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High-throughput sequencing analysis of BMMs and BMMs-derived osteoclasts

ABSTRACT: High-throughput sequencing analysis of BMMs and BMMs-derived osteoclasts

PROVIDER: PRJNA555428 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9702111_1.fastq.gz	Fastqsanger.gz
	SRR9702111_2.fastq.gz	Fastqsanger.gz
	SRR9702112_1.fastq.gz	Fastqsanger.gz
	SRR9702112_2.fastq.gz	Fastqsanger.gz

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High-throughput sequencing analysis of BMMs and BMMs-derived osteoclasts

Project description:Long noncoding RNAs (lncRNAs) have emerged as integral regulators of physiology and disease, but specific roles of lncRNAs in bone resorption remain largely unknown. Here, RNA-seq analysis indicated that lncRNA Nron was a candidate funtional regulators of bone resorption. Our further experimental analysis showed that Nron regulates bone resorption in mice. We further identified a functional motif of Nron. Consequently, delivery of Nron functional motif specifically in osteoclasts reversed bone loss without obvious side-effects in vivo. Together, our study indicate that Nron is a key bone resorption suppressor and reveal Nron functional motif might be a potential therapeutic strategy to combat excessive bone absorption in osteoporosis.

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Project description:Osteoclasts (OCs) are bone-resorbing cells differentiated from macrophage/monocyte precursors in response to M-CSF and RANKL. In vitro models are principally based on primary bone marrow macrophages, but RAW 264.7 cells are frequently used because they are widely available, easy to culture, and more amenable to genetic manipulation than primary cells. Increasing evidence, however, has shown that the vastly different origins of these two cell types may have important effects on cell behavior. In particular, M-CSF is prerequisite for the differentiation of BMMs, by promoting survival and proliferation and priming the cells for RANKL induction. RAW 264.7 cells readily form OCs in the presence of RANKL, but M-CSF is not required. Based on these key differences, we sought to understand their functional implications and how it might affect osteoclast differentiation and related signaling pathways. Using a robust and high-throughput proteomics strategy, we quantified the global protein changes in OCs derived from bone marrow macrophages and RAW 264.7 cells at 1, 3, and 5 days of differentiation. Correlation analysis of the proteomes demonstrated low concordance between the two cell types (R2 ≈ 0.13). Bioinformatics analysis indicate that RANKL-dependent signaling was intact in RAW 264.7 cells, but biological processes known to be dependent on M-CSF were significantly different; including cell cycle control, cytoskeletal organization, and apoptosis. RAW 264.7 cells exhibited constitutive activation of Erk and Akt that was dependent on the activity of Abelson tyrosine kinase, and the timing of Erk and Akt activation was significantly different between BMMs and RAW 264.7 cells. Our findings provide the first evidence for major differences between BMMs and RAW 264.7 cells, indicating that careful consideration is needed when using the RAW 264.7 cell line when studying M-CSF-dependent signaling and functions.

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