Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA262555 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR1592190_1.fastq.gz | Fastqsanger.gz | |||
| SRR1592190_2.fastq.gz | Fastqsanger.gz | |||
| SRR1592185_1.fastq.gz | Fastqsanger.gz | |||
| SRR1592185_2.fastq.gz | Fastqsanger.gz | |||
| SRR1592186_1.fastq.gz | Fastqsanger.gz |
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Project description:miR-155 has recently emerged as an important promoter of antitumor immunity through its functions in T lymphocytes. However, the impact of T cell expressed miR-155 on immune cell dynamics in solid tumors remains unclear. In the present study, we used single-cell RNA-sequencing to define the CD45+ immune cell populations within B16F10 murine melanoma tumors growing in either wild-type (WT) or miR-155 T cell conditional knockout (TCKO) mice at different timepoints. miR-155 was required for optimal T cell activation and reinforced the T cell response at the expense of infiltrating myeloid cells. Further, myeloid cells from tumors growing in TCKO mice were defined by an increase in wound healing genes and a decreased IFNg response gene signature. Finally, we found that miR-155 expression predicted a favorable outcome in human melanoma patients and was associated with a strong immune signature. Moreover, gene expression and histological analysis of the Cancer Genome Atlas (TCGA) data revealed that miR-155 expression also correlates with an immune-enriched subtype in 29 other human solid tumor types. Together, our study provides an unprecedented analysis of the cell types and gene expression signatures by immune cells within experimental melanoma tumors and elucidates miR-155’s role in coordinating this dynamic response.
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Project description: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
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