^{Department of Epidemiology, Human Genetics and Environment Science Center for Infectious Diseases School of Public Health, the University of Texas HSC, Houston, TX, USA}

^{Department of Cancer Biology, Sidney Kimmel Cancer Center, Cancer Genomics Core Facility, Thomas Jefferson University, Philadelphia, PA, USA}

^{Capital Health Cancer Center, One Capital Way, Pennington, NJ, USA}

Correspondence to:Aejaz Sayeed, email: gro.grebmulbb@deeyas.zajeA

Received 2020 Feb 28; Accepted 2020 May 16.

Copyright: Sayeed et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

The human circulation contains cell-free DNA and non-coding microRNA (miRNA). Less is known about the presence of messenger RNA (mRNA). This report profiles the human circulating mRNA transcriptome in people with liver cirrhosis (LC) and hepatocellular carcinoma (HCC) to determine whether mRNA analytes can be used as biomarkers of liver disease. Using RNAseq and RT-qPCR, we investigate circulating mRNA in plasma from HCC and LC patients and demonstrate detection of transcripts representing more than 19,000 different protein coding genes. Remarkably, the circulating mRNA expression levels were similar from person to person over the 21 individuals whose samples were analyzed by RNAseq. Liver derived circulating transcripts such as albumin (ALB), apolipoprotein (APO) A1, A2 & H, serpin A1 & E1, ferritin light chain (FTL) and fibrinogen like 1 (FGL1) were significantly upregulated in HCC patient samples. Higher levels of some of these liver-specific transcripts in the plasma of HCC patients were confirmed by RT-qPCR in another cohort of 20 individuals. Several less abundant circulating transcripts associated with cancer were detected in most HCC samples, but not in healthy subjects. Liver specificity of circulating transcripts was confirmed by investigating their expression in HCC tumor and liver cancer cell lines. Liver specific mRNA sequences in the plasma were predominantly present outside circulating extracellular vesicles.

Conclusions: The circulating “mRNA” transcriptome is remarkably consistent in diversity and expression from person to person. Detection of transcripts corresponding to disease selective polypeptides suggests the possibility that circulating mRNA can work as a biomarker analyte for cancer detection.

Keywords: hepatocellular carcinoma, circulating transcripts, biomarkers, liver cirrhosis, extracellular vesicles

ACKNOWLEDGMENTS

We thank Dr. Chari Cohen (The Baruch S. Blumberg Institute) and Abhar Nissar for reviewing this manuscript. We are also thankful to Nancy Young and Judith Marchand for administrative assistance.

Abbreviations

HCC	Hepatocellular carcinoma
LC	Liver cirrhosis
miRNA	microRNA
mRNA	messenger RNA
RNAseq	RNA sequencing
RT-qPCR	Reverse transcription-quantitative polymerase chain reaction
NHC	Normal healthy control
EVs	Extracellular vesicles
MVs	Microvesicles

Contributed by

Author contributions

AS and TB conceptualized and designed the study. AS, AJ, and BD developed methodology, carried out experiments, and acquired data. AS, TB, BD, AE, and UV performed analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis). AS and TB wrote the manuscript. DK, LYH, and CD provided the clinical plasma samples and reviewed the manuscript. JK provided interpretive insights.

Availability of data and materials

Data generated or analyzed during this study are included in this published article and its supporting supplementary information files. Additional datasets are available from the corresponding author on reasonable request.

CONFLICTS OF INTEREST

The authors have declared that no conflicts of interest exists for this work. Timothy Block is on the Board of Directors of Hepion, an early phase company developing therapeutics for hepatitis B and other liver diseases. Cirna therapeutics is a startup company created by The Baruch S. Blumberg Institute developing early detection of cancer diagnostics that plans to license technology described in this work. None of the authors have any ownership in Cirna Therapeutics.

FUNDING

This study was supported by a Commonwealth University Research Enhancement Program grant with the Pennsylvania Department of Health (TB); the Department specifically disclaims responsibility for any analyses, interpretations, or conclusions. This work was also supported by NIH R01 CA166111 (DEK).

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