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.