Advanced

Exploring the effects of platinum compounds on RNA, adduct formation, thermal melting properties and siRNA down regulation capacity of gene targets associated

Al Shiekh, Alak LU (2012) KEMT30 20112
Department of Chemistry
Abstract
Since its discovery, RNA interference (RNAi) has turned into an invaluable research tool for regulating gene expression. Furthermore, this naturally occurring phenomenon in mammalian cells is of interest in cancer research, both as a possible therapeutic approach and a target for cancer treatment. Platinum anticancer agents like cisplatin are considered one of the most active cancer drugs used either alone or in combination therapy. Cisplatin has made its way from the accidental discovery in 1965 to the first patient treated in 1971. The validated target of platinum cancer treatment is DNA. Other targets include RNA and protein. In this work, the aim was to shed more light on the interaction between platinum based drugs and RNA,... (More)
Since its discovery, RNA interference (RNAi) has turned into an invaluable research tool for regulating gene expression. Furthermore, this naturally occurring phenomenon in mammalian cells is of interest in cancer research, both as a possible therapeutic approach and a target for cancer treatment. Platinum anticancer agents like cisplatin are considered one of the most active cancer drugs used either alone or in combination therapy. Cisplatin has made its way from the accidental discovery in 1965 to the first patient treated in 1971. The validated target of platinum cancer treatment is DNA. Other targets include RNA and protein. In this work, the aim was to shed more light on the interaction between platinum based drugs and RNA, investigating the interaction with double stranded RNAs that are 20 nucleotides in length. The effect of platination on RNA thermodynamic stability and function in a cellular environment was studied. Purther knowledge in this area will be helpful in developing siRNA-based therapies, and to elucidate possible interactions that might arise from the combination of platinum based treatments and small interfering RNA (siRNA) based therapeutics e.g. the possibility of synergistic effects. Endogenous miRNA play a major role in the cancerous cell environment. A doser look at the interplay between platinum-based treatment and oncomiRs could be verified as additional therapeutic targets of platinum drugs. Several synthetic siRNA sequences that were designed to target regions of 3'-UTR of the proteins BRCAI and Wnt5a were studied in the present work, both proteins have a strong association with human cancers. The ehosen sequences were evaluated by thermal melting studies and platination reactions. Purification of platination products was performed to investigate the effects of cisplatin and oxaliplatin on both duplex stability and down regulating capacity in a cellular environment. siRNAs were studied by denaturing poly acrylamide gel electrophoresis (PAGE).

The down regulating ability was investigated in a cellular system consisting of MCF-7 breast cancer cells by using two model protein systems. The effect of siRNA on endogenous BRCAI protein levels was evaluated by Western blot. The effect of platination on siRNA's downregulation capacity was investigated by Luciferase assays with Wnt5a 3 '-UTR as atarget. Purified platination products or native siRNAs were transfected into MCF-7 cells and
comparison of luciferase productian by luminometry was measured. lntroduction of oxaliplatin in concentrations below previously determined IC50 values in MCF-7 celllines after transfection ofunplatinated siRNAsserved to evaluate the effect on down regulation capacity. (Less)
Please use this url to cite or link to this publication:
author
Al Shiekh, Alak LU
supervisor
organization
course
KEMT30 20112
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Proteinvetenskap
language
English
id
2857072
date added to LUP
2012-06-29 14:50:06
date last changed
2012-06-29 14:50:06
@misc{2857072,
  abstract     = {Since its discovery, RNA interference (RNAi) has turned into an invaluable research tool for regulating gene expression. Furthermore, this naturally occurring phenomenon in mammalian cells is of interest in cancer research, both as a possible therapeutic approach and a target for cancer treatment. Platinum anticancer agents like cisplatin are considered one of the most active cancer drugs used either alone or in combination therapy. Cisplatin has made its way from the accidental discovery in 1965 to the first patient treated in 1971. The validated target of platinum cancer treatment is DNA. Other targets include RNA and protein. In this work, the aim was to shed more light on the interaction between platinum based drugs and RNA, investigating the interaction with double stranded RNAs that are 20 nucleotides in length. The effect of platination on RNA thermodynamic stability and function in a cellular environment was studied. Purther knowledge in this area will be helpful in developing siRNA-based therapies, and to elucidate possible interactions that might arise from the combination of platinum based treatments and small interfering RNA (siRNA) based therapeutics e.g. the possibility of synergistic effects. Endogenous miRNA play a major role in the cancerous cell environment. A doser look at the interplay between platinum-based treatment and oncomiRs could be verified as additional therapeutic targets of platinum drugs. Several synthetic siRNA sequences that were designed to target regions of 3'-UTR of the proteins BRCAI and Wnt5a were studied in the present work, both proteins have a strong association with human cancers. The ehosen sequences were evaluated by thermal melting studies and platination reactions. Purification of platination products was performed to investigate the effects of cisplatin and oxaliplatin on both duplex stability and down regulating capacity in a cellular environment. siRNAs were studied by denaturing poly acrylamide gel electrophoresis (PAGE).

The down regulating ability was investigated in a cellular system consisting of MCF-7 breast cancer cells by using two model protein systems. The effect of siRNA on endogenous BRCAI protein levels was evaluated by Western blot. The effect of platination on siRNA's downregulation capacity was investigated by Luciferase assays with Wnt5a 3 '-UTR as atarget. Purified platination products or native siRNAs were transfected into MCF-7 cells and
comparison of luciferase productian by luminometry was measured. lntroduction of oxaliplatin in concentrations below previously determined IC50 values in MCF-7 celllines after transfection ofunplatinated siRNAsserved to evaluate the effect on down regulation capacity.},
  author       = {Al Shiekh, Alak},
  keyword      = {Proteinvetenskap},
  language     = {eng},
  note         = {Student Paper},
  title        = {Exploring the effects of platinum compounds on RNA, adduct formation, thermal melting properties and siRNA down regulation capacity of gene targets associated},
  year         = {2012},
}