Application of Liquid Biopsy for Hepatocellular Carcinomas using Plasma Circulating p28/Gankyrin RNA and miRNA-s

Abstract

Background:

Gankyrin/PSMD10  plays  an  important  role  in  tumorogenes,  especially  hepatocellular  carcinogenesis. Protein  expression  significantly  increased  in  Hepatocelular  Carcinoma  (HCC).   Gankyrin  and miRNA-s may  be  used as a diagnostic and prognostic marker for the HCC and for monitoring of tumor recurrence. 

 Aim:

To investigate the feasibility of plasma circulating Gankyrin mRNA and miRNA as a HCC biomarker.

Methods and Materials:

Total DNA/RNA was extracted from FFPE of tumor and adjacent liver tissues n=35. Control samples: cirrhotic (n=10) and normal (n=10) liver. Plasma samples were isolated from 4 mL of peripheral blood from patient with HCC (n=32), HCC with MTS  (n=5), Cirrhosis - (n=7), HCV positive - (n=5), and healthy individuals. Fresh Total DNA/RNA/miRNA was extracted from blood plasma using RecoverAll™ Total Nucleic Acid Isolation Kit. RT-qPCR was performed using TaqPath 1-Step Multiplex Master Mix. Human 18S ribosomal RNA was used as an internal reference gene. Expression of Gankyrin/PSMD10 mRNA and  122 miRNA, 130 miRNA, 15 miRNA, 152 miRNA, 200A miRNA, 200B miRNA, ,21 miRNA, 26 miRNA, 9 miRNA was evaluated. The relative expression levels were calculated using 2−ΔΔCq method using SAS-Studio Mann-Whitney U test was used to determine the statistical significance for comparisons between groups.

Results

In the blood plasma of the patients with cirrhosis, HCV without cirrhosis and healthy individuals cell free RNA of Gankyrin/PSMD10 was not detected. While significant expression was observed in patients with  HCC. As for the tissue samples, the following results were obtained. Garkirin/PSMD1 RNA expression was lower  in  healthy  liver  tissue.  While  a  3.2  folde  change  was  observed  in  cirrhotic  liver  sampes, compared  to  a  healthy  one.  In  the  HCC  tissue  samples  and  its  surrounding  liver  tissue  significant expression was identified – 120.7 and 68.5 fold change relatively.  Also miRNA expression profiles was determine for different tissues.

Conclusion

The obtained data indicate that plasma Garkirin/PSMD10 RNA can be used as a diagnostic as well as  prognostic and monitoring tool for HCC recurrence. Garkirin/PSMD10 RNA expression data in tissue indicate that gene upregulation begins in cirrhosis. It was confirmed also by miRNA expression profiles.  It  reaches a maximum in HCC. In non-cancer tissue surrounding the tumor (also cirrhotic)   the expression  is significantly higher. There is not clear it is consequence  of “malignant” process, or its  upregulation is a first step of “malignant” transformation. If later will be confirmed – we will have good prediction marker for patient with cirrhosis for determine of HCC risk.

Our findings indicates the potential role of Garkirin/PSMD10 expression in liver biopsy to predict cancer risk in cirrhotic patients.