Plasma Small Extracellular Vesicles Derived miR-21-5p and miR-92a-3p as Potential Biomarkers for Hepatocellular Carcinoma Screening

Andrei Sorop; Razvan Iacob; Speranta Iacob; Diana Constantinescu; Leona Chitoiu; Tudor Emanuel Fertig; Anca Dinischiotu; Mihaela Chivu-Economescu; Nicolae Bacalbasa; Lorand Savu; Liliana Gheorghe; Simona Dima; Irinel Popescu

doi:10.3389/fgene.2020.00712

Plasma Small Extracellular Vesicles Derived miR-21-5p and miR-92a-3p as Potential Biomarkers for Hepatocellular Carcinoma Screening

Front Genet. 2020 Jul 23:11:712. doi: 10.3389/fgene.2020.00712. eCollection 2020.

Authors

Andrei Sorop^{1

2}, Razvan Iacob^{1

3

4}, Speranta Iacob^{1

3

4}, Diana Constantinescu¹, Leona Chitoiu⁵, Tudor Emanuel Fertig^{3

5}, Anca Dinischiotu², Mihaela Chivu-Economescu^{2

6}, Nicolae Bacalbasa^{1

3}, Lorand Savu^{1

7}, Liliana Gheorghe^{1

3

4}, Simona Dima^{1

4}, Irinel Popescu^{1

4

7}

Affiliations

¹ Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest, Romania.
² Faculty of Biology, University of Bucharest, Bucharest, Romania.
³ "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania.
⁴ Digestive Diseases and Liver Transplantation Center, Fundeni Clinical Institute, Bucharest, Romania.
⁵ Ultrastructural Pathology Laboratory, Victor Babeş National Institute, Bucharest, Romania.
⁶ Department of Cellular and Molecular Pathology, Stefan S. Nicolau Institute of Virology, Bucharest, Romania.
⁷ "Titu Maiorescu" University of Medicine and Pharmacy, Bucharest, Romania.

Abstract

Introduction: Liquid biopsy using circulating microvesicles and exosomes is emerging as a new diagnostic tool that could improve hepatocellular carcinoma (HCC) early diagnosis and screening protocols. Our study aimed to investigate the utility of plasma exosomal miR-21-5p and miR-92-3p for HCC diagnosis during screening protocols.

Methods: The study group included 106 subjects: 48 patients diagnosed with HCC during screening, who underwent a potentially curative treatment (surgical resection or liver transplantation), 38 patients with liver cirrhosis (LC) on the waiting list for liver transplantation, and 20 healthy volunteers. The exosomes were isolated by precipitation with a reagent based on polyethylene glycol and were characterized based on morphological aspects (i.e., diameter); molecular weight; CD63, CD9, and CD81 protein markers; and exosomal miR-21-5p and miR-92a-3p expression levels.

Results: We first demonstrate that the exosome population isolated with the commercially available Total Exosome Isolation kit respects the same size ranging, morphological, and protein expression aspects compared to the traditional ultracentrifugation technique. The analysis of the expression profile indicates that miR-21-5p was upregulated (p = 0.017), and miR-92a-3p was downregulated (p = 0.0005) in plasma-derived exosomes from HCC subjects, independently from the patient's characteristics. AUROC for HCC diagnosis based on AFP (alpha-fetoprotein) was 0.72. By integrating AFP and the relative expression of exosomal miR-21-5p and miR-92a-3p in a logistic regression equation for HCC diagnosis, the combined AUROC of the new exosomal miR HCC score was 0.85-significantly better than serum AFP alone (p = 0.0007).

Conclusion: Together with serum AFP, plasma exosomal miR-21-5p and miR-92a-3p could be used as potential biomarkers for HCC diagnosis in patients with LC subjected to screening and surveillance.

Keywords: exosomes; hepatocellular carcinoma; microRNA; screening; serum biomarkers.