Unknown,Transcriptomics,Genomics,Proteomics

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MiR-125b: A Rheostat of Growth versus Stress-Induced MAPKs That Governs Stemness and OncomiR Addiction in Skin

ABSTRACT: We analyze the globel gene expression changes in the tumor initiating cells of regressing miR-125b addicted tumors after oncomiR withdrawal For Histone-H2BGFP expression in DTG tumor cells, the U6 promoter was deleted in the PLKO-PGK-H2B-GFP vector throughNdeI-AgeI digestion, Quick Blunting Kit treatment (NEB), and self-ligation. The resulting PLNA-PGK-H2BGFP plasmid was packaged into lentivirus and used to infect ~1x106freshly sorted α6hiβ1hi DTG tumor cells. After 30 min, cells were then extensively washed and immediately engrafted onto backskins of Nude mice by intradermal transplantation. GFP+α6hiβ1hicells were FACS-isolated from resulting tumors and then serially transplanted as above. For RNA seq analysis of the miR-125b addicted tumor regression process, H2BGFP labeled DTG tumor cells were intradermally engrafted onto backskins of Nude mice (1x104cells/site). Tumors were allowed to grow to ~1cm diameter. Mice were then taken Off Dox by transferring them to regular food for 0, 4, 7 days. GFP+α6hiβ1hiwere then FACS-isolated from the tumors. Two or three independent replicates were collected for each time point. Total RNAswere extracted from the FACS-sorted cells using the miRNeasy Mini Kit (Qiagen) according to the vender’s protocol. Expression of miR-125b in each sample was quantified by RT-PCR using TaqMan MicroRNA Assays (Applied Biosystems). For RNA seq, RNA samples were submitted to the Genomics Resources Core Facility of the Weill Cornell Medical College for library construction using IlluminaTruSeq Stranded mRNA Sample Prep Kit and then sequencing using Illumina HiSeq2000. Resultswere analyzed via the Galaxy web platform using TopHat for initiate mapping and Cufflinks for transcripts assembling and expression level estimation (Computing FPKM: fragments per kilobase of exon per million fragments mapped). The MM9 genome assembly (UCSC Genome Browser) was used as reference genome for all analyses. Low expression genes

ORGANISM(S): Mus musculus

SUBMITTER: liang zhang

PROVIDER: E-GEOD-54704 | 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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MiR-125b: A Rheostat of Growth versus Stress-Induced MAPKs That Governs Stemness and OncomiR Addiction in Skin

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