……… 3 ……… 4 - 16 1. TGF-β 2. TTF-1 TGF-β 3. Nrf2 4. RNA 5. ………. 17 - 31 ………. 32 - 63 1. TGF-β RBM47 2. TGF-β TTF-1 RBM47 3. RBM47 RBM47 4. RBM47 side population
5. RBM47
……… 64 – 71
……… 72 – 73
……… 74 – 84
TGF-β RNA RBM47
Nrf2 KEAP1 CUL3 mRNA RBM47
RBM47 knockdown KEAP1 CUL3
Nrf2 RBM47 knockdown
side population
1. TGF-β
TGF-β (transforming growth factor-β) 25 kDa
TGF-β TGF-β 1 TGF-β 2 TGF-β
epithelial to mesenchymal transition (EMT)
3 TGF-β sphere 4 TGF-β 5 TGF-β 70-80% TGF-β1~3 3
TGF-β1~3
TGF-β
TGF-β type I receptor (TβRI) type II
receptor (TβRII)
TGF-β
TβRII TβRI GS
TβRI Smad
Smad 8
R-Smad (receptor-regulated Smad) Co-Smad (common-mediator Smad) I-Smad
(inhibitory Smad) 3 TβRI R-Smad
Smad2/3 C Smad2/3
Co-Smad Smad4
( 1 ) 6,7
p300 CBP Smad
I-Smad Smad7 TGF-β TβRI
R-Smad
8,9
R-Smad Smad 7
E3
Smurf1, Smurf2 R-Smad TβRI
TGF-β-Smad 10
E3 Arkadia I-Smad c-Ski,
SnoN TGF-β
11
Smad TGF-β
Smad MAPK, S6K, RhoA
non-Smad ( 1 ) 12
1. TGF-β
TGF-β type I receptor (TβRI) type II receptor (TβRII)
TβRI GS Smad Smad
Smad non-Smad Smad
Smad2/3 Smad4
( )
Smad MAPK, S6K, RhoA
2. TTF-1 TGF-β
thyroid transcription factor 1 (TTF-1)
13 TTF-1 TTF-1 TTF-1 72% 89% TTF-1 14 TTF-1 TTF-1 EGF 15,16 TGF-β EMT 17 TTF-1 3. Nrf2
NF-E2-related factor 2 (Nrf2) (BZIP)
Nrf2 Kelch-like ECH-associated
protein 1 (KEAP1) Cullin3 (CUL3) - RING-box
protein 1 (RBX1) 18
Nrf2 small musculoaponeurotic
fibrosarcoma (sMaf) (MafF, MafG, MafK)
antioxidant/electrophile response element (ARE/EpRE)
( 2A) Nrf2
Nrf2
19
Nrf2 7 Nrf2–ECH homology (Neh)
( 2B) N Neh2 CUL3 RBX1
Nrf2 KEAP1 C
Neh1 sMaf DNA ARE
Nrf2 Neh2 Nrf2 KEAP1 624
( 2B) KEAP1 N CUL3
Bric-à-brac (BTB) C Nrf2 6 Kelch
BACK (IVR) KEAP1
27 Cys
21
CUL C CUL homology (CH)
CUL1, 2, 3, 4A, 4B, 5 6 DOC1
(destruction of cyclin B) IBR (in-between-ring) CUL7
CUL9 ( 2B) CUL RBX1 RBX2 CUL-RBX 22 CUL3 BTB DNA 23
2. Nrf2 KEAP1 CUL3 Nrf2
A Nrf2 Nrf2
KEAP1 CUL3 RBX1 E3
Nrf2 sMaf
ARE
B Nrf2 KEAP1 CUL3 (Neh : Nrf2–ECH homology BTB :
KEAP1 Nrf2 Nrf2 Nrf2 (11.4%) (6.3%) (8.0%) (13.0%) 24 KEAP1 (11.1%), (8.9%) (7.8%) (4.6%) (2.0%) (1.3%) 26
KEAP1 BACK Kelch
Nrf2 KEAP1 Neh2
24,25
KEAP1 Nrf2 Nrf2 KEAP1
Nrf2 ( 3 )
p21/CDKN1A Nrf2 Neh2 KEAP1
Nrf2 KEAP1 Nrf2 ( 3 ) 27,28 KEAP1 CpG KEAP1 Nrf2 ( 3 ) 29 Nrf2 sMaf ARE ( 3 )
( 3 ) 30 4. RNA DNA RNA 3. Nrf2 Nrf2 KEAP1 Nrf2 KEAP1 KEAP1 CUL3 Nrf2 Nrf2 sMaf ARE
31,32
microRNA (miRNA)
non-coding RNA RNA
33
RNA RNA RNA
34
RNA RNA RNA
(RNP: ribonucleoprotein complexes) RNA RNA RNA RNA 5’ ( 4) 35 RNA 36 RNA RNA
RNA RNA RNA RRM
(RNA recognition motif) KH (K-homology) dsRBM (double-stranded
RNA binding motif) PAZ (Piwi/Argonaute/Zwille domain)
RRM RNA 37
α β
RRM RNA RNA
RNA 34
RRM
RNA HuR
mRNA 3’UTR mRNA
HuR Cyclin D1, c-myc, VEGF Bcl2,
Mcl-1, SIRT1, p21/CDKN1A MMP-9 Snail
EMT mRNA
32
RRM RNA RNA
4. RNA
RNA DNA RNA
5’
RNA
RNA
RBP RNA binding protein 5’ cap eIF4E
3’ A poly(A)-binding protein (PABP) RBP
5.
TGF-β RNA
RNA
Nrf2 RNA
RNA Nrf2
TGF-β3 (R&D Systems) 2.5 mg/ml bovine serum albumin (BSA; Sigma-Aldrich)
4 mM HCl
α-tubulin (Sigma-Aldrich, DM1A)
TTF-1 (Novus Biologicals, 8G7G3/1)
RBM47 (Sigma-Aldrich, HPA006347)
Nrf2 (Abcam, EP1808Y)
KEAP1 (Proteintech, 10503-2-AP)
CUL3 (Abcam, EPR3196Y)
HDAC1 (Millipore, 2E10)
ATP5L (Abcam, ab126181)
NDUFS6 (Abcam, ab156099)
A549
A549 HEK293T 10% fetal bovine serum
(FBS) 100 U/ml penicillin G 100 μg/ml streptomycin Dulbecco's modified
Eagle's medium (DMEM #11965; Thermo Scientific) H441 10% FBS
100 U/ml penicillin G 100 μg/ml streptomycin RPMI 1640 (#11875; Thermo
Scientific) 37℃ 5% CO2
(chromatin immunoprecipitation, ChIP)
A549 10 cm (SARSTEDT) H441 15 cm (SARSTEDT) siRNA 1% 10 0.125 M 5 PBS (-) 2 PBS (-) 1 ml 4℃ 2,000 rpm 5
1 ml SDS lysis buffer (1% SDS 50 mM Tris-HCl [pH 8.0] 10 mM
EDTA protease inhibitor (P8340; Sigma-Aldrich))
”high” 30 3 30
8℃ 14,000 rpm 10 40 μl
800 μl ChIP dilution buffer (20
mM Tris-HCl [pH 8.0] 150 mM NaCl 2 mM EDTA 1% TritonX-100 Complete
EDTA-free protease inhibitors (Roche Diagnostics) ) 10 8 ml
2-methacryloxyethyl phosphorylcholine polymer-treated 15 ml polypropylene
tube ( ) 0.5% BSA PBS
(-) 4℃ 5 μg Dynabeads (Invitrogen)
4℃ 2,000 rpm 5 7 ml
Dynabeads 1.5 ml
Wash buffer (50 mM HEPES-KOH [pH 7.0] 0.5 M LiCl (Sigma-Aldrich) 1 mM EDTA
0.7% sodium deoxycholate 1% NP-40) 6 TE buffer (10 mM Tris-HCl [pH 8.0]
1 mM EDTA) 1 Magnetic Particle Concentrator (Dynal) Dynabeads
Dynabeads SDS lysis buffer 200 μl/tube
65℃ PCR purification
kit (Qiagen) nuclease-free ChIP-PCR
Green Master (Roche Applied Science) HBB (haemoglobin beta) 2 1 ChIP-seq RBM47 TTF-1 Smad3
ChIP-seq (GEO accession no: GSE51510)
38
siRNAs RNA
TTF-1 small interfering RNA (siRNA)
Thermo Scientific 17
RBM47 siRNA control siRNA
(Cat. 12935-112, ) Thermo Scientific
H441 siRNA Lipofectamine RNAiMAX reagent (Thermo
Scientific)
siRBM47#1 CCUUCGUCAUGUACUGCCACAAGCA UGCUUGUGGCAGUACAUGACGAAGG siRBM47#2 UAAAUAGUCAAGCGUUCCUUCAGUG CACUGAAGGAACGCUUGACUAUUUA RNA RT-PCR
TriPure (Roche Diagnostics) RNA 0.5
1 μg total RNA Oligo (dT)20 Prime Script II 1
st
strand cDNA
synthesis kit (Takara) cDNA RT-PCR
StepOnePlus™ Real-Time PCR Systems (Thermo Scientific) Fast SYBR ® Green
Master Mix (Thermo Scientific) mRNA
GAPDH (glyceraldehyde-3-phosphate dehydrogenase)
2 2
1. PCR qRT-PCR
Gene Forward Reverse
GAPDH TCTTTTGCGTCGCCAGCCGAG TGACCAGGCGCCCAATACGAC
RBM47 CCTCATGATGGACTTTGACG GCGGATCTCGTAGTTGTTGAG
KEAP1 GTCCCCTACAGCCAAGGTC GGGGTTCCAGAAGATAAGCA
CUL3 CTTGGAATGATCATCAAACAGC TGTTGTACATACACACGGTCCA
AKR1C1 GCCTTGGAAAGGTCACTGAA TCTTTTGGGATCACTTCCTCA
ALDH3A1 CGCAGACCTGCACAAGAAT TGTACTCGATCTCCTCTAGGACGT
A GPX2 CTGGTGGTCCTTGGCTTC TGTTCAGGATCTCCTCATTCTG SRXN1 GAGCCCAGAAGACACACCTG AAGAGAATGCACCCCTGCTA ATP5L TCAAACAGCTCACAGTTAAGGA A CTCCGACATAAAACCACATCAA
NDUFS6 AGAAGGTCACGCACACTGG CTCTTTCTGACGACCTACAAACC
qChIP-PCR
Gene Forward Reverse
GPX2 CATAGATATCAATTGGCCCTTCC CAGGATGACTTAGCAAAAACAGG T SRXN1 AATGTTCGGAACGACTCCAG CAGGGTGAGTCGGCAAAG HMOX1 CCCTGCTGAGTAATCCTTTCCCG A ATGTCCCGACTCCAGACTCCA HBB GGGCTGAGGGTTTGAAGTCC CATGGTGTCTGTTTGAGGTTGC
RBM47 promoter reporter cDNA
LacZ TTF-1
17
RBM47 promoter reporter PrimeSTAR GXL DNA
polymerase (Takara Bio) H441 DNA RBM47
5’ (−1950 to +50 bp)
PCR annealing buffer ( 10 mM Tris-HCl [pH 7.0] 50 mM
(Promega) HindIII-EcoRI PCR
Forward 1: AGCT TACACAGCTAGGAAGTCACCTACAACATCTT
Reverse 1: CATTAAGCATAGCAGATGGGTGATTGCTGGG
Forward 2: TACACAGCTAGGAAGTCACCTACAACATCTT
Reverse 2: AATT CATTAAGCATAGCAGATGGGTGATTGCTGGG
H441 12-well (Nunc) LacZ TTF-1
24 RBM47
(RBM47 promoter-Luc) CMV
(pGL4.75-SV40-hRluc)
DNA pcDNA3 FuGENE HD (Promega)
24
Dual-Luciferase Reporter Assay System (Promega)
SDS-PAGE
PBS (-) 1% NP-40 lysis buffer (1% Nonidet
P-40 20 mM Tris-HCl [pH 7.5] 150 mM NaCl cOmpleteTM
EDTA-free protease inhibitor
cocktail (Roche Diagnostics) )
NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific)
BCA Protein Assay Kit
(Thermo Scientific) SDS sample buffer (170 mM Tris-HCl [pH 8.8] 44%
glycerol 8.7% SDS 22 mM 1,4-dithiothreitol 0.05% Bromophenol Blue)
98℃ 3 SDS-PAGE
Fluoro Trans W Membrane (Pall)
blocking buffer (5% skim milk ( ) ) 30
1 4℃
HRP- 1
A549 EF luciferase H1 shRNA
luciferase shRNA
A549-Luc luciferase shRNA
75 cm2
(IWAKI) 7×106
293FT
pCAG-HIV-gp
pCMV-VSV-G-RSV-Rev negative control (shCT)
39 Lipofectamine 2000 reagent 2 RBM47 shRNA (shRBM47#1, shRBM47#2) shRBM47#1 Forward:GATCCCCGGGCTCAGTATTCCATGTTTCACGTGTGCTGTCCGTGAAACAT GGAATACTGAGCCC TTTTTGGAAAT Reverse:CTAGATTTCCAAAAAGGGCTCAGTATTCCATGTTTCACGGACAGCACACG TGAAACATGGAATACTGAGC CCGGG
shRBM47#2 Forward:GATCCCCGGATCTCTCCTTAAGCCAACAACGTGTGCTGTCCGTTGTTGGC TTAAGGAGAGATCC TTTTTGGAAAT Reverse:CTAGATTTCCAAAAAGGATCTCTCCTTAAGCCAACAACGGACAGCACAC GTTGTTGGCTTAAGGAGAGAT CCGGG RNA-seq
cDNA RNeasy Mini Kit with On-Column DNase Digestion Set
(QIAGEN) RNA-seq
Dynabeads mRNA DIRECT Purification Kit Ion Total RNA-Seq Kit v2 (Thermo
Scientific) cDNA Ion
PROTON Ion PI Template OT2 200 Kit v3 Ion PI Sequencing 200 Kit v3
RNA-seq Gene Set Enrichment
Analysis (GSEA) 40
mRNA
RP MRP
4 Balb/c nude mice 1.0×107 A549-Luc-shCT A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 = ( ) × ( )2 = (( ) × ( )2 )/2 4 Balb/c nude mice 3.0×107 A549-Luc-shCT A549-Luc-shRBM47#1
D-luciferin potassium salt (Wako
Chemicals) photon counting (LB983, Berthold Technologies)
t- in vitro Mann-Whitney-Wilcoxon test
(Mann-Whitney U test) in vivo (http://www.r-project.org/)
RIP-seq
A549 10 cm RIP™ RNA-Binding Protein
Immunoprecipitation Kit (Millipore) RIP assay
Rabbit control IgG
SNRNP70 Ion Total RNA-Seq
Kit v2 RIP cDNA
Ion PROTON Ion PI Template OT2 200
Kit v3 Ion PI Sequencing 200 Kit v3
TopHat2 human reference transcriptome (NCBI Build 37,
hg19) RNA Cufflinks cuffdiff
PBS (-) 2% FBS 25 mM
solution (HBSS) 1.0×106
1–12 μg/ml Hoechst33342 (Invitrogen) 37℃ 60
Fumitremorgin C (FTC) ( 10 μM)
Hoechst33342 PBS (-) Propidium iodide (PI)
MoFlo Astrios cell sorter (Beckman Coulter)
Forward Scatter (FSC) Side Scatter (SSC)
PI 350 nm
UV 405/BP30 (Hoechst blue)
570/BP20 (Hoechst red) 450 nm 675 nm
FTC side population (SP) FTC
non-side population (non-SP)
Flow Jo software (Treestar, Ashland, OR, USA)
OCR (oxygen consumption ratio) XF cell mitochondrial stress kit Seahorse
A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 24-well (XFe24 ) 1 1.0×105 10% FBS DMEM 37℃ 5% CO2 12 TGF-β A549 6-well 1 1.0×105 24 TGF-β 2.5 ng/ml 24 24-well (XFe24 ) 1 1.0×105 TGF-β 2.5 ng/ml 10% FBS DMEM 37℃ 5% CO2 12 (DMEM
(Sigma D5030), 1.85 g/L NaCl, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 mM glucose)
37°C CO2 30
37°C CO2 ATP
Oligomycin ( 1 μM)
Carbonyl cyanide
p-trifluoromethoxyphenyl hydrazine (FCCP) ( 12 μM) Antimycin
A ( 1 μM)
Antimycin A
Accession Numbers
RNA-seq RIP-seq FPKM fold-enrichment GEO
(GSE61910) 26 9 11 DNA TGF-β
1. TGF-β RBM47 (NMuMG ) 24 TGF-β EMT RNA-seq 41 NMuMG TGF-β EMT RNA Rbm35b (Esrp2) 42
TGF-β Rbm35A (ESRP1) ESRP2
43,44 TGF-β RNA RNA NMuMG TGF-β 24 RNA-seq RNA ( 2) 2. TGF-β RNA
Gene Symbol (-) (FPKM) TGF-β (FPKM) fold
Rbm47 19.9 2.9 0.146 Tst 21.0 7.5 0.356 Wdhd1 25.3 9.7 0.383 Dhfr 17.8 7.3 0.407 Ythdc2 9.1 4.1 0.448 Rad51ap1 12.4 5.7 0.460 Synj2 7.2 14.4 2.017 Rbfox2 11.7 24.3 2.068 Ccrn4l 3.0 6.4 2.104 Pcbp4 13.1 27.7 2.123
Mex3d 8.0 17.2 2.143
Rbms2 9.8 24.9 2.548
Mex3b 2.4 7.2 2.972
Adarb1 0.9 10.6 12.378
Esrp2 (Rbm35b) 17.7 11.0 0.621
GO:0003723 (RNA binding) 653 records
RNA binding 653 RNA-seq
NMuMG (FPKM 5 )
TGF-β 2 14 Rbm47
TGF-β RNA
Esrp2 0.621 Rbm47
RBM47 80 RNA recognition motifs (RRM) 3
RNA RBM47 N RRM C alanine-rich ( 5A) RBM47 45,46 RBM47 RBM47 RBM47 RBM47 TGF-β ( 5B, C) RBM47 TGF-β
5. RBM47 RBM47
A RBM47 N RNA Recognition Motif (RRM) 3
C alanine-rich
B H441
RBM47 α-tubulin
HDAC1
C FLAG-RBM47 pcDNA3 HEK293T
RBM47 α-tubulin
HDAC1
22 1st strand cDNA
RBM47 (A549 H441 ) (MCF7 MDA-MB-231 ) (MKN OCUM ) ( 6A) ( ) RBM47 RBM47 ( 6B) 47,48 RBM47 ( 3) 6. RBM47 A 22 mRNA PCR RBM47
B RBM47 ( ) ( ) (
) Kaplan-Meier 2 Log-rank test
3.
Gene
Symbol P value Hazard Ratio
Lung cancer Histology 0.0077 1.31 (1.07 - 1.59) Stage 0 1.5 (1.31 - 1.71) RBM47 0.0127 0.73 (0.57 - 0.93) Breast cancer ESR1 0 0.64 (0.56 - 0.74) HER2 (ERBB2) 0.0001 1.32 (1.15 - 1.52) RBM47 0.0813 0.9 (0.79 - 1.01) 2. TGF-β TTF-1 RBM47 A549 H441 TGF-β 24 RBM47 RT-PCR RBM47 A549 H441 TGF-β RBM47
( 7A, B) NMuMG A549 H441
RBM47 TGF-β
Isogaya H441 Smad3 ChIP-seq
38
Control siRNA H441 TTF-1
siRNA (siTTF-1) H441 Smad3
( 7C) Isogaya TTF-1 siRNA (siTTF-1) Smad RBM47 Smad 7. TGF-β RBM47 Smad3 A A549 H441 2.5 ng/ml TGF-β 24 PCR RBM47 mRNA GAPDH B A549 H441 2.5 ng/ml TGF-β 24 RBM47 α-tubulin ImageJ 1.00
C TTF-1 siRNA (siTTF-1) siRNA (siCT)
TTF-1 15,16 17 TTF-1 RBM47 RBM47 TTF-1 Isogaya H441 TTF-1 ChIP-seq 38 TTF-1 RBM47 ( 8A) RBM47 (+700 bp ~ +704 bp +1110 bp ~ +1114 bp ) TTF-1 AAGTG 38 H441 TTF-1 knockdown RBM47 TTF-1 RBM47 TTF-1 RBM47 ( 8B) RT-PCR TGF-β TTF-1 TTF-1 RBM47 -1950 bp ~ +50 bp pGL4.10 (RBM47 promoter-Luc) LacZ TTF-1 A549 luciferase TTF-1
A549 RBM47 ( 8C) TTF-1 RBM47 promoter RBM47 8. TTF-1 RBM47 TTF-1 A H441 TTF-1 ChIP-seq RBM47
B TTF-1 siRNA (siTTF-1) siRNA (siCT)
H441 RBM47 α-tubulin C FLAG-LacZ FLAG-TTF-1 A549 RBM47 promoter-Luc 48
A549 TTF-1
3. RBM47 RBM47
RBM47 RBM47 knockdown
luciferase A549
luciferase A549 (A549-Luc ) 2
RBM47 shRNA (shRBM47#1, shRBM47#2)
A549-Luc RBM47
knockdown A549 (A549-Luc-shCT A549-Luc-shRBM47#1
A549-Luc-shRBM47#2 )
RBM47 A549-Luc-shCT A549-Luc-shRBM47#1
A549-Luc-shRBM47#2 RBM47 (
9A) shRNA knockdown RBM47
RNA-seq A549-Luc-shCT
A549-Luc-shRBM47#1
A549-Luc-shCT A549-Luc-shRBM47#1
fragments per kilobases of exon per million sequence read (FPKM)
A549-Luc-shRBM47#1 Nrf2
GSEA Molecular Signatures Database (MSigDB) C6:
oncogenic signatures 2 normalized
enrichment score (NES) ( 9B, 4)
49
Nrf2 aldo-keto reductase family 1, member C1 (AKR1C1)
aldehyde dehydrogenase 3 family, member A1 (ALDH3A1) glutathione peroxidase 2
(GPX2) sulfiredoxin 1 (SRXN1) RT-PCR
mRNA TGF-β A549-Luc-shRBM47#1
A549-Luc-shRBM47#2 mRNA ( 9C)
shRNA A549 (shRBM47#1 A549-Luc-shRBM47#1
shRBM47#2 A549-Luc-shRBM47#2 )
RBM47 α-tubulin
B RNA-seq GSEA
NFE2L2 (Nrf2) enrichment plot
C A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1)
A549-Luc-shRBM47#2 (shRBM47#2) RNA PCR
Nrf2 AKR1C1 ALDH3A1 GPX2 SRXN1 mRNA
GAPDH
4. RBM47 knockdown
Name size NES FDR q-val
CSR_LATE_UP.V1_DN 52 2.02 0.0053 NFE2L2.V2 142 1.85 0.0360 MEK_UP.V1_UP 99 1.83 0.0358 MEL18_DN.V1_UP 60 1.72 0.0735 AKT_UP.V1_UP 66 1.72 0.0646 RB_P130_DN.V1_UP 59 1.69 0.0705 STK33_SKM_UP 73 1.59 0.0955 ESC_V6.5_UP_LATE.V1_UP 64 1.59 0.0918 ERB2_UP.V1_UP 84 1.56 0.0870 ATF2_UP.V1_DN 50 1.56 0.0834 P53_DN.V1_DN 69 1.54 0.0965 ESC_J1_UP_EARLY.V1_DN 75 1.52 0.1044 PRC2_EDD_UP.V1_UP 101 1.52 0.1045 EGFR_UP.V1_UP 92 1.51 0.1053 BMI1_DN.V1_UP 64 1.50 0.1082 ESC_J1_UP_LATE.V1_DN 76 1.50 0.1054
RNA-seq A549-Luc-shCT A549-Luc-shRBM47#1
2 TGF-β A549-Luc-shCT
2 ( 10A) RBM47
RBM47 knockdown 2 TGF-β 2
215 RBM47 knockdown 2
TGF-β 2 48 RBM47 knockdown 2
TGF-β 2 68 RBM47
knockdown 2 TGF-β 2 215
Nrf2 Maf family GSEA
2 Nrf2
RBM47 knockdown 2 TGF-β 2
215 RBM47 knockdown 2
TGF-β 2 68 9
EMT Snail Slug
RT-PCR TGF-β Snail Slug
mRNA RBM47 knockdown TGF-β Snail
10. RBM47 knockdown TGF-β
A A549-Luc-shCT A549-Luc-shRBM47#1 (shRBM47#1)
TGF-β 24 RNA RNA-seq mRNA
A549-Luc-shCT A549-Luc-shRBM47#1 2 A549-Luc-shCT TGF-β 2 B A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1) A549-Luc-shRBM47#2 (shRBM47#2) TGF-β
24 mRNA RT-PCR Snail Slug mRNA
GAPDH
RBM47 Nrf2
RNA immunoprecipitation (RIP) A549
RNA RBM47 RNA
A549-Luc-shCT FPKM 10 5730
rabbit IgG RBM47 RIP 4 fold
enrichment 165 Nrf2 Nrf2
KEAP1 CUL3 RBX1 p21/CDKN1A small Mafs RIP-seq
RBM47 KEAP1 CUL3 mRNA
mRNA ( 11)
11. RBM47 Nrf2 KEAP1 CUL3 RBX1 mRNA
A549 RNA RBM47 RBM47 RIP
RIP RNA deep sequencing
RBM47 Nrf2 KEAP1 CUL3 RBX1 mRNA RBM47
RIP biological replicates 2
(RBM47_1, RBM47_2) IgG
RBM47 KEAP1 CUL3 mRNA
RT-PCR KEAP1 CUL3 mRNA
A549-Luc-shRBM47#2 KEAP1 mRNA
( 12A) A549-Luc-shRBM47#1 A549-Luc-shRBM47#2
CUL3 mRNA ( 12A) Actinomycin D
mRNA KEAP1 CUL3 mRNA
RT-PCR A549-Luc-shRBM47#1 A549-Luc-shRBM47#2
Actinomycin D 3 6 KEAP1 mRNA
Actinomycin D 12 KEAP1 mRNA
( 12B) A549-Luc-shRBM47#1 A549-Luc-shRBM47#2
Actinomycin D 3 CUL3 mRNA
Actinomycin D 6 12 CUL3 mRNA
( 12B) KEAP1 CUL3
A549-Luc-shRBM47#1
A549-Luc-shRBM47#2 KEAP1 CUL3
( 12C)
Nrf2 A549-Luc-shRBM47#1
A549-Luc-shRBM47#2 Nrf2
24
KEAP1 Nrf2
Nrf2
50
RBM47 KEAP1 CUL3 mRNA
KEAP1 CUL3 Nrf2
12. RBM47 knockdown KEAP1 CUL3
A A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1)
A549-Luc-shRBM47#2 (shRBM47#2) RNA PCR
KEAP1, CUL3 mRNA GAPDH
B A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1)
A549-Luc-shRBM47#2 (shRBM47#2) Actinomycin D
3 6 12 RNA PCR
KEAP1, CUL3 α-tubulin ImageJ shCT 1.00 D A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1) A549-Luc-shRBM47#2 (shRBM47#2) Nrf2 α-tubulin Nrf2 ARE RNA-seq RBM47 shRNA A549-Luc-shRBM47#1 Nrf2 GPX2 SRXN1 HMOX1 mRNA 51,52 Nrf2 ChIP-qPCR GPX2 SRXN1 5’ ARE A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 Nrf2 HMOX1 5’ ARE A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 Nrf2
( 13A) RBM47 siRNA H441 A549
Nrf2 ChIP-qPCR GPX2 HMOX1 5’
ARE RBM47 siRNA
13. RBM47 knockdown Nrf2
A A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1)
A549-Luc-shRBM47#2 (shRBM47#2) Nrf2 ChIP
GPX2, HMOX1, HBB
PCR HBB
B (siCT) RBM47 (siRBM47#1, #2) siRNA
H441 Nrf2 ChIP GPX2,
HMOX1, HBB
PCR HBB
RBM47 Nrf2 KEAP1, CUL3 mRNA
RBM47 knockdown KEAP1, CUL3
Nrf2 ARE
Nrf2, KEAP1, CUL3, RBX1 Nrf2 p21/CDKN1A
MafK RBM47 RIP-seq
RBM47 p21/CDKN1A MafK mRNA RNA-seq
p21/CDKN1A MafK mRNA
A549-Luc-shRBM47#1 p21/CDKN1A MafK mRNA
TGF-β p21/CDKN1A MafK
28,53
RBM47 A549-Luc TGF-β
p21/CDKN1A MafK mRNA
( 14) RBM47 p21/CDKN1A MafK mRNA
p21/CDKN1A MafK
RBM47 KEAP1 CUL3 p21/CDKN1A MafK
Nrf2
mRNA
RNA-seq RBM47 knockdown Maf family (MafF,
MafG, MafK) p21/CDKN1A mRNA
RBM47 Nrf2 mRNA
RIP-seq RBM47 ATP5L NDUFS6
mRNA ( 15A) RT-PCR ATP5L
NDUFS6 mRNA A549-Luc-shRBM47#1 ATP5L mRNA
A549-Luc-shRBM47#2 ATP5L mRNA
A549-Luc-shRBM47#1 NDUFS6 mRNA
A549-Luc-shRBM47#2 NDUFS6 mRNA
( 15B) ATP5L
NDUFS6 A549-Luc-shRBM47#1
A549-Luc-shRBM47#2 ATP5L NDUFS6
15. RBM47 ATP5L NDUFS6 mRNA RBM47
knockdown ATP5L NDUFS6
A A549 RNA RBM47 RBM47
RIP RIP RNA deep sequencing
RBM47 ATP5L NDUFS6 mRNA RBM47
RIP 2 (RBM47_1, RBM47_2)
IgG
B A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1)
A549-Luc-shRBM47#2 (shRBM47#2) mRNA RT-PCR
ATP5L NDUFS6 mRNA GAPDH
C A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1) A549-Luc-shRBM47#2 (shRBM47#2) ATP5L NDUFS6 α-tubulin ImageJ shCT 1.00 RNA-seq RBM47 knockdown Nrf2 RBM47 knockdown EGF Nrf2 EGF
RBM47 Wnt 46 RBM47 4. RBM47 side population RBM47 knockdown Nrf2 54 (OCR) RBM47
Mito stress test A549-Luc-shCT
A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 ( 16A) Oligomycin A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 ( 16B) RBM47 knockdown A549 Oligomycin A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 RBM47 knockdown A549 ATP FCCP
A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 RBM47 knockdown A549 A549 A549 Antimycin A A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 RBM47 knockdown RBM47 A549 16. RBM47 knockdown A A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1) A549-Luc-shRBM47#2 (shRBM47#2) (OCR) OCR Oligomycin (Oligo)
Antimycin A (AA) 1 μmol/L carbonyl cyanide
p-trifluoromethoxyphenyl hydrazine (FCCP) 16 μmol/L
B Oligomycin A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1) A549-Luc-shRBM47#2 (shRBM47#2) OCR A549-Luc-shCT A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 t- * p < 0.05 TGF-β A549 ( 17A) Oligomycin TGF-β A549 ( 17B) TGF-β A549 Oligomycin TGF-β A549 TGF-β A549 ATP FCCP TGF-β A549 TGF-β A549 Antimycin A TGF-β A549
RBM47 TGF-β
17. TGF-β
A A549 TGF-β
24 A549 OCR OCR
Oligomycin (Oligo)
Antimycin A (AA) 1 μmol/L carbonyl cyanide
p-trifluoromethoxyphenyl hydrazine (FCCP) 16 μmol/L
B Oligomycin A549 TGF-β 24 A549 OCR A549 TGF-β t- * p<0.05 RBM47 knockdown Nrf2 side population 55 A549
side population A549 side population
56,57
Hoechest33342 A549
450 nm (Hoechst Blue) 675 nm (Hoechst Red)
side population ABC
ABCG2 side popultion
Fumitremorgin C (FTC) 58,59
A549-Luc-shRBM47#1
A549-Luc-shRBM47#2 A549-Luc-shCT side population
( 18A) TGF-β
A549 side population ( 18B)
RBM47 side population
RBM47 side population TGF-β
18. RBM47 knockdown A549 side population
TGF-β A549 side population
A Hoechest33342 A549-Luc-shCT (shCT) A549-Luc-shRBM47#1
(shRBM47#1) A549-Luc-shRBM47#2 (shRBM47#2)
450 nm (Hoechst Blue) 675 nm (Hoechst Red)
A549-Luc-shCT Fumitremorgin C (FTC)
side population A549-Luc-shCT
A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 side population
B Hoechest33342 A549 (NTC) TGF-β 24
A549 (TGF-β) 450 nm
(Hoechst Blue) 675 nm (Hoechst Red) A549
Fumitremorgin C (FTC) side population
A549 TGF-β A549 side population
5. RBM47
A549-Luc-shCT A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 6 cm dish 2 4 A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 A549-Luc-shCT ( 19) 19. RBM47 knockdown A549 A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1) A549-Luc-shRBM47#2 (shRBM47#2) 2 6 cm dish 100,000 (0 ) 2 4 cell count 2 6 cm dish A549-Luc-shCT A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 4 A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 A549-Luc-shCT
( 19)
RBM47 knockdown side population
( 18) RNA-seq RBM47
knockdown fibrinogen alpha chain fibrinogen beta chain matrix metalloproteinase-7
vitronectin
20. RBM47 knockdown A549
A A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1)
B A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1) 9 24 C A549-Luc-shCT (shCT) A549-Luc-shRBM47#2 (shRBM47#2) 24 D A549-Luc-shCT (shCT) A549-Luc-shRBM47#2 (shRBM47#1) 6 25 A549-Luc-shCT A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 7 photon count A549-Luc-shRBM47#1 A549-Luc-shRBM47#2 ( 21A, B) RBM47 knockdown Kaplan-Meier RBM47 ( 6B)
21. RBM47 knockdown A549
A A549-Luc-shCT (shCT) A549-Luc-shRBM47#1 (shRBM47#1)
6
photon count ph/s photon
counts/sec
B A549-Luc-shCT (shCT)
6 A549-Luc-shRBM47#1 (shRBM47#1)
10
RBM47 KEAP1 CUL3 mRNA KEAP1 CUL3
p21/CDKN1A MafK mRNA
Nrf2 ( 9, 11 12, 13, 14)
RBM47 side population
( 20A, B, C, D 21A, B) RIP-seq Nrf2
RBM47 knockdown side population ( 18)
A549 side population non-side
population
57
TGF-β RNA RBM47 RBM47 RBM47 RBM47 60 RBM47 47 RBM47 RBM47
RBM47 KEAP1 CUL3 mRNA RBM47
knockdown KEAP1 CUL3
RBM47 KEAP1 CUL3 mRNA KEAP1 CUL3
Nrf2 KEAP1 CUL3
RBM47
p21/CDKN1A MafK mRNA RBM47 knockdown
p21/CDKN1A MafK RBM47
knockdown Nrf2 side population
( 22) Nrf2 Nrf2 KEAP1 Nrf2 24,25 A549 KEAP1 Nrf2 24 RBM47
knockdown KEAP1 CUL3 Nrf2 ARE
KEAP1 Nrf2
RBM47 KEAP1 CUL3
22. RBM47
RBM47 KEAP1 CUL3 mRNA
p21/CDKN1A MafK Nrf2
TGF-β TGF-β
Smad RNA
Davis Smad miRNA
DROSHA miRNA 61 TGF-β T CCL22 miR-34a miRNA 62 miRNA TGF-β RNA
ESRP1 ESRP2 TGF-β fibroblast growth
factor receptor 2 (FGFR2) mRNA mRNA
EMT 42
TGF-β RBM47
RBM47 KEAP1 CUL3 mRNA KEAP1
CUL3 Nrf2 NMuMG TGF-β RNA RBM47 ESRP2 TGF-β ( 2) A549-Luc-shRBM47 TGF-β ( 10A) RBM47 TGF-β
TGF-β Snail Slug RBM47 knockdown
TGF-β RBM47
( 10B) Nrf2 side
population Nrf2 RBM47
knockdown TGF-β
Cancer Genome Atlas Research Network KEAP1
63
Hast KEAP1 18 A549
G333C KEAP1 CUL3 Nrf2 50 RBM47 knockdown KEAP1 CUL3 Nrf2 RBM47 knockdown Nrf2 Nrf2 KEAP1 Nrf2 50
RBM47 knockdown A549 HMOX1 ARE
Nrf2 HMOX1
RBM47 knockdown H441 HMOX1 ARE Nrf2
( 13A, B) Nrf2 NQO1 RBM47 RBM47 knockdown A549 TGF-β Smad 38,64,65 TGF-β Bach1 Maf family Nrf2
A549 53
RBM47 knockdown
Nrf2 Nrf2
Nrf2
Cancer Genome Atlas Research Network RTK-RAS PI3K-mTOR
63 Nrf2 Nrf2 Nrf2 Nrf2 Nrf2 RBM47 RBM47 ES Nanog mRNA 66 RBM47 RBM47 Wnt 46 RBM47 RBM47 Wnt RBM47 60
Vanharanta deep sequencing cross-linking immunoprecipitation
Vanharanta RBM47 Ray “GAUGAU” 67 HITS-CLIP RBM47 RIP-seq RBM47 ATP5L
NDUFS6 mRNA mRNA ( 15A, B, C)
RBM47
RBM47
Fossat RBM47 APOBEC1 RNA
45
RBM47 RNA
APOBEC RNA
68,69
A549 RNA-seq ChIP-seq
KEAP1 CUL3 RBM47 RNA
RNA-seq APOBEC1
A549 APOBEC1
TGF-β NMuMG RNA RBM47 RBM47 RBM47 NMuMG A549 H441 TGF-β RBM47 H441 TTF-1 RBM47 RBM47
RBM47 knockdowon A549 A549
RBM47 RIP-seq A549 RBM47
Nrf2 KEAP1 CUL3 mRNA Nrf2
RBM47 Nrf2 KEAP1
CUL3 Nrf2 p21/CDKN1A MafK
mRNA A549 RBM47 Nrf2
ARE Nrf2
side population RBM47
RBM47 side population
RBM47 A549
RBM47
1. Roberts AB, Anzano MA, Wakefield LM, Roche NS, Stern DF, Sporn MB. Type beta
transforming growth factor: a bifunctional regulator of cellular growth. Proc Natl
Acad Sci U S A. 1985; 82(1): 119-123.
2. Roberts AB, Wakefield LM. The two faces of transforming growth factor beta in
carcinogenesis. Proc Natl Acad Sci U S A. 2003; 100(15): 8621-8623.
3. Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal
transition. Nat Rev Mol Cell Biol. 2014; 15(3): 178-196.
4. Ikushima H, Todo T, Ino Y, Takahashi M, Miyazawa K, Miyazono K. Autocrine
TGF-beta signaling maintains tumorigenicity of glioma-initiating cells through
Sry-related HMG-box factors. Cell Stem Cell. 2009;v5(5):v504-514.
5. Hoshino Y, Katsuno Y, Ehata S, Miyazono K. Autocrine TGF-β protects breast cancer
cells from apoptosis through reduction of BH3-only protein, Bim. J Biochem. 2011;
149(1): 55-65.
6. Shi Y, Massagué J. Mechanisms of TGF-beta signaling from cell membrane to the
nucleus. Cell. 2003; 113(6): 685-700.
pathways in TGF-beta superfamily signalling. Genes Cells. 2002; 7(12): 1191-1204.
8. Nakao A, Afrakhte M, Morén A, et al. Identification of Smad7, a TGFbeta-inducible
antagonist of TGF-beta signalling. Nature. 1997; 389(6651): 631-635.
9. Hayashi H, Abdollah S, Qiu Y, et al. The MAD-related protein Smad7 associates with
the TGFbeta receptor and functions as an antagonist of TGFbeta signaling. Cell. 1997;
89(7): 1165-1173.
10. Izzi L, Attisano L. Regulation of the TGFbeta signalling pathway by
ubiquitin-mediated degradation. Oncogene. 2004; 23(11): 2071-2078.
11. Nagano Y, Mavrakis KJ, Lee KL, et al. Arkadia induces degradation of SnoN and
c-Ski to enhance transforming growth factor-beta signaling. J Biol Chem. 2007;
282(28): 20492-20501.
12. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in
TGF-beta family signalling. Nature. 2003; 425(6958): 577-584.
13. Stahlman MT, Gray ME, Whitsett JA. Expression of thyroid transcription factor-1
(TTF-1) in fetal and neonatal human lung. J Histochem Cytochem. 1996; 44(7):
673-678.
adenocarcinomas. Am J Surg Pathol. 2002; 26(6): 767-773.
15. Yamaguchi T, Yanagisawa K, Sugiyama R, et al. NKX2-1/TITF1/TTF-1-Induced
ROR1 is required to sustain EGFR survival signaling in lung adenocarcinoma. Cancer
Cell. 2012; 21(3): 348-361.
16. Yamaguchi T, Hosono Y, Yanagisawa K, Takahashi T. NKX2-1/TTF-1: an enigmatic
oncogene that functions as a double-edged sword for cancer cell survival and
progression. Cancer Cell. 2013; 23(6): 718-723.
17. Saito RA, Watabe T, Horiguchi K, et al. Thyroid transcription factor-1 inhibits
transforming growth factor-beta-mediated epithelial-to-mesenchymal transition in
lung adenocarcinoma cells. Cancer Res. 2009; 69(7): 2783-2791.
18. Kobayashi A, Kang MI, Okawa H, et al. Oxidative stress sensor Keap1 functions as an
adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol
Cell Biol. 2004; 24(16): 7130-7139.
19. Itoh K, Chiba T, Takahashi S, et al. An Nrf2/small Maf heterodimer mediates the
induction of phase II detoxifying enzyme genes through antioxidant response elements.
Biochem Biophys Res Commun. 1997; 236(2): 313-322.
Nrf2 engage in crosstalk for cytoprotection. Trends Pharmacol Sci. 2015; 36(1): 6-14.
21. Hayes JD, McMahon M. NRF2 and KEAP1 mutations: permanent activation of an
adaptive response in cancer. Trends Biochem Sci. 2009; 34(4): 176-188.
22. Chen Z, Sui J, Zhang F, Zhang C. Cullin family proteins and tumorigenesis: genetic
association and molecular mechanisms. J Cancer. 2015; 6(3): 233-242.
23. Zhao Y, Sun Y. Cullin-RING Ligases as attractive anti-cancer targets. Curr Pharm.
2013; 19(18): 3215-3225.
24. Singh A, Misra V, Thimmulappa RK, et al. Dysfunctional KEAP1-NRF2 interaction
in non-small-cell lung cancer. PLoS Med. 2006; 3(10): e420.
25. Shibata T, Kokubu A, Gotoh M, et al. Genetic alteration of Keap1 confers constitutive
Nrf2 activation and resistance to chemotherapy in gallbladder cancer.
Gastroenterology. 2008; 135(4): 1358-1368.
26. Yoo NJ, Kim HR, Kim YR, An CH, Lee SH. Somatic mutations of the KEAP1 gene
in common solid cancers. Histopathology. 2012; 60(6): 943-952.
27. Chen W, Sun Z, Wang XJ, et al. Direct interaction between Nrf2 and
p21(Cip1/WAF1) upregulates the Nrf2-mediated antioxidant response. Mol Cell.
28. Oshimori N, Oristian D, Fuchs E. TGF-β promotes heterogeneity and drug resistance
in squamous cell carcinoma. Cell. 2015; 160(5): 963-976.
29. Wang R, An J, Ji F, Jiao H, Sun H, Zhou D. Hypermethylation of the Keap1 gene in
human lung cancer cell lines and lung cancer tissues. Biochem Biophys Res Commun.
2008; 373(1): 151-154.
30. Mitsuishi Y, Taguchi K, Kawatani Y, et al. Nrf2 redirects glucose and glutamine into
anabolic pathways in metabolic reprogramming. Cancer Cell. 2012; 22(1): 66-79.
31. Silvera D, Formenti SC, Schneider RJ. Translational control in cancer. Nat Rev
Cancer. 2010; 10(4): 254-266.
32. Wurth L. Versatility of RNA-Binding Proteins in Cancer. Comp Funct Genomics.
2012; 2012: 178525.
33. Suzuki HI, Katsura A, Matsuyama H, Miyazono K. MicroRNA regulons in tumor
microenvironment. Oncogene. 2015; 34(24): 3085-3094.
34. Lukong KE, Chang KW, Khandjian EW, Richard S. RNA-binding proteins in human
genetic disease. Trends Genet. 2008; 24(8): 416-425.
35. Kim MY, Hur J, Jeong S. Emerging roles of RNA and RNA-binding protein network
36. Abdelmohsen K, Gorospe M. Posttranscriptional regulation of cancer traits by HuR.
Wiley Interdiscip Rev RNA. 2010; 1(2): 214-229.
37. Lunde BM, Moore C, Varani G. RNA-binding proteins: modular design for efficient
function. Nat Rev Mol Cell Biol. 2007; 8(6): 479-490.
38. Isogaya K, Koinuma D, Tsutsumi S, et al. A Smad3 and TTF-1/NKX2-1 complex
regulates Smad4-independent gene expression. Cell Res. 2014; 24(8): 994-1008.
39. Hoshino Y, Nishida J, Katsuno Y, et al. Smad4 Decreases the Population of Pancreatic
Cancer-Initiating Cells through Transcriptional Repression of ALDH1A1. Am J
Pathol. 2015; 185(5): 1457-1470.
40. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a
knowledge-based approach for interpreting genome-wide expression profiles. Proc
Natl Acad Sci U S A. 2005; 102(43): 15545-15550.
41. Arase M, Horiguchi K, Ehata S, et al. Transforming growth factor-β-induced
lncRNA-Smad7 inhibits apoptosis of mouse breast cancer JygMC(A) cells. Cancer Sci.
2014; 105(8): 974-982.
42. Horiguchi K, Sakamoto K, Koinuma D, et al. TGF-β drives epithelial-mesenchymal
3190-3201.
43. Warzecha CC, Sato TK, Nabet B, Hogenesch JB, Carstens RP. ESRP1 and ESRP2 are
epithelial cell-type-specific regulators of FGFR2 splicing. Mol Cell. 2009; 33(5):
591-601.
44. Yae T, Tsuchihashi K, Ishimoto T, et al. Alternative splicing of CD44 mRNA by
ESRP1 enhances lung colonization of metastatic cancer cell. Nat Commun. 2012; 3:
883.
45. Fossat N, Tourle K, Radziewic T, et al. C to U RNA editing mediated by APOBEC1
requires RNA-binding protein RBM47. EMBO reports. 2014; 15(8): 903-910.
46. Guan R, El-Rass S, Spillane D, et al. rbm47, a novel RNA binding protein, regulates
zebrafish head development. Dev Dyn. 2013; 242(12): 1395-1404.
47. Gyorffy B, Lanczky A, Eklund AC, et al. An online survival analysis tool to rapidly
assess the effect of 22,277 genes on breast cancer prognosis using microarray data of
1,809 patients. Breast Cancer Res. Treat. 2010; 123(3): 725-731.
48. Gyorffy B, Surowiak P, Budczies J, Lanczky A. Online survival analysis software to
assess the prognostic value of biomarkers using transcriptomic data in non-small-cell
49. Malhotra D, Portales-Casamar E, Singh A, et al. Global mapping of binding sites for
Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and
network analysis. Nucleic Acids Res. 2010; 38(17): 5718-5734.
50. Hast BE, Cloer EW, Goldfarb D, et al. Cancer-derived mutations in KEAP1 impair
NRF2 degradation but not ubiquitination. Cancer Res. 2014; 74(3): 808-817.
51. Singh A, Ling G, Suhasini AN, et al. Nrf2-dependent sulfiredoxin-1 expression
protects against cigarette smoke-induced oxidative stress in lungs. Free Radic Biol
Med. 2009; 46(3): 376-386.
52. Singh A, Rangasamy T, Thimmulappa RK, et al. Glutathione peroxidase 2, the major
cigarette smoke-inducible isoform of GPX in lungs, is regulated by Nrf2. Am J Respir
Cell Mol Biol. 2006; 35(6): 639-650.
53. Okita Y, Kamoshida A, Suzuki H, et al. Transforming growth factor-β induces
transcription factors MafK and Bach1 to suppress expression of the heme oxygenase-1
gene. J Biol Chem. 2013; 288(28): 20658-20667.
54. Hayes JD, Ashford ML. Nrf2 orchestrates fuel partitioning for cell proliferation. Cell
Metab. 2012; 16(2): 139-141.
is regulated by Nrf2 in cancer cells that confers side population and chemoresistance
phenotype. Mol Cancer Ther. 2010; 9(8): 2365-2376.
56. Sung JM, Cho HJ, Yi H, et al. Characterization of a stem cell population in lung
cancer A549 cells. Biochem Biophys Res Commun. 2008; 371(1): 163-167.
57. Akunuru S, Palumbo J, Zhai QJ, Zheng Y. Rac1 targeting suppresses human
non-small cell lung adenocarcinoma cancer stem cell activity. PLoS One. 2011; 6(2):
e16951.
58. Rabindran SK, Ross DD, Doyle LA, Yang W, Greenberger LM. Fumitremorgin C
reverses multidrug resistance in cells transfected with the breast cancer resistance
protein. Cancer Res. 2000; 60(1): 47-50.
59. Ehata S, Johansson E, Katayama R, et al. Transforming growth factor-β decreases the
cancer-initiating cell population within diffuse-type gastric carcinoma cells. Oncogene.
2011; 30(14): 1693-1705.
60. Vanharanta S, Marney CB, Shu W, et al. Loss of the multifunctional RNA-binding
protein RBM47 as a source of selectable metastatic traits in breast cancer. eLife. 2014;
3.
DROSHA-mediated microRNA maturation. Nature. 2008; 454(7200): 56-61.
62. Yang P, Li QJ, Feng Y, et al. TGF-β-miR-34a-CCL22 signaling-induced Treg cell
recruitment promotes venous metastases of HBV-positive hepatocellular carcinoma.
Cancer Cell. 2012; 22(3): 291-303.
63. Network CGAR. Comprehensive molecular profiling of lung adenocarcinoma. Nature.
2014; 511(7511): 543-550.
64. Koinuma D, Tsutsumi S, Kamimura N, et al. Chromatin immunoprecipitation on
microarray analysis of Smad2/3 binding sites reveals roles of ETS1 and TFAP2A in
transforming growth factor beta signaling. Mol Cell Biol. 2009; 29(1): 172-186.
65. Morikawa M, Koinuma D, Miyazono K, Heldin CH. Genome-wide mechanisms of
Smad binding. Oncogene. 2013; 32(13): 1609-1615.
66. Yeganeh M, Seyedjafari E, Kamrani FA, Ghaemi N. RNA-binding protein Rbm47
binds to Nanog in mouse embryonic stem cells. Mol Biol Rep. 2013; 40(7):
4391-4396.
67. Ray D, Kazan H, Cook KB, et al. A compendium of RNA-binding motifs for
decoding gene regulation. Nature. 2013; 499(7457): 172-177.
retroviral and cellular RNA in vitro and in vivo. J Mol Biol. 2009; 385(1): 65-78.
69. Valdmanis PN, Roy-Chaudhuri B, Kim HK, et al. Upregulation of the microRNA
cluster at the Dlk1-Dio3 locus in lung adenocarcinoma. Oncogene. 2015; 34(1):
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