Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA314818 | ENA |
REPOSITORIES: ENA
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Project description:Purpose: Previous work has demonstrated that miR-33 is an important regulator of lipid metabolism and atherogenesis. By performing bone marrow transplant experiments into LDLR-/- mice, our work demonstrates that the effects of miR-33 in macrophages play a major role in its ability to reduced atherosclerotic plaque burdon. To have performed extensive additional characterization of the effects of miR-33 deficiency in macrophages icluding RNA-seq analysis of peritoneal macrophages from wildtype, miR-33-/-, LDLR-/-, and miR33-/-/LDLR-/- animals. Methods: Thioglycolate elicited peritoneal macrophages from WT and miR-33-/- mice were harvested by peritoneal lavage. Cells were then plated for 2hr, then washed to remove non-adherant cells. Macrophages were then scraped, pelleted and frozen at -80◦C. Total RNA from WT and miR-33-/- thioglycollate-elicited peritoneal macrophages was extracted and purified using a RNA isolation Kit (Qiagen) followed by DNAse treatment to remove genomic contamination using RNA MinElute Cleanup (Qiagen). The purity and integrity of total RNA sample was verified using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). rRNA was depleted from RNA samples using Ribo-Zero rRNA Removal Kit (Illumina). RNA libraries from WT BMDMs were performed TrueSeq Small RNA Library preparation (Illumina) and were sequenced for 45 cycles on Illumina HiSeq 2000 platformm (1 x 75bp read length). The reads obtained from the sequencer are trimmed for quality using in-house developed scripts. The trimmed reads are aligned to the reference genome using TopHat2. The transcript abundances and differences calculated using cuffdiff. The results were plotted using R and cummeRbund using in-house developed scripts. Results: Our RNA-seq analysis has allowed us to identify genes and pathways that are altered in miR-33 deficient peritoneal macrophages under hyperlipidemic conditions (LDLR-/- vs. miR33-/-/LDLR-/-). Further analysis of gene expression changes that occur between wildtype and LDLR-/- animals has allowed us to identify which of these changes are likely due to differences in lipid loading and which are independent of these effects.
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Project description:Mouse peritoneal macrophages were transfected with 80-120 nM miRIDIAN miRNA mimics (miR-mimic-33/miR-mimic-33*) or with 80-120 nM miRIDIAN miRNA inhibitors (anti-miR-33 ASO/anti-miR-33*ASO) Control samples were treated with an equal concentration of a non-targeting control mimics sequence (control mimic) or inhibitor negative control sequence (control aso), to control for non-specific effects in miRNA experiments.
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Project description:Using the highly sensitive miRNA microarray, we screened 107 up-regulated microRNAs and 33 down-regulated microRNAs in the INT-HA-induced M2-like macrophages and we explored the functions of these miRNAs in macrophage polarization. The enrichment results indicated that these miRNAs might participate in the process of macrophage polarization. Furthermore, the quantitative real-time polymerase chain reaction results showed that miR-935 may play an important role in M2 macrophage polarization which was activated by INT-HA.
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Project description:Peritoneal metastasis (PM) has a suppressive tumor immune microenvironment (TIME), which limits the effects of immunotherapy. This study aims to investigate the immunomodulatory effects of intraperitoneal administration of IL-33 on PM-associated TIME. Immunocompetent mice were used to investigate the role of IL-33 in development of abdominal dissemination and host outcome. Murine (m) and human (h) gastric cancer cells were tested for their response to IL-33 by qRT-PCR, flow cytometry, and immunofluorescence. Survival was significantly prolonged in patients with high Il-33 mRNA expression. Intraperitoneal administration of IL-33 could induce the celiac inflammatory environment, activate immunologic effector cells and reverse the immunosuppressive tumor microenvironment, which delayed tumor progression and peritoneal metastasis of gastric cancer. Mechanistically, IL-33 could induce M2 polarization by activating p38-GATA-binding protein 3 (GATA3) signaling pathway. IL-33 combined with anti-CSF1R or p38 inhibitor to regulate tumor-associated macrophages (TAMs) showed synergistic anti-tumor effect. Intraperitoneal administration of IL-33 inducing local inflammatory milieu provided a novel approach for the treatment of metastatic peritoneal malignancies, which combined with TAMs reprogramming to reshape TIME could achieve better treatment efficacy.
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