter plot of principal components analysis. All the data were log₁₀ transformed to carry out analysis. Scatter plot of first two principal component of miRNAs variables including miR-122, -572, -575, -638 and -744 (C), and liver function parameters including ALT, AST, GGT, ALP, TBIL and bile acid (D) in CHB (n = 24), NASH (n = 20) and Ctrls (n = 24).