1. Effect of RNAi knockdown of PIK3CA in U87-EGFP cells
and under different toxic conditions
Problem Statement
Hypothesis
Procedure
Data
Results
Discussion
Conclusion
Reference
1. It is hypothesized that PIK3ca siRNA transfection will not affect cell viability
and proliferation.
2. It is hypothesized that after siRNA transfection, PIK3ca and MGMT
expression will be downregulated significantly in U87 cancer cell line.
3. It is hypothesized that pharmacologic agents, TMZ or GNE-317, will
decrease cell viability and proliferation of PI3Kca transfected cells.
0
0.05
0.1
0.15
0.2
0.25
EXP Neg Control
RelativeexpressionlevelofPIK3CA
U87-EGFP
PIK3CA expression post siRNA transfection
0
0.01
0.02
0.03
0.04
0.05
0.06
EXP Neg Control
RelativeexpressionlevelofMGMT
U87-EGFP
MGMT expression post siRNA transfection
3.6
3.65
3.7
3.75
3.8
3.85
3.9
3.95
4
4.05
Experimental Negative Control
CellProliferation(A=450nm)
U87-EGFP
U87-EGFP Cell Viability post siRNA
transfection
• Reject null hypothesis and accept alternate hypothesis.
• siRNA transfection decreased cell viability
• Decreased absorbance in experimental group and negative group correlates with
decrease in cell viability
Specific
Aim1
• Rejects null hypothesis and accept alternate hypothesis
• PIK3CA siRNA transfection shows significant less PIK3CA
protein expression
Specific Aim
2 –PIK3CA
• Accepts null hypothesis and reject alternate hypothesis
• PIK3CA siRNA transfection increased MGMT protein expression
• In order to overcome PIK3CA silence, U87-EGFP may upregulate different pathway
to increase production of MGMT
Specific Aim
2 - MGMT
• No vehicular control for transfection
• Western blot’s sample size was n=1
• In the process of finishing clonogenic assay
Limitations
• One hour incubation for CCK-8 might be too long
• Possible uneven amount of protein loaded to gels for gel
electrophoresis
Possible
Errors
Pilot Study
Specific Aim 1:
CCK-8 (n=3)
Specific Aim 2:
Western Blot (n=1)
Specific Aim 3:
Clonogenic Assay
(n=3)
Data Analysis
siRNA Transfection
OptimizationPilot
siRNA TransfectionTransfection
• Control
• Experimental
• Scramble
CCK-8 AssayAim 1
• Control
• Experimental
• Scramble
Western BlotAim 2
• Control
• Experimental
• Scramble
• House Keeping Gene
Clonogenic AssayAim 3
• Each test groups w/
• TMZ (Conc. = 0, 50, 100, and 200μM)
• GNE-317 (Conc. = 0, 1, 5, and 10μM)
• 80,000 cell/ml showed best confluency after 72 hours
incubationPilot Study
• Both groups of transfected U87-EGFP cells had lower
optical density after 72 hours of transfection compared
to normal U87-EGFP cells.
CCK-8
Assay
• The experimental group expressed significantly less PIK3CA
protein expression.
• The experimental group expressed more MGMT protein
expression.
Western
Blot
PI3K
Akt
NF-κβ
MGMT
• This experiment will study the cytotoxic effects after PI3K inhibition via
PIK3ca-siRNA transfection of U87-EGFP and whether the expression of
PIK3ca may affect cell viability under different toxic condition.
Greater Importance
• Down regulating PI3K pathway does not reduce MGMT levels and therefore
does not affect U87-EGFP cell viability.
• MGMT protein levels are higher with lower levels of PIK3CA protein.
Future Studies
• Investigate the kinetics of PI3K pathway with PIK3CA inhibition in
downregulating MGMT production
• Experiment targeting the PTEN gene promotor to downregulate MGMT
production only in U87-EGFP cells via retroviral transfection(~3-6 months
project)
A B C
D E F
A B
C
• Glioblastoma multiforme constitutes
more than 34% of all malignant brain
tumors.1
• Current treatment combines radiations
therapy with chemotherapeutic drug,
Temozolomide, TMZ.2
• Lack of MGMT promoter methylation is
associated with TMZ resistance.3
• MGMT is a downward signaling protein
of PI3K pathway.4
Figure 1. Images from a pilot study of the proliferation of U87-EGFP cells at different concentrati
ons after 72 hours These images show U87 cell proliferation with initial cell counts at 10000(A), 25
000(B), 50000(C), 80000(D), 100000(E), and 200000(F) after 72 hours. It was recommended by life
technologies to transfect with an initial count of 80000 cells, and through this pilot study seeing ap
proximately 70% confluency for the 80000 cell count, it was confirmed that 80000 was the most op
timal cell count for transfection.
Figure 2. Absorbance reading of U87 cells after administration of CCK-8 Cytotoxicity Assay
on control, negative, and experimental groups 5000 cells of each sample group (n = 3) were
plated and administered 100µL of CCK-8 to test the cell viability of each sample group. After
incubation of 1.5 hours the absorbance was assessed through a microplate reader. The
absorbance levels directly correlate to cell viability and the control, negative, and
experimental groups had a reading of 3.982, 3.834, and 3.809 respectively. A one-way
ANOVA was performed to find statistical significance between the control and negative
groups (p =1.9945e-05 ) and between the control and experimental groups (p = 2.8028e-06 )
with significance of p<0.05.
Figure 3. Adjusted Densities quantified from Western Blot of proteins PIK3CA and MGMT post transfection of negative and experimental groups 10 µg
of protein from the control, negative, and experimental were used to run through gel electrophoresis. After attaching the antibodies to evaluate the
expression of proteins PIK3CA and MGMT separately, the bands were imaged and quantified through the ImageJ software. The adjusted densities, which
correlate to the amount of protein expression in the samples, for PIK3CA (A) were 0.2335, 0.1802, and 0.0056 for the control, negative, and experimental
groups respectively. The adjusted densities for MGMT(B) values were 0.0272, 0.0288, and 0.0482 for the control, negative, and experimental groups
respectively. The Western Blot images (C) show the two different proteins PIK3CA and MGMT compared to the loading control GAPDH. One can see
fainter bands for PIK3CA with the experimental and negative group lanes which corresponds to lower expressions of the protein and a lower adjusted
density compared to the control lane which fluoresces more. As for MGMT one can see that the experimental group is the most fluorescent compared to
the negative and control lanes and that the the negative lane is more fluorescent than the control lane.
BMED 3610 Team Apocalypse: Chunghee Kim, Jordan Marshall, and Hee Young “Matt” Yoon
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Gangliogliomas. Springer Science+Business Media. 40(2):13-24.
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A., . . . Phillips, Heidi S. (2012). Targeting the PI3K Pathway in the Brain—Efficacy of a PI3K Inhibitor
Optimized to Cross the Blood–Brain Barrier. Clinical Cancer Research, 18(22), 6239-6248. doi:
10.1158/1078-0432.ccr-12-0720
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N. (2009). Induction of MGMT expression is associated with temozolomide resistance in glioblastoma
xenografts. Neuro-Oncology, 11(3), 281-291. doi: 10.1215/15228517-2008-090
4. Zhou, Xin-Ke, Tang, Sheng-Song, Yi, Gao, Hou, Min, Chen, Jin-Hui, Yang, Bo, . . . He, Zhi-Min. (2011).
RNAi knockdown of PIK3CA preferentially inhibits invasion of mutant PIK3CA cells. World Journal of
Gastroenterology : WJG, 17(32), 3700-3708. doi: 10.3748/wjg.v17.i32.3700