Unknown,Transcriptomics,Genomics,Proteomics

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MicroRNA-203 represses selection and expansion of oncogenic HRas transformed tumor initiating cells [array]

ABSTRACT: In many mouse models of skin cancer, only a few tumors typically form although many cells competent for tumorigenesis receive the same oncogenic mutations. These observations suggest a selection process for defining tumor initiating cells. Here we use quantitative mRNA- and miR-Seq to determine the impact of HRasG12V on the transcriptome of keratinocytes. We discover that microRNA-203 is downregulated by HRasG12V. Using a knockout mouse model, we demonstrate that loss of microRNA-203 promotes selection and expansion of tumor-initiating cells. Conversely, restoration of microRNA-203 with an inducible model potently inhibits proliferation of these cells. We comprehensively identify microRNA-203 targets required for HRas-initiated tumorigenesis. These targets include important effectors of the Ras pathway and essential genes required for cell division. Together, this study establishes a role for the loss of microRNA-203 in promoting selection and expansion of HRas mutated cells and identifies a mechanism through which microRNA-203 antagonizes HRas-mediated tumorigenesis. Identifying mRNA and microRNA networks regulated by oncogenic HRasG12V in primary keratinocytes through the use of 3Seq and small-RNA-Sequencing. Additionally we utilize ribosome-profiling, 3Seq, Microarray and Ago2-HITS-CLIP approaches to identify novel miR-203 target genes.

ORGANISM(S): Mus musculus

SUBMITTER: Kent Riemondy

PROVIDER: E-GEOD-66055 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Increasing evidence suggests that microRNAs may play important roles in regulating self-renewal and differentiation in mammalian stem cells (SCs). Here, we explore this issue in skin. We first characterize microRNA expression profiles of skin SCs versus their committed proliferative progenies and identify a microRNA subset associating with “stemness”. Of these, miR-125b is dramatically downregulated in early SC-progeny. We engineer an inducible mice system and show that when miR-125b is sustained in SC-progenies, tissue balance is reversibly skewed towards stemness at the expense of epidermal, oil-gland and HF differentiation. Using gain-and-loss of function in vitro, we further implicate miR-125b as a repressor of SC differentiation. In vivo, transcripts repressed upon miR-125b induction are enriched >700% for predicted miR-125b targets normally downregulated upon SC-lineage commitment. We verify some of these miR-125b targets, and show that Blimp1 and VDR in particular can account for many tissue imbalances we see when miR-125b is deregulated. We used microarrays to compare the global miRNA expression profile of P4 stage hair follicle ORS cells from DTG (K14-rtTA,TRE-miR-125b) and control littermates. Hair follicle cells were isolated from P4 Backskin (Dox since P3 for 24hrs) of DTG (K14-rtTA/TRE-miR-125b/K14-H2BGFP), TRE (TRE-miR-125b/K14-H2BGFP), KrtA (K14-rtTA/K14-H2BGFP) as following: interfollicular epidermis sheet was pealed from hair follicle & dermis after dispase treatment.The hair follicle & dermis were first digested by collagenase (Sigma). Intact hair follicles were separated from dermal cells by low speed spinning (20g). The hair follicles were then digested by Trypsin and filtered by 40 µm cell strainers. The isolated hair follicle cells were FACS sorted. During FACS, cells were first gated against CD34 (endothelial cells), CD45 (immune cells), CD114 (melanocytes) and DAPI (dead cells). ORS cells were sorted from the remaining cells as α6HiGFPHi.

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MicroRNA-203 represses selection and expansion of oncogenic HRas transformed tumor initiating cells [array]

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