Circulating miR-106b-3p, miR-101-3p and miR-1246 as diagnostic biomarkers of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the most common liver cancer and second leading cause of cancer related death worldwide. Most HCCs occur in a damaged cirrhotic background and it may be difficult to discriminate between regenerative nodules and early HCCs. No dependable molecular biomarker exists for the early detection of HCC. MicroRNAs (miRNAs) have attracted attention as potential blood-based biomarkers. To identify circulating miRNAs with diagnostic potential in HCC, we performed preliminary RNAseq studies on plasma samples from a small set of HCC patients, cirrhotic patients and healthy controls. Then, out of the identified miRNAs, we investigated miR-101-3p, miR-106b-3p, miR-1246 and miR-411-5p in plasma of independent HCC patients' cohorts. The use of droplet digital PCR (ddPCR) confirmed the aberrant levels of these miRNAs. The diagnostic performances of each miRNA and their combinations were measured using Receiver Operating Characteristic (ROC) curve analyses: a classifier consisting of miR-101-3p, miR-1246 and miR-106b-3p produced the best diagnostic precision in plasma of HCC vs. cirrhotic patients (AUC = 0.99). A similar performance was found when the levels of miRNAs of HCC patients were compared to healthy controls (AUC = 1.00). We extended the analyses of the same miRNAs to serum samples. In serum of HCC vs. cirrhotic patients, the combination of miR-101-3p and miR-106b-3p exhibited the best diagnostic accuracy with an AUC = 0.96. Thus, circulating miR-101-3p, miR-106b-3p and miR-1246, either individually or in combination, exhibit a considerable potential value as diagnostic biomarkers of HCC.

Keywords: circulating microRNA; cirrhosis; diagnostic biomarkers; hepatocellular carcinoma.

Figure 1. Validation of differential levels of plasma miR-101-3p, miR-1246, miR-106b-3p and miR-411-5p using droplet digital PCR absolute quantification in various cohorts of HCC vs. cirrhosis and HCC vs. healthy control

Mann-Whitney U test; ^*p value < 0.05, ^**p value < 0.005, ^***p value < 0.001, ns: p value ≥ 0.05.

Figure 2. Distribution of levels and ROC curve analysis of plasma miR-101-3p, miR-1246, miR-106b-3p in combined cohorts (1 + 2) of HCC vs. cirrhosis

AUC: Area Under the ROC Curve. CI: Confidence Interval.

Figure 3. Distribution of levels and ROC curve analysis of plasma miR-101-3p, miR-1246, miR-106b-3p in combined cohorts (1 + 3) of HCC vs. healthy control

AUC: Area Under the ROC Curve. CI: Confidence Interval.

Figure 4. Comparison of ROC Curves display the diagnostic precision of miRNA classifiers

(A) Plasma miRNA classifiers HCC vs. cirrhosis. (B) Plasma miRNA classifiers HCC vs. healthy control. (C) Serum miRNA classifier HCC vs. cirrhosis.

Figure 5. Distribution of levels and ROC curve analysis of serum miRNAs miR-101-3p, miR-1246 and miR-106b-3p, in HCC vs. cirrhosis patients

AUC: Area Under the ROC Curve. CI: Confidence Interval.

Figure 6. Differential levels of miR-122-5p in HCC patients versus controls (cirrhosis patients or healthy controls) in plasma and serum samples

Mann-Whitney U test; ^*p value < 0.05, ^****p value < 0.0001, ns p value ≥ 0.05.