Profiling the circulating mRNA transcriptome in human liver disease.

Aejaz Sayeed,Timothy Block,Brielle E Dalvano,Alhaji H Janneh,David E Kaplan,Usha Viswanathan,John Kulp,Lu-Yu Hwang,Cataldo Doria,Adam Ertel

doi:10.18632/oncotarget.27617

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.

Highlights

We are interested in using messenger RNA (mRNA) in the blood as a source of biomarkers of liver disease
To evaluate the presence and diversity of mRNA transcripts in the human circulation, we performed RNA sequencing (RNAseq) analysis on “total” cell-free RNA from the plasma of 10 individuals with hepatocellular carcinoma (HCC) and liver cirrhosis (LC), 5 with LC alone, and 6 “normal” healthy controls (NHC), bringing the total number of samples analyzed by RNAseq to 21 (Table 1)
Both genders and those with HCC and LC with and without chronic viral hepatitis were included in all liver disease categories, balance was not achieved with this opportunistic set

Summary

Introduction

We are interested in using mRNA in the blood as a source of biomarkers of liver disease. Detection and understanding of circulating mRNA sequences is in a much earlier research phase. Several laboratories have demonstrated the existence of circulating mRNA in healthy subjects and those with a disease diagnosis [3,4,5,6,7,8,9,10,11,12,13]. Albumin (ALB) mRNA has been detected in the circulation of advanced stage HCC patients and was reported to predict recurrence of HCC after transplantation [18, 19]. During the preparation of this manuscript, circulating mRNA in cardiac disease patients was reported [3]. The extent to which circulating mRNA is derived from “non-” blood tissue as opposed to blood or endothelial cells that line the blood is not clear. The extent to which circulating mRNA is derived from “non-” blood tissue as opposed to blood or endothelial cells that line the blood is not clear. mRNA sequences in the blood, originating from non-blood cells, may be rare and/or may be representative of pathological events

Methods

Results

Conclusion