Transient strong reduction of PTEN expression by specific RNAi induces loss of adhesion of the cells.
The tumor suppressor gene pten encodes a lipid phosphatase that dephosphorylates D3 of phosphatidylinositol(3,4,5)trisphosphate, producing phosphatidylinositol(4,5)bisphosphate. Although PTEN has been implicated in cell adhesion and migration, the underlying molecular mechanism is unknown. To investigate the role of PTEN in cell adhesion, we designed three different siRNAs (siRNA PTEN-a, siRNA PTEN-b, and siRNA PTEN-c) and transfected into 293T cells. Two days later, only the cells transfected with siRNA PTEN-b became round and detached from the culture dishes, whereas cells transfected with a control siRNA against GFP or the two other siRNAs against PTEN did not. Evaluation of the RNAi effect revealed that siRNA PTEN-b inhibited >95% of PTEN expression, the most effective among the three siRNAs. To check for non-specific effects such as interferon response and inhibition of off-target genes, we then used quantitative PCR analysis and DNA microarray analysis. None was detected, indicating that the RNAi system was highly specific. Immunofluorescence studies using PTEN-knockdown HeLa cells revealed that the loss of adhesion was accompanied by a reduction in the number of focal adhesion plaques and disorganization of the actin cytoskeleton. Transient and near-complete loss of PTEN expression induces loss of adhesion of the cells.
Mise-Omata
Setsuko
S
Technology and Development Team for BioSignal Program, Subteam for BioSignal Integration, RIKEN Bioresource Center, RIKEN Tsukuba Institute, 3-1-1 Koyadai Tsukuba, Ibaraki 305-0074, Japan.
Obata
Yuichi
Y
Iwase
Shigeru
S
Mise
Nathan
N
Doi
Takahiro S
TS
D017209:Apoptosis / Q000502:physiology; D002448:Cell Adhesion / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000378:metabolism*; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics*; D013997:Time Factors; D014162:Transfection / Q000379:methods; D025521:Tumor Suppressor Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*
D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human