Candidate Anti-cancer shRNAs Identified by Mortalin Staining Reporter

摘要：

　　RNA interference,a phenomenon whereby small double-stranded RNA knocks down the expression of a sequence-specific gene,is widely used as a therapeutic tool for many diseases including cancers.In case of the latter,selection of gene targets poses one of the major hurdles and hence requires an effective screening method.Most of the screening methods rely on the upregulation or enrichment of gene targets in cancers.Here we report a cancer target screening method based on the change in subcellular distribution of a hsp70 family protein,mortalin/mthsp70.Besides its upregulation in cancers,its staining pattern differs in the normal and cancer cells.Whereas it is pancytoplasmic in normal cells,a large variety of cancer cells show its perinuclear staining.Furthermore,induction of senescence by a variety of chemicals and stress conditions in cancer cells was seen to cause shift in the mortalin staining pattern from the perinuclear type to pancytoplasmic type.We used mortalin staining as a model reporter and screened shRNAs for anticancer activity.Cancer cells were stably transfected with the shRNA expressing plasmids in 96-well plates,immunostained with anti-mortalin antibody and screened under the automated scanning microscope (Axiovert 200M,ZEISS).By four rounds of screening,we identified candidate shRNAs that caused shift in mortalin staining from the perinuclear to the pancytoplasmic pattern and induced senescence in cancer cells.We investigated the molecular mechanism of induced senescence by (i) examining the p53 and pl6 activities and (ii) adopting bioinformatics approach.We reported that the DNA damagesignaling pathway could be a good candidate target for anticancer therapy.

关键词： Anti-cancer shRNA Bioinformatics Immunolocalization Mortalin ShRNA screening Visual reporter assay

作者: Ran GAO Tetsuro ISHII Sunil C KAUL Renu WADHWA

作者单位: National Institute of Advanced Industrial Science & Technology(AIST),Central 4,Tsukuba,Japan,305-856 Graduate school of Life & Environmental Sciences,University of Tsukuba,Ibaraki,Japan,305-8575 National Institute of Advanced Industrial Science & Technology(AIST),Central 4,Tsukuba,Japan,305-856

会议类型: 国际会议

会议名称: The 2nd China-Japan Graduate Student Forum -- Life, Environment and Energy (第二届中日研究生论坛--生命,环境与能源)

会议地点: 北京

会议语种:英文

页码: 12-16

在线出版日期: 2009-09-24（万方平台首次上网日期，不代表论文的发表时间）