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Short-hairpin RNA-mediated stable silencing of Grb2 impairs cell growth and DNA synthesis.

ABSTRACT: Grb2 is an SH2-SH3 protein adaptor responsible for linking growth factor receptors with intracellular signaling cascades. To study the role of Grb2 in cell growth, we have generated a new COS7 cell line (COS7(shGrb2)), based on RNAi technology, as null mutations in mammalian Grb2 genes are lethal in early development. This novel cell line continuously expresses a short hairpin RNA that targets endogenous Grb2. Stable COS7(shGrb2) cells had the shGrb2 integrated into the genomic DNA and carried on <10% of normal levels of Grb2. Silencing Grb2 expression reduced, but did not eliminate, basal cell growth rate. This could be reversed by either the addition of neomycin to the cell cultures or by rescuing with an Xpress-Grb2(SiL) construct (made refractory to the shRNA-mediated interference), but not with an SH2-deficient mutant (R86K). Thus, a viable knock-down and rescue protocol has demonstrated that Grb2 is crucial for cell proliferation.

SUBMITTER: Di Fulvio M

PROVIDER: S-EPMC2247433 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Short-hairpin RNA-mediated stable silencing of Grb2 impairs cell growth and DNA synthesis.

Di Fulvio Mauricio M Henkels Karen M KM Gomez-Cambronero Julian J

Biochemical and biophysical research communications 20070410 3

Grb2 is an SH2-SH3 protein adaptor responsible for linking growth factor receptors with intracellular signaling cascades. To study the role of Grb2 in cell growth, we have generated a new COS7 cell line (COS7(shGrb2)), based on RNAi technology, as null mutations in mammalian Grb2 genes are lethal in early development. This novel cell line continuously expresses a short hairpin RNA that targets endogenous Grb2. Stable COS7(shGrb2) cells had the shGrb2 integrated into the genomic DNA and carried o ...[more]

PMID: 17445773

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Project description:Keratin 17 (KRT17) has been demonstrated to be a potential biological marker for the prediction of prognosis in particular types of cancer. The aim of the present study was to investigate the molecular mechanisms underlying the function of KRT17 in the pancreatic cancer (PAC) cell line PANC-1 and the potential of KRT17 as a therapeutic target for PAC. KRT17 expression levels were analyzed using quantitative PCR and compared with histological data using bioinformatics tools in PAC samples and three human PAC cell lines. Cell proliferation was determined using an MTT assay, in addition to cell cycle distribution and apoptosis analysis using flow cytometry, colony formation assay using Giemsa staining and cell motility analysis using a Transwell migration assay. Tumor growth was evaluated in vivo in nude mice. The expression levels of a number of signaling molecules were measured to establish the potential mechanism by which silencing KRT17 expression affected PAC PANC-1 cells. Increased levels of KRT17 expression were observed in human PAC compared with normal tissues, as well as in three human PAC cell lines (MIA PaCa-2, PANC-1 and KP-3 cells) compared with the H6c7 human immortal pancreatic duct epithelial cell line. High expression levels of KRT17 in PAC samples were associated with poor overall survival (P=0.036) and disease-free survival (P=0.017). Lentivirus-mediated KRT17 silencing inhibited cell proliferation, colony formation and migration, but promoted apoptosis and resulted in cell cycle arrest in the G0/G1 phase in PANC-1 cells. In addition, KRT17 knockdown inhibited in vivo tumor growth. KRT17 knockdown induced dysregulation of ERK1/2 and upregulation of the pro-apoptotic Bcl-2 protein Bad. In conclusion, the present study demonstrated that elevated KRT17 levels are positively associated with pancreatic cancer progression; KRT17 knockdown suppressed cell growth, colony formation, migration and tumor growth, and induced apoptosis and cell cycle arrest, affecting ERK1/2/Bad signaling. Therefore, the results of the present study suggested that KRT17 may be a potential target for the treatment of pancreatic cancer.

Dataset Information

Short-hairpin RNA-mediated stable silencing of Grb2 impairs cell growth and DNA synthesis.

Publications

Short-hairpin RNA-mediated stable silencing of Grb2 impairs cell growth and DNA synthesis.

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