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approaches is urgently required.

REGULATION OF ALTERNATIVE PRE-MRNA SPLICING
Alternative splicing (AS) is a process by which multiple messenger RNAs (mRNAs) are generated from a single
pre-mRNA, resulting in functionally distinct protein products that may have different or even opposing
[5]
roles . Genome-wide studies showed that nearly all multi-exon genes in human undergo alternative splicing
and produce multiple mRNA isoforms from a single pre-mRNA in a tissue or developmental stage-specific
[6,7]
manner . Thus, AS is an important mechanism to vastly expand transcriptomic and proteomic diversity
[8]
from a finite genome . This is accomplished by the differential recognition of splice sites by RNA binding
[9]
splicing factors in the pre-mRNA . The different types of AS are shown schematically in Figure 1. The most
common type of AS consists of a single cassette exon that is either included or skipped in the mRNA. Other
forms of AS include alternative selection of 5’ and 3’ splice sites, selection of mutually exclusive exon, and in-
tron retention. Different cis-regulatory elements in the pre-mRNA play a critical role in alternative selection
of splice sites by binding to splicing regulatory proteins. Based on the location of binding in the pre-mRNA
and function, there are four cis-regulatory elements: exonic splicing enhancers, exonic splicing silencers,
intronic splicing enhancers and intronic splicing silencers. These cis-regulatory elements which are present
within the alternative exon itself or upstream/downstream intron sequences bind trans-regulatory splic-
ing factors and either promote or inhibit the usage of the alternative exon(s). Though there are a number of
RNA-binding proteins that regulate alternative pre-mRNA splicing, two of the well-studied families are ser-
[10]
ine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) . Other less com-
mon families include the CELF/BRUNOL family, and the RBM family [10-12] . Both SR proteins and hnRNPs
[10]
can promote or inhibit exon recognition depending on location of the binding and sequence context .

ALTERNATIVE SPLICING AND HCC
Alternative splicing is a major post-transcriptional regulatory event that can modulate key aspects of cancer
cell biology including cell proliferation, metabolism, apoptosis, survival, invasiveness, angiogenesis, drug-re-
sistance, and metastasis [13-15] , thus playing a very critical role in the development and progression of cancers.
In case of HCC, splicing alterations of genes such as DNA methyltransferase 3b (DNMT3b), Aurora kinase B
[16]
(AURKB), E3 ubiquitin ligase (MDM2), TENSIN2, MAD1, SVH, TP53, and Fibronectin1 (FN1) have long
been reported. Recent studies have shown that the list of tumor-specific aberrantly spliced mRNAs is in-
[17]
creasing and implicated in HCC .

Alternative splicing facilitates the development of HCC either by generating oncogenic variants or by inac-
tivating the tumor suppressors. For example, an alternative POLDIP3 transcript promotes HCC progres-
[18]
sion . POLDIP3 is a target of ribosomal protein S6 kinase 1, and regulates DNA replication and mRNA
translation. The alternative POLDIP3 transcript (POLDIP3-β), which lacks exon 3, was found to be signifi-
cantly up-regulated in clinical HCC tissue compared to paired adjacent noncancerous hepatic tissue. This
POLDIP3-β isoform has been shown to increase HCC cell proliferation, inhibit HCC cell apoptosis, enhance
HCC cell migration, and promote xenograft growth. Another example is the cell fate determinant protein,
Numb, which is aberrantly spliced in HCC and produces an isoform that contains a long proline-rich region
[19]
(PRRL) . In HCC cell lines, PRRL generally promotes and PRRS (short proline-rich region) suppresses pro-
liferation, migration, invasion, and colony formation. PRRL-Numb expression has been shown to increase in
[19]
HCC and be associated with early recurrence and thus reduces overall survival after surgery .
It was observed that, in HCC cell lines and tumors, insulin receptor (IR) is aberrantly spliced and promotes
expression of the mitogenic isoform of insulin receptor (IR-A) that is generally expressed in the embryonic
tissues but not in the adult liver. In contrary to the isoform IR-B that is normally expressed in the adult
liver and promotes metabolic effects of insulin, IR-A signals proliferative effects via binding to insulin-like