Transcript RNA interference RNAi relation to p53

RNA INTERFERENCE RNAI
RELATION TO P53
U S I N G R N A I TO S I L E N C E M U TA N T
P53 IN BLADDER CANCER CELLS &
SIRNA BAR CODE SCREENING
By: Chelsey Maag
RNAI RECAP
Defense mechanism
Triggered by dsRNA
Causes a strong
suppression of gene
expression
P53 RECAP
SILENCING MUTANT P53
IN HUMAN BLADDER
CANCER CELLS
WHAT IS KNOWN..
P53 most frequently
mutated tumorsuppressor gene in
human cancers
Mutated p53 also
contribute to gain of
novel-cancer related
functions
Leads to oncogenesis
Focuses on Bladder
Cancer
Using two known lineages
bladder cancer cells
T24 & 5637
Both have p53 mutations
WHAT WE WANT TO FIND OUT…
Conditional knockdown of
p53 mutants by small
interfering RNAs in
various Bladder cancer
cell lines will induce cell
cycle arrest and cell
apoptosis
Knockdown of p53 mutant
with RNAi will cooperate
with cisplatin in the
inhibition and apoptosis
of these cancer cells.
EFFECTS OF P53-TARGETING SIRNA
ON THE EXPRESSION OF MUTANT
P53 IN 2 LINES OF HUMAN BLADDER
CANCER CELLS
KNOCKDOWN OF MUTANT P53 INHIBITION OF
GROWTH AND VIABILITY
Less dense more
dead cells
Lost viability
SILENCING OF MUTANT P53 COOPERATES WITH
CISPLATIN IN INHIBITING BLADDER CANCER CELLS
Conflicting on whether p53 mutation presents
increased responsiveness or increased resistance to
cisplatin-based chemotherapy in advanced bladder
cancer
Cisplatin chemotherapy only gets a median survival
rate of 14 months and side effects caused by lack of
specificity of tumors remain a problem
Test using MTT assay to determine the combined
effects of siP53 and cisplatin on bladder cancer cells
SILENCING OF MUTANT P53 COOPERATES WITH
CISPLATIN IN INHIBITING BLADDER CANCER
CELLS
average
reduction in cell
viability was
72.3%
SILENCING OF MUTANT P53 COOPERATES WITH
CISPLATIN IN INDUCING APOPTOSIS
The percentage of cells
in G1, S, or G2 phase
equal to number of
cells in each phase
Both cisplatin & siP53
had increase in the
sub-G0/G1 population,
indicating apoptotic
cells
SILENCING OF MUTANT P53 COOPERATES WITH
CISPLATIN IN INDUCING APOPTOSIS
When siP53
transfected cells
were treated with
cisplatin the
proportion of subG0/G1 cells
increased to 51.51%
Suggesting that the
p53-targeting siRNA
can co-operate with
cisplatin in the
inhibition of bladder
cancer cells
CONCLUSION
-The study indicated that knockdown of mutant p53
by siRNA was able to induce cell cycle arrest and
apoptosis in 5637 and T24 human bladder cancer
cells
-Plus, this strategy cooperated with cisplatin in the
inhibition and apoptosis of bladder cancer cells.
-The results provide evidence that targeting mutant
p53 by RNAi may serve as a promising
therapeutic strategy for treatment of advanced
bladder cancer with p53 mutations
FUTURE
APPLICATIONS
Biggest challenges that remain in
achieving application of RNAi
therapeutics are the delivery
and target validation
Further studies are needed to
depict the exact mechanisms
effectiveness for future clinical
uses
SiRNA bar coding could be a
solution
SIRNA BAR-CODE SCREENS
Mammalian cells screens have been troubled by the lack of
suitable tools that can used on a large scale.
So recently, developed expression vectors to direct the synthesis
of short hairpin RNAs that act as short interfering RNA like
molecule to stably suppress gene expression
Since, typical RNAi screening is time consuming so alternative
strategy used to rapidly screen complex shRNA vector libraries
known as siRNA bar-code screens
SIRNA BAR CODE SCREEN
siRNA bar-code
screens, takes
advantage that
each hairpin vector
contains a unique
gene specific
molecular identifier:
19-mer targeting
sequence Using
DNA microarrays
that contain the
oligonucleotides
ULTIMATELY
SiRNA Bar Code Screen
could be and effective
way to screen RNAi
therapeutics against
p53
REFERENCES
Berns, Katrien, and Roderick L. Beijersbergen. "A Large-scale RNAi Screen in Human
Cells Identifies New Components of the P53 Pathway." Nature.com. Nature
Publishing Group,
25 Mar. 2004. Web. 16 Nov. 2014.
<http://www.nature.com/nature/journal/v428/n6981/full/nature02371.h
tml>
Bin Zhu,, Hai-, and Kai Yang. "Silencing of Mutant P53 by SiRNA Induces Cell Cycle
Arrest and Apoptosis in Human Bladder Cancer Cells." WJSO. World Journal of
Surgical
Oncology, 28 Jan. 2013. Web. 16 Nov. 2014.
<http://www.wjso.com/content/11/1/22>
http://biol1020-2011-2.blogspot.com/2011/09/rnai-interference-potency-factorsand.html