{"id":{"repo_id":"umkc","oai_identifier":"oai:mospace.umsystem.edu:10355/33194"},"canonical_url":"https://search.dev.ndltd.org/etd/umkc/oai:mospace.umsystem.edu:10355/33194","repository":{"repo_id":"umkc","name":"University of Missouri - Kansas City","base_url":"https://mospace.umsystem.edu/oai/request"},"display":{"title":"Evaluation of therapeutic effect of IKK epsilon SiRNA on breast cancer cells and development of a peptide-based SiRNA delivery system","abstract":"The objective of this dissertation is to develop siRNA-based cancer therapeutics. In chapters 1 and 2, we introduced the mechanism, challenge and promise of RNAi-based therapy, as well as various siRNA delivery systems. In Chapter 3, we examined the therapeutic potential of IKKε siRNA on human breast cancer cells. Eight siRNAs targeting different regions of the IKKε mRNA were designed, and the silencing effect was screened by quantitative real time RT-PCR. Silencing of IKKε in human breast cancer cells resulted in decrease of focus formation potential and clonogenicity as well as in vitro cell migration/invasion capabilities. Moreover, knockdown of IKKε suppressed cell proliferation. Cell cycle assay showed that the anti-proliferation effect of IKKε siRNA was mediated by arresting cells in G0/G1 phase, which was caused by downregulation of cyclin D1. Furthermore, we demonstrated that silencing of IKKε inhibited the NF-κB basal activity and the Bcl-2 expression. Significant apoptosis was not observed in breast cancer cells upon the silencing of IKKε. In chapter 4, cholesterol was conjugated to a series of peptides composed of lysine and histidine residues. Our results indicate that the amphiphilic cholesteryl peptides can self-assemble to form a micelle-like structure in aqueous solutions. Formation of the micelle structure significantly promotes siRNA condensation capability of the peptides. The cholesteryl peptides can form stable complex with siRNA and effectively protect siRNA from enzymatic degradation in rat serum. Furthermore, the cholesteryl peptides can efficiently transfect siRNA into different cancer cells and mediate potent gene silencing effect, whereas peptides without cholesterol modification fail to deliver siRNA into the cells. In addition, one of the cholesteryl peptide Chol-H3K2s displayed comparable cellular uptake and gene silencing effect but less cytotoxicity compared with bPEI and lipofectamine-2000. In chapter 5, we introduced folic acid and a disulfide linker into the Chol-H3K2s. The reducible cholesteryl peptides efficiently condense siRNA, and the resultant complexes can release siRNA under reductive condition. PEGylation of the reducible cholesteryl peptide significantly decreased nonspecific cellular uptake. Conjugation of folic acid via a PEG spacer resulted in high cellular uptake and gene silencing effect. In Chapter 6, we identified a prostate cancer specific peptide using a phage display library and evaluate its potential application in targeted drug delivery. One phage clone with a high binding affinity to LNCaP cells was identified. Cell phage ELISA and immunostaining demonstrated high specificity of this phage to LNCaP cells. The fluorescein labeled KYL peptide exhibited higher binding to LNCaP cells in comparison to other cells. The fusion peptide composed of the KYL peptide and the proapoptotic peptide induced cell death in LNCaP cells, but not in PC-3 cells. The KYL peptide-protamine conjugate also efficiently delivered a fluorescein labeled siRNA into LNCaP cells.","abstract_html":"The objective of this dissertation is to develop siRNA-based cancer therapeutics. In chapters 1 and 2, we introduced the mechanism, challenge and promise of RNAi-based therapy, as well as various siRNA delivery systems. In Chapter 3, we examined the therapeutic potential of IKKε siRNA on human breast cancer cells. Eight siRNAs targeting different regions of the IKKε mRNA were designed, and the silencing effect was screened by quantitative real time RT-PCR. Silencing of IKKε in human breast cancer cells resulted in decrease of focus formation potential and clonogenicity as well as in vitro cell migration/invasion capabilities. Moreover, knockdown of IKKε suppressed cell proliferation. Cell cycle assay showed that the anti-proliferation effect of IKKε siRNA was mediated by arresting cells in G0/G1 phase, which was caused by downregulation of cyclin D1. Furthermore, we demonstrated that silencing of IKKε inhibited the NF-κB basal activity and the Bcl-2 expression. Significant apoptosis was not observed in breast cancer cells upon the silencing of IKKε. In chapter 4, cholesterol was conjugated to a series of peptides composed of lysine and histidine residues. Our results indicate that the amphiphilic cholesteryl peptides can self-assemble to form a micelle-like structure in aqueous solutions. Formation of the micelle structure significantly promotes siRNA condensation capability of the peptides. The cholesteryl peptides can form stable complex with siRNA and effectively protect siRNA from enzymatic degradation in rat serum. Furthermore, the cholesteryl peptides can efficiently transfect siRNA into different cancer cells and mediate potent gene silencing effect, whereas peptides without cholesterol modification fail to deliver siRNA into the cells. In addition, one of the cholesteryl peptide Chol-H3K2s displayed comparable cellular uptake and gene silencing effect but less cytotoxicity compared with bPEI and lipofectamine-2000. In chapter 5, we introduced folic acid and a disulfide linker into the Chol-H3K2s. The reducible cholesteryl peptides efficiently condense siRNA, and the resultant complexes can release siRNA under reductive condition. PEGylation of the reducible cholesteryl peptide significantly decreased nonspecific cellular uptake. Conjugation of folic acid via a PEG spacer resulted in high cellular uptake and gene silencing effect. In Chapter 6, we identified a prostate cancer specific peptide using a phage display library and evaluate its potential application in targeted drug delivery. One phage clone with a high binding affinity to LNCaP cells was identified. Cell phage ELISA and immunostaining demonstrated high specificity of this phage to LNCaP cells. The fluorescein labeled KYL peptide exhibited higher binding to LNCaP cells in comparison to other cells. The fusion peptide composed of the KYL peptide and the proapoptotic peptide induced cell death in LNCaP cells, but not in PC-3 cells. The KYL peptide-protamine conjugate also efficiently delivered a fluorescein labeled siRNA into LNCaP cells.","abstract_has_math":false,"creators":["Qin, Bin"],"institution":"University of Missouri--Kansas City","degree_name":"Ph.D.","degree_level":"Doctoral","degree_discipline":"Pharmacy (UMKC)","degree_department":null,"school":null,"contributors":[],"advisors":["Cheng, Kun (Professor)"],"committee_chairs":[],"committee_members":[],"year":2012,"date_issued":"2012","date_published":"2012","updated_at":"2026-07-24T05:19:15Z","subjects":[],"languages":["en_US"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10355/33194","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Cheng, Kun (Professor)"]},{"key":"dc:creator","label":"Author","values":["Qin, Bin"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2013-03-12T15:15:34Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2015-03-12T11:15:05Z"]},{"key":"dc:date.issued","label":"Date","values":["2012"]},{"key":"dc:publisher","label":"Institution","values":["University of Missouri--Kansas City"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Pharmacy (UMKC)","Chemistry (UMKC)"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Missouri--Kansas City"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en_US"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10355/33194"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Title from PDF of title page, viewed on March 12, 2013","Dissertation advisor: Kun Cheng","Vita","Includes bibliographic references (p.192-174)","Thesis (Ph.D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2012"]},{"key":"dc:description.abstract","label":"Abstract","values":["The objective of this dissertation is to develop siRNA-based cancer therapeutics. In chapters 1 and 2, we introduced the mechanism, challenge and promise of RNAi-based therapy, as well as various siRNA delivery systems. In Chapter 3, we examined the therapeutic potential of IKKε siRNA on human breast cancer cells. Eight siRNAs targeting different regions of the IKKε mRNA were designed, and the silencing effect was screened by quantitative real time RT-PCR. Silencing of IKKε in human breast cancer cells resulted in decrease of focus formation potential and clonogenicity as well as in vitro cell migration/invasion capabilities. Moreover, knockdown of IKKε suppressed cell proliferation. Cell cycle assay showed that the anti-proliferation effect of IKKε siRNA was mediated by arresting cells in G0/G1 phase, which was caused by downregulation of cyclin D1. Furthermore, we demonstrated that silencing of IKKε inhibited the NF-κB basal activity and the Bcl-2 expression. Significant apoptosis was not observed in breast cancer cells upon the silencing of IKKε. In chapter 4, cholesterol was conjugated to a series of peptides composed of lysine and histidine residues. Our results indicate that the amphiphilic cholesteryl peptides can self-assemble to form a micelle-like structure in aqueous solutions. Formation of the micelle structure significantly promotes siRNA condensation capability of the peptides. The cholesteryl peptides can form stable complex with siRNA and effectively protect siRNA from enzymatic degradation in rat serum. Furthermore, the cholesteryl peptides can efficiently transfect siRNA into different cancer cells and mediate potent gene silencing effect, whereas peptides without cholesterol modification fail to deliver siRNA into the cells. In addition, one of the cholesteryl peptide Chol-H3K2s displayed comparable cellular uptake and gene silencing effect but less cytotoxicity compared with bPEI and lipofectamine-2000. In chapter 5, we introduced folic acid and a disulfide linker into the Chol-H3K2s. The reducible cholesteryl peptides efficiently condense siRNA, and the resultant complexes can release siRNA under reductive condition. PEGylation of the reducible cholesteryl peptide significantly decreased nonspecific cellular uptake. Conjugation of folic acid via a PEG spacer resulted in high cellular uptake and gene silencing effect. In Chapter 6, we identified a prostate cancer specific peptide using a phage display library and evaluate its potential application in targeted drug delivery. One phage clone with a high binding affinity to LNCaP cells was identified. Cell phage ELISA and immunostaining demonstrated high specificity of this phage to LNCaP cells. The fluorescein labeled KYL peptide exhibited higher binding to LNCaP cells in comparison to other cells. The fusion peptide composed of the KYL peptide and the proapoptotic peptide induced cell death in LNCaP cells, but not in PC-3 cells. The KYL peptide-protamine conjugate also efficiently delivered a fluorescein labeled siRNA into LNCaP cells."]},{"key":"dc:title","label":"Title","values":["Evaluation of therapeutic effect of IKK epsilon SiRNA on breast cancer cells and development of a peptide-based SiRNA delivery system"]}]}],"canonical_facts":{"dc:contributor.advisor":["Cheng, Kun (Professor)"],"dc:creator":["Qin, Bin"],"dc:date.accessioned":["2013-03-12T15:15:34Z"],"dc:date.available":["2015-03-12T11:15:05Z"],"dc:date.issued":["2012"],"dc:description":["Title from PDF of title page, viewed on March 12, 2013","Dissertation advisor: Kun Cheng","Vita","Includes bibliographic references (p.192-174)","Thesis (Ph.D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2012"],"dc:description.abstract":["The objective of this dissertation is to develop siRNA-based cancer therapeutics. In chapters 1 and 2, we introduced the mechanism, challenge and promise of RNAi-based therapy, as well as various siRNA delivery systems. In Chapter 3, we examined the therapeutic potential of IKKε siRNA on human breast cancer cells. Eight siRNAs targeting different regions of the IKKε mRNA were designed, and the silencing effect was screened by quantitative real time RT-PCR. Silencing of IKKε in human breast cancer cells resulted in decrease of focus formation potential and clonogenicity as well as in vitro cell migration/invasion capabilities. Moreover, knockdown of IKKε suppressed cell proliferation. Cell cycle assay showed that the anti-proliferation effect of IKKε siRNA was mediated by arresting cells in G0/G1 phase, which was caused by downregulation of cyclin D1. Furthermore, we demonstrated that silencing of IKKε inhibited the NF-κB basal activity and the Bcl-2 expression. Significant apoptosis was not observed in breast cancer cells upon the silencing of IKKε. In chapter 4, cholesterol was conjugated to a series of peptides composed of lysine and histidine residues. Our results indicate that the amphiphilic cholesteryl peptides can self-assemble to form a micelle-like structure in aqueous solutions. Formation of the micelle structure significantly promotes siRNA condensation capability of the peptides. The cholesteryl peptides can form stable complex with siRNA and effectively protect siRNA from enzymatic degradation in rat serum. Furthermore, the cholesteryl peptides can efficiently transfect siRNA into different cancer cells and mediate potent gene silencing effect, whereas peptides without cholesterol modification fail to deliver siRNA into the cells. In addition, one of the cholesteryl peptide Chol-H3K2s displayed comparable cellular uptake and gene silencing effect but less cytotoxicity compared with bPEI and lipofectamine-2000. In chapter 5, we introduced folic acid and a disulfide linker into the Chol-H3K2s. The reducible cholesteryl peptides efficiently condense siRNA, and the resultant complexes can release siRNA under reductive condition. PEGylation of the reducible cholesteryl peptide significantly decreased nonspecific cellular uptake. Conjugation of folic acid via a PEG spacer resulted in high cellular uptake and gene silencing effect. In Chapter 6, we identified a prostate cancer specific peptide using a phage display library and evaluate its potential application in targeted drug delivery. One phage clone with a high binding affinity to LNCaP cells was identified. Cell phage ELISA and immunostaining demonstrated high specificity of this phage to LNCaP cells. The fluorescein labeled KYL peptide exhibited higher binding to LNCaP cells in comparison to other cells. The fusion peptide composed of the KYL peptide and the proapoptotic peptide induced cell death in LNCaP cells, but not in PC-3 cells. The KYL peptide-protamine conjugate also efficiently delivered a fluorescein labeled siRNA into LNCaP cells."],"dc:identifier.uri":["http://hdl.handle.net/10355/33194"],"dc:language.iso":["en_US"],"dc:publisher":["University of Missouri--Kansas City"],"dc:title":["Evaluation of therapeutic effect of IKK epsilon SiRNA on breast cancer cells and development of a peptide-based SiRNA delivery system"],"dc:type":["Thesis"],"thesis:degree_discipline":["Pharmacy (UMKC)","Chemistry (UMKC)"],"thesis:degree_level":["Doctoral"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Missouri--Kansas City"]},"updated_at":"2026-07-24T05:19:15Z"}