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Page 2 of 17 Matsushita et al. Hepatoma Res 2018;4:61 I http://dx.doi.org/10.20517/2394-5079.2018.81

Keywords: Hepatocellular carcinoma, hepatitis B virus, covalently closed circular DNA, far upstream element-
binding protein-interacting repressor, poly (U)-binding-splicing factor, F-box and W (Typ) D (Asp) repeat domain-
containing 7, natural product depository array, U2AF homology motif, U2AF homology motif ligand motif

INTRODUCTION
[1,2]
C-Myc is overexpressed in the majority of colorectal cancers and is required for tumor maintenance . The far
upstream element (FUSE) is a sequence required for proper expression of the human c-Myc gene. The FUSE is
located 1.5 kb upstream of c-Myc promoter P1, and binds the FUSE binding protein1 (FUBP1), a transcription
[3,4]
factor stimulating c-Myc expression in a FUSE dependent manner . FUBP1 is overexpressed and regulates
[5-7]
proliferation and migration of hepatoma cells . Yeast two-hybrid analysis revealed that FUBP1 binds to a
protein that has transcriptional inhibitory activity termed the FUBP1-interacting repressor (FIR), and FIR
was found to engage the transcriptional factor IIH [TFIIH/p89/xeroderma pigmentosum type B (XPB)]
[8]
helicase and repress c-Myc transcription . FIR induces apoptosis via c-Myc suppression, and is thus a suitable
cancer therapy [9,10] . Adenovirus-FIR or Sendai virus-FIR vectors gene therapy for nasopharyngeal cancer
[15]
were reported [11-14] . Up to 60% of all human genes present at least one alternative splice variant . Disturbed
alternative splicing (AS) in cancer cells or hepatitis B virus (HBV) virus affect host’s immune response [16,17] . AS
has been documented to play a significant role in human disease and DNA repair in cancers [18-21] . A splicing
variant of FIR that lacks exon2, FIRΔexon2, failed to repress c-Myc and inhibited FIR-induced apoptosis
[22]
suggesting FIRΔexon2 is a dominant negative of FIR in human cancers . On the other hand, FIR is a splicing
variant form of poly(U)-binding-splicing factor (PUF60) [23,24] . Anti-PUF60 autoantibodies are reported to
be detected in the sera of autoimmune diseases such as dermatomyositis, Sjogren’s syndrome or idiopathic
inflammatory myopathy [25,26] . Further, the combination of anti-FIRs antibodies with other clinically available
tumor markers such as anti-p53 antibodies, CEA, and CA19-9 further improved the specificity and accuracy
of diagnosis [27,28] . Besides, haploinsufficiency of FIR mouse model promoted p53-dependent T-cell acute
[29]
lymphoblastic leukemia progression . SAP155, a subunit of the essential splicing factor 3B (SF3B) subcomplex
in the spliceosome, is required for proper P27Kip1 pre-mRNA splicing, and P27Kip1 arrests cells at G1 [30,31] .
Moreover, spliceostatin A (SSA) or pladienolide, a natural SF3B inhibitor, markedly inhibited P27 expression
by disrupting its pre-mRNA splicing with striking cell killing effects [32,33] . Further, FIR/PUF60 is required for
[34]
transcriptional and post-transcriptional regulation of HBV pgRNA expression . To develop novel diagnosis
and therapy for hepatoma targeting FIR (PUF60)/FIRΔexon2, small molecular chemical compounds against
FIRΔexon2 were screened among 2,3275 chemicals by natural product depository (NPDepo) array at RIKEN
(Japan) to develop anti-cancer drugs [35-37] . Finally, small inhibitory chemicals against FIR/FIRΔexon2 for
hepatoma therapy will be discussed.

METHODS

Cancer cell lines
Human cervical SCCs (HeLa cells), gastric cancer cells (NUGC4), HLE cells and HLF cells were purchased
from the American Type Culture Collection (https://www.atcc.org/). These cells were treated as described
[18]
previously . All cell lines were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal
calf serum (FCS; Invitrogen, Tokyo, Japan) and 1% penicillin-streptomycin, and they were cultured at 37 °C in
a humidified atmosphere containing 5% CO .
2

Protein extraction, western blotting and antibodies
Culture medium was removed, and the cells were washed twice with cold (4 °C) phosphate buffered saline
(PBS), lysed with 1:20 β-mercaptoethanol and 2x sample buffer, and incubated at 100 °C for 5-min. Whole-cell
lysates were assayed for protein content (Bio-Rad, Hercules, CA, USA), and 10 μg of proteins were separated