Circulating microRNAs, miR-939, miR-595, miR-519d and miR-494, Identify Cirrhotic Patients with HCC - PubMed (original) (raw)

. 2015 Oct 28;10(10):e0141448.

doi: 10.1371/journal.pone.0141448. eCollection 2015.

Manuela Ferracin 2, Davide Trerè 3, Maddalena Milazzo 1, Sara Marinelli 1, Marzia Galassi 1, Laura Venerandi 1, Daniela Pollutri 1, Clarissa Patrizi 1, Alberto Borghi 1, Francesco G Foschi 4, Giuseppe F Stefanini 4, Massimo Negrini 2, Luigi Bolondi 1, Laura Gramantieri 1

Affiliations

Circulating microRNAs, miR-939, miR-595, miR-519d and miR-494, Identify Cirrhotic Patients with HCC

Francesca Fornari et al. PLoS One. 2015.

Abstract

The performance of circulating biomarkers for the diagnosis of hepatocellular carcinoma (HCC) is sub-optimal. In this study we tested circulating microRNAs as biomarkers for HCC in cirrhotic patients by performing a two stage study: a discovery phase conducted by microarray and a validation phase performed by qRT-PCR in an independent series of 118 patients. Beside miRNAs emerged from the discovery phase, miR-21, miR-221, miR-519d were also tested in the validation setting on the basis of literary and tissue findings. Deregulated microRNAs were assayed in HCC-derived cells in the intracellular compartment, cell culture supernatant and exosomal fraction. Serum and tissue microRNA levels were compared in 14 patients surgically treated for HCC. From the discovery study, it emerged that seven circulating microRNAs were differentially expressed in cirrhotic patients with and without HCC. In the validation set, miR-939, miR-595 and miR-519d were shown to differentiate cirrhotic patients with and without HCC. MiR-939 and miR-595 are independent factors for HCC. ROC curves of miR-939, miR-595 and miR-519d displayed that AUC was higher than AFP. An exosomal secretion of miR-519d, miR-21, miR-221 and miR-1228 and a correlation between circulating and tissue levels of miR-519d, miR-494 and miR-21 were found in HCC patients. Therefore, we show that circulating microRNAs deserve attention as non-invasive biomarkers in the diagnostic setting of HCC and that exosomal secretion contributes to discharging a subset of microRNAs into the extracellular compartment.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Microarray analysis.

Genome-wide circulating miRNA expression analysis in serum of cirrhotic patients with and without HCC. As reported in the expression color-bar, for each miRNA, red color means a higher expression value than its average expression across all samples while green color means a lower expression level than its average expression across all samples.

Fig 2

Fig 2. Real Time PCR validation analysis.

Box-plot representation of circulating levels of selected miRNAs (miR-939, miR-595, miR-519d, miR-494, miR-21, miR-221) in patients with liver cirrhosis (LC) without nodular liver lesions, or with early HCCs (unifocal HCCs less than 2 cm in the main diameter) or intermediate/advanced HCCs. Graphic representation and statistical analysis were performed after Log2 transformation of RT-qPCR data.

Fig 3

Fig 3. MiRNAs performance as biomarkers of diagnosis.

Receiver operating characteristic curve (ROC) analysis for hepatocellular carcinoma diagnosis. Area under the curve (AUC) estimation for miR-595 (A), miR-939 (B), miR-519d (C) and alpha-feto-protein (D).

Fig 4

Fig 4. Exosome characterization in HCC cell lines.

(A) Correlation graphs between intracellular levels and exosomal fraction of miR-519d, miR-494, miR-21, miR-221 in 7 HCC-derived cell lines. Numbers in x- and y-axes represent 2-ΔΔCt values from the Real Time PCR analysis, transformed in a logarithmic form. U6RNA was used as housekeeping gene for the quantification of intracellular miRNAs levels, whereas cel-miR-39 was used to normalize data derived from exosome miRNA analysis. (B) Western blot analysis of exosomes-specific markers, Alix and CD63, in cell culture supernatant, exosomes-depleted fraction and exosomal fraction derived from Huh-7 cell line. (C) Correlation graphs between cell culture supernatant and exosomal fraction of AFP and Albumin (ALB) mRNA levels in HCC-derived cell lines. β-actin was used as housekeeping gene. Numbers in the y-axis represent 2-ΔΔCt values from the Real Time PCR analysis reported in a linear scale.

Fig 5

Fig 5. Exosomal and tissue miRNAs quantification in HCC patients.

(A) Box plot graphs showing miRNA levels in the exosomal-depleted and enriched fractions. Numbers in the y-axis represent 2-ΔΔCt values from the Real Time PCR analysis, transformed in a logarithmic form. ** p<0.01; *** p<0.0001. (B) Correlation graphs between intra-tumor and serum levels of miR-519d, miR-494 and miR-21 in 14 HCC patients. Graphic representation and statistical analysis were performed after Log2 transformation of RT-qPCR data. Numbers in x- and y-axes represent 2-ΔΔCt values from the Real Time PCR analysis, transformed in a logarithmic form. U6RNA was used as housekeeping gene for the quantification of tissue miRNAs levels, whereas cel-miR-39 was used to normalize data derived from serum miRNA analysis.

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