The NFkB pathway was identified as important for high CCL2 expression in the glioma cell line U105MG. Using a transcription factor siRNA array to knock down 42 transcription factors, RELA (a subunit of NFkB) was found to significantly lower CCL2 expression levels. Knocking down RELA also enhanced the effect of BCNU (carmustine) treatment, indicating that targeting the NFkB pathway may help sensitize tumor cells to chemotherapy in glioma.

1. siRNA array study identified the NFkB pathway as an important controller
for high CCL2 expression in glioma cell line U105MG
Song Tian1, Alexander Bank1, Jiaqiang Huang1, Teizo Yoshimura2, Xiao Zeng1.
Frederick, MD 21703, 2National Cancer Institute at Frederick, Frederick, MD 21702
Performance of SureSilencing siRNA Array for TFs
20
Figure 3. Performance of siRNA array. A. Summary of remaining gene expression after
treatment with Transcription Factor siRNA array, compared with NC control group.
Validation showed siRNA in the array can knock down 42 transcription factors at more
than 70%. B. High efficiency of the siRNA array system. With the format of siRNA array
but with siRNA that targets MAPK1 only, reverse transfected with SureFECT
transfection reagent, it can efficiently knock down MAPK1 in nine popular cell lines.
U105MG has a high CCL2 expression
CCL2
40
0 .6
(ACTB=1)
Relative gene expression
45
0 .8
35
0 .4
3
3
2
2
1
1
1
Figure 6. Identification of the NFkB as the main player for the high expression of CCL2 in
U105MG cells with two different siRNA arrays. A. RelA showed the most effective player
in CCL2 transcription by Human Transcription Factor siRNA array. Knockdown RelA
significantly inhibited the CCL2 expression. B. RelA was confirmed to be the important
TFs for CCL2 expression in U105MG cells by NFkB siRNA array.
B
A
C
1 .0
2
CCL2 relative level in supernatant
(NC=100%)
A
2
RELA
H SF1
S MA D9
TP53
HIF1A
HD AC1
FO XA2
N FAT5
SMAD 4
NFATC 3
ATF1
CR EB1
REL
S P1
SMAD3
JU ND
ATF3
ATF2
STAT5A
C RE BBP
ID 1
RELB
CEBPB
S MA D2
CTNNB1
PPARA
TBP
H AND1
NFATC 1
N FKB2
MYC
N FKB 1
STAT5B
MEF2A
STAT3
AR
FOS
N EG
ELK1
CEBPG
YY1
RB1
E2F1
HepG2
HCT116
PC3
SKBR3
293H
MB-231
Hela
42 Gene Targets
MCF7
A549
0
0
strong ac tivating
f ac tor
120%
120%
80%
RELA
NC
40%
80%
DMSO
BCNA
40%
0%
72h
0%
96h
NC
0 .2
RELA
30
Non-related TFs
str ong inhibiting
f ac tor
1.0
CCL2 (ng/ml)
XCL1
ACTB
TLR 4
TCP10
TREM1
TNFR SF1A
IL8
SDF2
NFKB1
IL16
IL1A
MMP7
LTB4R
TYMP
HIF1A
C XCL6
GPR 31
C XCL3
CXCR 6
CXCR 3
C XCL1
CXCL11
CXCL13
CX3CL1
C CR6
C CR8
CMTM3
CMTM1
CCR L2
CMKLR 1
CC L4
CC L7
C CR4
CC L2
C CR2
C CR1
CC L1
C CL18
C CL13
C CL16
C5AR1
AGTRL1
B
Relative gene expression
(ACTB=1)
non-r elated fac tor
25
Figure 7. inhibition of RELA showed inhibited CCL2 secretion and it will sensitize the
tumor cell growth inhibition with the treatment. M105MG cells were first treated with
BCMU for 1 hour then treated with RELA siRNA or control. A. Curcumin treatment result.
B. Staurosporine treatment result. These treatments showed only marginal changes in
gene expression. No anti-apoptosis genes were significantly up-regulated or downregulated by either STS or CUR treatment. p<0.05 compared with controls
20
15
0.8
0.6
10
0.4
CCL2
0.2
5
×
NC
CXCR 5
0 .0
inhibiting
protein
The SureSilencing™ siRNA Array is the latest technological
innovation for conducting target identification
through RNA
interference (RNAi). The Transcription factor focused siRNA array
has siRNAs for 42 key transfection factors with appropriate RNAi
controls are arrayed on a 96-well cell culture plate. Following a
simple reverse transfection protocol with the siRNA, people can
directly determine the transcription regulator for your gene of
interest in cells on the same plate with real-time PCR based
assays. Using the SureSilencing siRNA Arrays, you can quickly
identify genes involved in a biological process or disease state, the
targets of chemical compounds, and the biological impact of your
genes of interest.
Figure 2. Introduction of siRNA array. 42 function or pathway
focused key targets listed on each plate. Last row provides critical
controls required for reliable interpretation of the RNA interference
data in different phenotype assays. B. How the siRNA array works.
Reverse transfection produces equivalent or improved transfection
efficiencies over standard pre-plated methods and saves an entire
day in the process. The simplicity and reliability of the siRNA Array
allow you to quickly analyze gene function in your pathways of
interest.
XCL1
ACTB
TREM1
TLR4
SDF2
TCP10
TNFRSF1A
NFKB1
IL8
IL1A
MMP7
LTB4R
IL16
HIF1A
GPR31
TYMP
CXCL6
CXCR6
CXCR3
CXCL3
CXCL1
CXCL13
CXCL11
CX3CL1
CMKLR1
CCR8
CCR6
CMTM3
CCRL2
CMTM1
CCR2
CCR4
CCL7
CCL2
CCL4
CCR1
CCL1
CCL18
CCL16
CCL13
C5AR1
CXCR5
Figure 1. How the TF siRNA array help you to find the responsible transcription regulator.
When you knock down each individual transcription regulators, the one that responsible for
your target gene will be removed from the transcription machinery. That will change your
target gene’s transcription base on their function. Then your up-regulated gene expression
will be changed base on the function of the knocking down target.
AGTRL1
0.0
Identify the gene that responsible
for up-regulated transcription
3
Induction of CCL2 expression (
40
3
Inhibition of CCL2 expression (fold change)
60
1
20
Strong
NC
80
RELA
R IPK1
MYD88
TIC AM1
NFKBIB
IKBKE
PIK3C G
TRAF6
TR AF2*
TNFRSF1A
LTBR
TICAM2
IRAK1
FAD D
BCL10
RELB
CD40
TLR 2
TLR 4
IKBKB
MAP3K1
N FKB2
TLR1
N FKB1
IG F1R*
M ALT1
TNFRSF10A
MAP3K3
N EG
TNFR SF10B
PRKC Q
TLR3
TR ADD
MAP 3K7
NFKBIA
CHU K
IL1R1
TRAF3
EGFR
IRAK2
AKT1
IKBKG
ELK1
40
100
B
Cell viability
60
Strong
NC
Gene Knock-down Efficiency (%)
80
Induction of CCL2 expression (fold change)
A
100
SureSilencing™ siRNA Array for TFs
™
activating
protein
NFkB pathway is important for CCL1 expression in U105MG
Inhibition of CCL2 expression (fold change)
Introduction
Gliomas, in the form of astrocytomas, anaplastic astrocytomas and
glioblastomas, are the most common primary tumors of the central nervous
system in humans. Malignant gliomas are associated with a marked inflammatory
response that further promotes tumor growth. In the present study, we examined
the gene expression profiles of two human glioma cell lines, U-105MG and U373MG, using a PCR array. U-105MG is highly infiltrative, metastatic, and
apoptosis-resistant cell line compared with the U-373MG. We found high CCL2
expression in U-105MG. Since CCL2 facilitates tumor growth by recruiting tumorassociated macrophages, understanding the mechanism of CCL2 expression is
critical. To analyze the mechanisms, we performed a transcription factor siRNA
array screening. We used a siRNA Array to knock down the expression of 42 key
transcription regulator genes side-by-side in human U105MG cells, and evaluated
the CCL2 mRNA level by comparing it to that obtained with non-targeting siRNA
control samples, followed by real-time PCR. Knock-down of RELA expression
significantly lowered the secretion of CCL2 protein. Furthermore, we found that
knock-down of RELA enhanced the effect of BCNU treatment. Our results
indicated that high levels of CCL2 expression in U105MG were due to the
activation of NF-kB. These results demonstrate that the Transcription Factor
siRNA Array is an efficient approach to study the mechanisms of gene
expression. Further studies are necessary to better understand the mechanisms
regulating the development of brain tumors and to design new strategies for
treatment.
×
1SABiosciences,
0
U105MG
U373MG
Figure 4. U105MG cells expression high level of CCL2. U105MG and U373MG cells’ total
RNA was extracted and cDNA was reverse transcribed. Human Chemokines PCR array
was used to detect 84 chemokines and related gene expression level. A: U105MG cells
showed significant CCL2 high expression in mRNA level. B. U373MG cells showed
significant lower CCL2 expression under same detect. C. ELISA assay for cell culture
supernatant CCL2 .
CCL2 Promoter binding site analysis
Figure 5. The image displays the most relevant transcription factor binding sites of
CCL2 gene promoter as predicted by SABiosciences' Text Mining Application and the
UCSC Genome Browser. NFkB showed the first one that predicted to bind to CCL2
promoter for several locations.
Conclusion
•
The siRNA Array is a simple and powerful tool for carrying out
gene expression regulation screening.
•
Based on PCR Array analysis using the Human RT2 Profiler
PCR Array, CCL2 appears to express in a much higher level even
compare with another glioma cell line.
•
The siRNA Array is very useful for identifying the transcription
factors that plays a critical role in the up-regulated gene expression.
NFkB pathway is found to be essential for the high expression of
CCL2 in M105MG cells. It is a potential target for the glioma
treatment.
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