Serum miR-30e and miR-223 as Novel Noninvasive Biomarkers for Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers and is the third leading cause of all cancer-related death. Limited noninvasive biomarkers are available for HCC detection. Early detection is the key in improving the survival of HCC patients. In this study, we tested the hypothesis that serum miRNAs can be used as a potential biomarker for HCC. Quantitative RT-PCR for miRNA analysis was performed using 70 serum samples. Receiver operating characteristic analysis was performed to measure the prognostic power of the miRNAs. The miRNA expression level was also measured from liver biopsy samples. Our study revealed that two miRNAs, miR-30e and miR-223, were expressed at significantly lower levels (P < 0.003) in the sera of HCC patients compared with healthy volunteers. Furthermore, expression of these miRNAs was compared between sera from chronic liver disease and sera from HCC patients. miR-30e and miR-223 expression was significantly lower in HCC sera compared with sera from chronic liver disease patients. Both miRNA expression levels were lower in HCC liver biopsy specimens compared with normal liver RNA. Taken together, our results suggested that serum miR-30e and miR-223 are useful biomarkers of HCC, irrespective of etiology, and deserve further study for their diagnostic value.

Figure 1

Down-regulation of serum levels of miR-30e and miR-223. Scatter plot of serum miRNA levels of miR-30e (A) and miR-223 (B) in healthy volunteers (HVs) and hepatocellular carcinoma (HCC) patients. The line indicates the median value per group. Fold-regulation values are expressed as relative quantification on the basis of 2^ΔΔCt method. A U test was used to determine statistical significance. Receiver operating characteristic (ROC) curve was analyzed using serum miR-30e and miR-223 for discriminating HCC patients. ROC curves with corresponding area under the ROC curve (AUC) for miR-30e (C) and miR-223 (D) in discriminating HCC patients from HVs. Scatter plot of serum levels of miR-30e (E) and miR-223 (F) in chronic liver disease (CLD) and HCC patients. ROC curves with corresponding AUC for miR-30e (G) and miR-223 (H) in discriminating HCC patients from CLD. Data are presented as means ± SD. n = 14 (A and B, healthy volunteers); n = 39 (A, B, E, and F, HCC patients); n = 17 (E and F, CLD patients). ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 2

Down-regulation of serum levels of miR-30e and miR-223 in different etiologies of hepatocellular carcinoma (HCC) patients. A: Scatter plot of serum levels of miR-30e in healthy volunteers (HVs), hepatitis B virus (HBV)–induced HCC, hepatitis C virus (HCV)–induced HCC patients, and non–viral (NV)-induced HCC. B: Scatter plot of serum levels of miR-223 in HVs, HBV-induced HCC, HCV-induced HCC patients, and NV-induced HCC. C and D: Scatter plot of serum levels of miR-30e (C) and miR-223 (D) in chronic liver disease (CLD), HBV-induced HCC, HCV-induced HCC patients, and NV-induced HCC. The line indicates the median value per group. Fold-regulation values are expressed as relative quantification on the basis of 2^ΔΔCt method. One-way analysis of variance was used to determine statistical significance. Data are presented as means ± SD (A–D). n = 14 (A and B, healthy volunteers and HBV- and HCV-induced HCC patients; C and D, HBV- and HCV-induced HCC patients); n = 11 (A, C, and D, NV-induced HCC patients); n = 17 (C and D, CLD patients). ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 3

Reduced expression of miR-30e and miR-223 of liver biopsy samples compared with normal liver RNA. Relative expression of miR-30e (A) and miR-223 (B) from liver biopsy samples collected from hepatocellular carcinoma (HCC) patients. Normal liver RNA was purchased from Life Technologies. Fold-regulation values are expressed as relative quantification on the basis of the 2^ΔΔCt method. Data are presented as means ± SD (A and B). ^∗P < 0.05, ^∗∗P < 0.01. HCV, hepatitis C virus.