Dataset Information

The miR-183/96/182 Cluster Regulates Macrophage Functions in Response to Pseudomonas aeruginosa.

ABSTRACT: Macrophages (Mϕ) are an important component of the innate immune system; they play critical roles in the first line of defense to pathogen invasion and modulate adaptive immunity. MicroRNAs (miRNAs) are a newly recognized, important level of gene expression regulation. However, their roles in the regulation of Mϕ and the immune system are still not fully understood. In this report, we provide evidence that the conserved miR-183/96/182 cluster (miR-183/96/182) modulates Mϕ function in their production of reactive nitrogen (RNS) and oxygen species (ROS) and their inflammatory response to Pseudomonas aeruginosa (PA) infection and/or lipopolysaccharide (LPS) treatment. We show that knockdown of miR-183/96/182 results in decreased production of multiple proinflammatory cytokines in response to PA or LPS treatment in Mϕ-like Raw264.7 cells. Consistently, peritoneal Mϕ from miR-183/96/182-knockout versus wild-type mice are less responsive to PA or LPS, although their basal levels of proinflammatory cytokines are increased. In addition, overexpression of miR-183/96/182 results in decreased production of nitrite and ROS in Raw264.7 cells. We also provide evidence that DAP12 and Nox2 are downstream target genes of miR-183/96/182. These data suggest that miR-183/96/182 imposes global regulation on various aspects of Mϕ function through different downstream target genes.

SUBMITTER: Muraleedharan CK

PROVIDER: S-EPMC6738186 | biostudies-other | 2019

REPOSITORIES: biostudies-other

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The miR-183/96/182 Cluster Regulates Macrophage Functions in Response to Pseudomonas aeruginosa.

Muraleedharan Chithra K CK McClellan Sharon A SA Ekanayaka Sandamali A SA Francis Rebecca R Zmejkoski Alex A Hazlett Linda D LD Xu Shunbin S

Journal of innate immunity 20190109 4

Macrophages (Mϕ) are an important component of the innate immune system; they play critical roles in the first line of defense to pathogen invasion and modulate adaptive immunity. MicroRNAs (miRNAs) are a newly recognized, important level of gene expression regulation. However, their roles in the regulation of Mϕ and the immune system are still not fully understood. In this report, we provide evidence that the conserved miR-183/96/182 cluster (miR-183/96/182) modulates Mϕ function in their produ ...[more]

PMID: 30625496

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Project description:Epstein-Barr virus (EBV) is an oncogenic human herpesvirus involved in the pathogenesis of Burkitt's lymphoma (BL) and various other lymphoproliferative disorders. In BL, EBV protein expression is restricted to EBV nuclear antigen 1 (EBNA1), but small noncoding RNAs such as EBV-encoded small RNAs (EBERs) and microRNAs (miRNAs) can also be detected. miRNAs play major roles in crucial processes such as proliferation, differentiation, and cell death. It has recently become clear that alterations in the expression profile of miRNAs contribute to the pathogenesis of a number of malignancies. During latent infection, EBV expresses 25 viral pre-miRNAs and modulates the expression of specific cellular miRNAs, such as miR-155 and miR-146, which potentially play a role in oncogenesis. Here, we established the small-RNA expression profiles of three BL cell lines. Using large-scale sequencing coupled to Northern blotting and real-time reverse transcription-PCR (RT-PCR) analysis validation, we demonstrated the differential expression of some cellular and viral miRNAs. High-level expression of the miR-183-96-182 cluster and EBV miR-BamHI A rightward transcript (miR-BART) cluster was significantly associated with EBV type I latency. This expression was not affected by viral reactivation since transforming growth factor ?1 (TGF-?1) stimulation did not significantly change the miRNA profiles. However, using several approaches, including de novo infection with a mutant virus, we present evidence that the expression of latent membrane protein 1 (LMP-1) triggered downregulation of the expression of the miR-183-96-182 cluster. We further show that this effect involves the Akt signaling pathway.In addition to expressing their own miRNAs, herpesviruses also impact the expression levels of cellular miRNAs. This regulation can be either positive or negative and usually results in the perturbation of pathways to create a cellular environment that is more "virus-friendly." For example, EBV induces the expression of miR-155, a well-characterized oncomiR, which leads to increased cell proliferation and decreased cell death. Here, we show that EBV-encoded LMP-1 is also involved in the downregulation of a cluster of three miRNAs, miR-183, -96, and -182, which are known to be also repressed in several cancers. We therefore identify yet another potential player in EBV-induced oncogenesis.

Project description:IntroductionThe miR-183/-96/-182 cluster is a conserved polycistronic microRNA (miRNA) cluster which is highly expressed in most breast cancers. Although there are some sporadic reports which demonstrate the importance of each miRNA in this cluster in breast cancer, the biological roles of this cluster as a whole and its regulation mechanisms in breast cancer are still unclear. We compared the expression of this cluster in different cancer types, analyzed the regulation mechanism of this cluster, identified new target genes, and examined the impact of this cluster on breast cancer cells.MethodsThe miRNA level was detected by LNA-based northern blot and Real-time PCR, and was also analyzed from TCGA dataset. Bioinformatics research and luciferase assay were applied to find the promoter regions and transcription factors. To investigate the biological effects of the miR-183/-96 /-182 cluster in breast cancer, we generated miR-96, miR-182 and miR-183 overexpression stable cell lines to check the overdose effects; we also used miR-Down™ antagomir for each miRNA as well as miR-183/-96 /-182 cluster sponge lentivirus to check the knockdown effects. Growth, migration, cell cycle profile and survival of these cells was then monitored by colony formation assay, MTT assay, cell wound healing assay, flow cytometry and microscopy. The target gene was validated by Real-time PCR, luciferase assay, Western blot and Phalloidin/DAPI counterstaining.ResultsThe miR-183/-96/-182 cluster was highly expressed in most breast cancers, and its transcription is disordered in breast cancer. The miR-183/-96/-182 cluster was transcribed in the same pri-miRNA and its transcription was regulated by ZEB1 and HSF2. It increased breast cell growth by promoting more rapid completion of mitosis, promoted cell migration and was essential for cell survival. MiR-183 targeted the RAB21 mRNA directly in breast cancer.ConclusionThe miR-183/-96/-182 cluster is up-regulated in most breast cancer. It functions as an oncogene in breast cancer as it increases cell proliferation and migration.

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The miR-183/96/182 Cluster Regulates Macrophage Functions in Response to Pseudomonas aeruginosa.

Publications

The miR-183/96/182 Cluster Regulates Macrophage Functions in Response to Pseudomonas aeruginosa.

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