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Exosomal MicroRNAs as Liquid Biopsy Biomarkers in Hepatocellular Carcinoma.

Author(s): Wang S, Yang Y, Sun L, Qiao G, Song Y, Liu B

Publication: Onco Targets Ther, 2020, Vol. 13, Page 2021-2030

PubMed ID: 32210570 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study to compare diagnostic accuracy using miRNA levels in plasma and isolated exosomes for HCC and cirrhosis by comparisons to healthy controls. Blood was collected from 50 patients with hepatocellular carcinoma, 50 patients with hepatic cirrhosis, and 50 healthy volunteers but details of blood collection including tube type were not provided. Whole blood was centrifuged at 3000 x g for 15 min to obtain plasma. To extract exosomes, ExoQuick was added to the plasma, stored for an unspecified duration at 4°C, and centrifuged at 1500 x g for 30 min at 4°C to precipitate the exosomes. The pellet was recentrifuged at 1500 x g for 5 min, dissolved in PBS, and then stored at -20°C. RNA was extracted from exosomes and plasma using the RecoverALL Total Nucleic Acid Isolation Kit and then stored at -70°C until analysis. RNA purity and yield were evaluated by spectrophotometer. RNA was reverse-transcribed using the TaqMan microRNA Reverse Transcription Kit and mIR-122, mIR-21, mIr-96, and U6 were quantified by real-time PCR. All miRNA levels were normalized to U6. Protein from exosomes and supernatant were extracted using RIPA lysis buffer, quantified using BCA protein assay, and analyzed by Western blot with anti-CD63.

   Summary of Findings:

   Isolation of exosomes was confirmed by Western blot analysis. Exosomal and plasma levels of miR-122 were lower and miR-21 higher in patients with HCC than in healthy patients or those with cirrhosis (P<0.001, all). Exosomal and plasma levels of miR-96 were higher in patients with HCC than in healthy patients (P<0.001, both). However, miR-122, miR21, and miR-96 diagnostic accuracy for HCC was higher when quantified in exosomes rather than plasma and was superior to using alpha-fetoprotein (AFP) levels (P<0.01). Further, diagnostic accuracy for cirrhosis using miR-122, miR21, and miR-96 levels was higher in exosomes than plasma (P<0.001, all). The panel of miR-122, miR-21, and miR-96 in exosomes had a sensitivity of 96% and specificity of 98% for discriminating between HCC and healthy controls and a sensitivity of 82% and specificity of 92% for discriminating between HCC and cirrhosis. Finally, high exosomal miR-21 or miR-96 were associated with poor survival (P=0.0092 and P=0.04, respectively) and high exosomal miR-122 was associated with better survival (P=0.04).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal
   • Cirrhosis

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   Protein	Western blot
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Cirrhosis HCC Healthy
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-21 miR-122 miR-96
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Plasma exosomes

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