Dataset Information

The loss of the RNA helicase Dhx15 impairs endothelial energy metabolism, lymphatic drainage and tumor metastasis in mice

ABSTRACT: DHX15 is an ATP-dependent RNA helicase involved in pre-mRNA splicing. We have recently reported that DHX15 is a downstream substrate for Akt1, which plays a significant role in vascular biology. Therefore, we aimed to explore the regulatory function of DHX15 over the vasculature, and the endothelial cell biology in different contexts: development, metabolism, ischemia and tumor growth. Methods: Deficient DHX15 mice and zebrafish were generated using transcription activator-like effector nuclease (TALEN) and Crispr/cas9 gene edition. Lymphatic functionality was evaluated by lymphangiography and magnetic resonance imaging. Mouse-induced tumor and metastasis model were generated by injection of syngeneic LLC1 tumor cells. DHX15 gene silencing in mouse liver endothelial cells (LEC) was performed by the lentiviral transduction of shRNA (Dharmacon). The changes in the transcriptome and the proteome resulting from the shRNA transduction were investigated by RNAseq and mass spectrometry, respectively. Results: Homozygous DHX15 gene deficiency was lethal in mouse and zebrafish embryos. DHX15-/- zebrafish also showed an undeveloped parachordal line, which leads to the formation of lymphatic structures in the trunk during the development. DHX15+/- mice and zebrafish were viable, although DHX15+/- gene deficiency triggered lower vascular network density and impaired lymphatic function postnatally in mice. Whole transcriptome and proteome analysis of DHX15 silenced LEC revealed differential expression of enzymes involved in the glycolysis and the gluconeogenesis pathways. The functional validation of these results demonstrated an uncoupling of the glycolysis with the oxidation of pyruvate into the mitochondria and lower activity of the Complex I in the mitochondrial membrane, resulting in lower cellular ATP production. Noteworthy, heterozygous DHX15 deficiency partially inhibited primary tumor growth and reduced lung metastasis after injection of syngeneic LLC1 tumor cells, compared to wild-type mice

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: guadalupe espadas

LAB HEAD: Eduard Sabidó

PROVIDER: PXD018104 | Pride | 2021-11-04

REPOSITORIES: Pride

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			Action	DRS
	161007_S_JRMM_01_02_2ug.raw	Raw
	161007_S_JRMM_01to10_5FDR.msf	Msf
	161007_S_JRMM_01to10_5FDR.pdResult	Other
	161007_S_JRMM_02_01_2ug.raw	Raw
	161007_S_JRMM_03_01_2ug.raw	Raw

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The loss of DHX15 impairs endothelial energy metabolism, lymphatic drainage and tumor metastasis in mice.

Ribera Jordi J Portolés Irene I Córdoba-Jover Bernat B Rodríguez-Vita Juan J Casals Gregori G González-de la Presa Bernardino B Graupera Mariona M Solsona-Vilarrasa Estel E Garcia-Ruiz Carmen C Fernández-Checa José C JC Soria Guadalupe G Tudela Raúl R Esteve-Codina Anna A Espadas Guadalupe G Sabidó Eduard E Jiménez Wladimiro W Sessa William C WC Morales-Ruiz Manuel M

Communications biology 20211015 1

DHX15 is a downstream substrate for Akt1, which is involved in key cellular processes affecting vascular biology. Here, we explored the vascular regulatory function of DHX15. Homozygous DHX15 gene deficiency was lethal in mouse and zebrafish embryos. DHX15<sup>-/-</sup> zebrafish also showed downregulation of VEGF-C and reduced formation of lymphatic structures during development. DHX15<sup>+/-</sup> mice depicted lower vascular density and impaired lymphatic function postnatally. RNAseq and pro ...[more]

PMID: 34654883

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Project description:The lymphatic vascular system plays important roles in the maintenance of interstitial fluid pressure, the afferent immune response and the absorption of dietary lipids. However, the molecular mechanisms that control lymphatic vessel network maturation and function remain largely unknown. To identify novel players in lymphatic vessel function, we isolated pure populations of lymphatic and blood vascular endothelial cells from mouse intestine using fluorescence-activated high-speed cell sorting and performed transcriptional profiling. We found that the axonal guidance molecules semaphorin 3A (Sema3A) and Sema3D were specifically expressed by lymphatic vessels. Quantitative PCR of ex vivo isolated cells and immunohistochemical analysis confirmed these results. Importantly, we found that the semaphorin receptor neuropilin-1 (Nrp-1) is expressed on the valves of collecting lymphatic vessels. Treatment of mice in utero (E12.5-E16.5) with an antibody that blocks Sema3A binding to Nrp-1, but not with an antibody that blocks VEGFA binding to Nrp-1, resulted in abnormal development of collecting lymphatic vessels and valves, and aberrant smooth muscle cell coverage. Conversely, Sema3A-deficient mice displayed branching defects of collecting lymphatic vessels as well as impaired valve development. Together, these results reveal an unanticipated role of Sema3A/Nrp-1 signaling in the maturation of the lymphatic vascular network. Colon single-cell suspensions were prepared by a fast protocol that minimizes the RNA degradation. Fluorescence-activated cell sorting (FACS) was used to sort blood vascular endothelial cells (BEC) and lymphatic endothelial cells (LEC). 4 animal-matched pairs of LEC and BEC were chosen based on the quality of extracted and amplified material to provide homogenous groups of biological replicates. This gave 8 samples to analyze. Samples present LEC and BEC isolated from 4 healthy normal mice. The 4 mice used present the 4 biological replicates.