Unknown,Transcriptomics,Genomics,Proteomics

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RNAi off-target analysis in Trp53-/- mouse embryonic fibroblasts transduced with miRNA-based shRNAs targeting Trp53.

ABSTRACT: We analyzed miRNA-based shRNA off-target effects by transducing Trp53-/- MEFs at single- and high-copy with six well-characterized, potent and weak Trp53 shRNAs. To advance RNAi therapy for KRAS-mutant cancer, we developed a functionally validated library of siRNAs against RAS pathway genes that minimize off-target effects and enable combination gene silencing at low dose. We developed an in vivo model for real-time tracking of nanoparticle-based siRNA delivery and offer proof-of-principle that siRNA-mediated inhibition of a single gene (KRAS) or combinations of genes (A/B/C-RAF or KRAS+PIK3C-A/B) can impair the growth of KRAS-mutant colorectal cancer xenografts. Trp53-/- MEFs were transduced with LMP expressing Trp53 shRNAs at single copy (11-21% infection efficiency) and high copy (>98% infection efficiency), selected on puromycin and grown in absence of the selection agent before harvest. Uninfected Trp53-/- MEFs and Trp53-/- MEFs infected with an empty vector control served as M-bM-^@M-^\no shRNAM-bM-^@M-^] reference.

ORGANISM(S): Mus musculus

SUBMITTER: Christof Fellmann

PROVIDER: E-GEOD-59952 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Densely ionizing radiation is a major component of the space radiation environment and has potentially greater carcinogenic effect compared to sparsely ionizing radiation that is prevalent in the terrestrial environment. It is unknown to what extent the irradiated microenvironment contributes to the differential carcinogenic potential of densely ionizing radiation. To address this gap, 10-week old BALB/c mice were irradiated with 100 cGy sparsely ionizing g-radiation or 10, 30, or 80 cGy of densely ionizing, 350 MeV/amu Si particles and transplanted 3 days later with syngeneic Trp53 null mammary fragments. Tumor appearance was monitored for 600 days. Tumors arising in Si-particle irradiated mice had a shorter median time to appearance, grew faster and were more likely to metastasize. Most tumors arising in sham-irradiated mice were ER-positive, pseudo-glandular and contained both basal keratin 14 and luminal keratin 8/18 cells (designated K14/18), while most tumors arising in irradiated hosts were K8/18 positive (designated K18) and ER negative. Comparison of K18 vs K14/18 tumor expression profiles showed that genes increased in K18 tumors were associated with ERBB2 and KRAS while decreased genes overlapped with those down regulated in metastasis and by loss of E-cadherin. Consistent with this, K18 tumors grew faster than K14/18 tumors and more mice with K18 tumors developed lung metastases compared to mice with K14/18 tumors. However, K18 tumors arising in Si-particle irradiated mice grew even faster and were more metastatic compared to control mice. A K18 Si-irradiated host profile was enriched in genes involved in mammary stem cells, stroma, and Notch signaling. Thus systemic responses to densely ionizing radiation enriches for a ER-negative, K18-positive tumor, whose biology is more aggressive compared to similar tumors arising in non-irradiated hosts. Key Words: ionizing radiation; breast cancer; heavy ion radiation;initiation; promotion 3 different dose of Si were used. Total RNA was extracted from mammary tumors derived from transplantations of non-irradiated p53null mammary fragments into irradiated hosts. We analyzed a total of 45 Trp53-null tumors: 18 from sham-irradiated hosts, 9 from 10 cGy Si-irradiated hosts, 10 from 30 cGy Si-irradiated hosts, and 8 from irradiated hosts.

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RNAi off-target analysis in Trp53-/- mouse embryonic fibroblasts transduced with miRNA-based shRNAs targeting Trp53.

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