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RNAi-based functional selection identifies novel cell migration determinants dependent on PI3K and AKT pathways.

ABSTRACT: Lentiviral short hairpin RNA (shRNA)-mediated genetic screening is a powerful tool for identifying loss-of-function phenotype in mammalian cells. Here, we report the identification of 91 cell migration-regulating genes using unbiased genome-wide functional genetic selection. Individual knockdown or cDNA overexpression of a set of 10 candidates reveals that most of these cell migration determinants are strongly dependent on the PI3K/PTEN/AKT pathway and on their downstream signals, such as FOXO1 and p70S6K1. ALK, one of the cell migration promoting genes, uniquely uses p55? regulatory subunit of PI3K, rather than more common p85 subunit, to trigger the activation of the PI3K-AKT pathway. Our method enables the rapid and cost-effective genome-wide selection of cell migration regulators. Our results emphasize the importance of the PI3K/PTEN/AKT pathway as a point of convergence for multiple regulators of cell migration.

SUBMITTER: Seo M

PROVIDER: S-EPMC6581447 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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RNAi-based functional selection identifies novel cell migration determinants dependent on PI3K and AKT pathways.

Seo Minchul M Lee Shinrye S Kim Jong-Heon JH Lee Won-Ha WH Hu Guang G Elledge Stephen J SJ Suk Kyoungho K

Nature communications 20141028

Lentiviral short hairpin RNA (shRNA)-mediated genetic screening is a powerful tool for identifying loss-of-function phenotype in mammalian cells. Here, we report the identification of 91 cell migration-regulating genes using unbiased genome-wide functional genetic selection. Individual knockdown or cDNA overexpression of a set of 10 candidates reveals that most of these cell migration determinants are strongly dependent on the PI3K/PTEN/AKT pathway and on their downstream signals, such as FOXO1 ...[more]

PMID: 25347953

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Project description:When skin tissue is damaged, the wound microenvironment is hypoxic or anoxic due to the rupture of blood vessels and the high oxygen consumption of related cells; so far, it is not clear whether such hypoxic microenvironment will promote epidermal cell migration and the underlying molecular regulatory mechanisms of this effect remains unclear. Studies to date have shown that, immortal keratinocyte cell line HaCaT and primary human keratinocytes are maintained under conditions of hypoxia (1% oxygen) or normoxia. The researchers would adopt methods such as live cell imaging system, Western blotting, transwell assays and wound scratch assays etc. to study the changes of cell migration. Expression profile of mRNAs in HaCaT cells from 3 hypoxia and 3 normoxic conditions were analyzed by the Clariom D microarray assay. Gene Ontology (GO) and KEGG genomics analyses were performed to identify significant functions, pathways, and the associations of differentially expressed mRNAs. Moreover, the potential mechanism is discussed and studied. According to relevant results, the epidermal cell migration is promoted in the early hypoxia. Furthermore, experiment showed that 1456 mRNAs showed differential expression between the hypoxia and normoxic conditions, which included 537 upregulated and 919 downregulated mRNAs. These results also shown that all G proteins are upregulated. At the same time, GO analysis indicated that most upregulated mRNA are in connection with cell inflammation and migration. Pathway analysis indicated that 20 pathways corresponded to the upregulated mRNA and 6 pathways corresponded to the upregulated mRNA. These pathway analyses have indicated that genes involved in inflammation, migration and PI3K-Akt signaling are potentially regulated. Combine with microarray assay, we further shown that G protein accelerates epidermal cell migration via activation of PI3K/Akt-dependent mTORC1 signaling under the condition of hypoxia. Based on this evidence, researchers can further reveal the molecular and cellular mechanisms of local wound hypoxia, and for improving the wound healing.

Dataset Information

RNAi-based functional selection identifies novel cell migration determinants dependent on PI3K and AKT pathways.

Publications

RNAi-based functional selection identifies novel cell migration determinants dependent on PI3K and AKT pathways.

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