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Yoon. Hepatoma Res 2018;4:42 I http://dx.doi.org/10.20517/2394-5079.2018.23 Page 5 of 9

[58]
fibrosis and NAFLD-related HCC . Additionally, transmembrane 6 superfamily 2 (TM6SF2) E167K and
glucokinase regulator (GCKR) rs780094 gene variants have been reported to be associated with a higher risk
[59]
for fatty liver and liver fibrosis . Although numerous factors that contribute to HCC from NAFLD have
been revealed, there remain several unsolved issues for the molecular mechanism of HCC in the context of
NAFLD, including the direct role of the gut microbiome, epigenetic regulation, identification of metabolo-
mics profiles, and function of cancer stem cells linked to lipid metabolism.

MOLECULAR MECHANISMS OF HCC BY SINGLE-CELL TRANSCRIPTOMIC ANALYSIS
Recent advances in NGS technologies have facilitated deeper insights into the molecular mechanisms of
tumor development and progression, thereby opening the way for a new era of personalized medicine. In
particular, NGS-based transcriptome analysis (RNA-seq) has become a powerful tool for both characteriz-
[60]
ing the transcriptomes of each cell and profiling alternative splicing variants associated with cell function .
To date, almost all genomic studies have been carried out using bulk samples. However, RNA-seq using
bulk tissue samples comprising various cell populations is inappropriate for comprehensively investigating
transcriptomic profiling because each cell in the tumor is constantly differentiating, proliferating, and het-
erogeneous. Thus, the newly developed single-cell RNA sequencing (scRNA-seq) technology is a powerful
approach to dynamically analyze the genetic and cytologic heterogeneity of each cell in specific tumor tissue,
providing a more comprehensive understanding of the molecular mechanism of carcinogenesis and the pro-
cess of cancer evolution. The heterogeneity of single cells is diversely manifested in morphologic and pheno-
typic characteristics, genomics, and proteomics. Proper targets that can be used to analyze the heterogeneity
of cancer cell using scRNA-seq include cancer stem cells (CSCs), circulating tumor DNA (ctDNA) and cell-
free DNA (cfDNA) [61-63] .

Recently, RNA-seq-based transcriptome analyses using tumor and non-tumor tissue from 10 HBV-related
[64]
HCCs were first reported by Huang et al. . Differentially expressed genes (DEGs; 1378) and differentially
expressed exons (DEEs; 24,338) were identified in their study. Comprehensive functional analyses demon-
strated that DEGs were most significantly enriched in cell growth-related, metabolism-related and immune-
related pathways, suggesting a very complicated mechanism for hepatocarcinogenesis. Furthermore, RNA-
seq data analyses at the exon level revealed a highly complex landscape of transcript-specific differential
expression in HCC. In particular, a novel, highly up-regulated exon-exon junction was detected in the
ATAD2 gene. This is the first study dealing with transcriptome profiles, including exon level expression
changes and novel splicing variants using RNA-seq, and represents the most comprehensive characterization
of HBV-related HCC transcriptomes as well as provides important clues for understanding the molecular
mechanisms of HCC pathogenesis at system-wide levels. More recently, to further explore the dynamic
mechanisms that simultaneously occur in genetic and epigenetic regulation on gene expression associated
with heterogeneity at the single cell level in cancer, single-cell triple omics sequencing (scTrio-seq) tech-
[65]
[66]
niques, including the genome, epigenome and transcriptome, have been developed . Recently, Hou et al.
using scTrio-seq technology, have demonstrated correlations between genomic (copy-number variations,
CNVs), transcriptomic, and methylomic data analyzed in the same individual cells in HCC. In addition,
they revealed that changes in the gene dosage of certain regions due to CNVs proportionally affect the RNA
[66]
expression levels of those corresponding regions .

Although few studies have reported on the heterogeneity of liver CSCs at the single-cell level in HCC, a re-
cent study showed that different CSC subpopulations contain distinct molecular signatures, suggesting that
CSC heterogeneity may contribute to the molecular and biological diversity of HCC cell groups and, conse-
[67]
quently, patient prognosis . Therefore, heterogeneity at the single cell level of liver CSCs may be critical for
tumor progression and prognosis in HCC and might be important for the development of targeted agents for
HCC.