RNA INTERFERENCE RNAI RELATION TO P53 USING RNAI TO SILENCE MUTANT 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

P53 most frequently mutated tumor- suppressor 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 IS KNOWN..

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 sub- G0/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 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 SIRNA BAR CODE SCREEN

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 Web. 16 Nov 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 Web. 16 Nov and.html