Dataset Information

Netrin-1 promotes retinoblastoma-associated angiogenesis.

ABSTRACT:

Background

Retinoblastoma (Rb) is the most common intraocular cancer of infancy and childhood, with an incidence of nearly 0.006% in all live births. Although a functional loss or inactivation of both alleles of the retinoblastoma 1 (RB1) gene during retinal development appears to be the predominant etiology for Rb, genes associated with tumor angiogenesis are also likely to be involved in the development of this condition. Netrin-1 is a factor that regulates pathological angiogenesis, while its role in Rb is largely unknown. The present study examined the role of netrin-1 in Rb.

Methods

The expression of netrin-1 in Rb was assessed using public databases and using clinical specimens by RT-qPCR for mRNA and by ELISA for protein. The expression of netrin-1 was suppressed in Rb by siRNA and the effects on cell growth were determined by a CCK-8 assay, while the effects on angiogenesis were examined in vitro using human umbilical vein endothelial cell (HUVEC) assays and in vivo by quantification of tumor vessel density.

Results

Analysis of published databases revealed that the netrin-1 gene is significantly upregulated in Rb, which was confirmed by immunohistochemistry on clinical specimens. Inhibition of netrin-1 in Rb cell lines significantly reduced their effects on angiogenesis in vitro using a HUVEC co-culture assay without affecting cell growth. Inhibition of netrin-1 expression in vivo suppressed the growth of grafted Rb, and this effect could be abolished by co-expression of vascular endothelial growth factor A (VEGF-A).

Conclusions

This data demonstrated a novel role for netrin-1 in the regulation of Rb-associated cancer vascularization and may represent a novel therapeutic target for patients with Rb.

SUBMITTER: Yang X

PROVIDER: S-EPMC8667090 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Publications

Netrin-1 promotes retinoblastoma-associated angiogenesis.

Yang Xiaosheng X Sun Hui H Tang Tianchi T Zhang Wenchuan W Li Yi Y

Annals of translational medicine 20211101 22

<h4>Background</h4>Retinoblastoma (Rb) is the most common intraocular cancer of infancy and childhood, with an incidence of nearly 0.006% in all live births. Although a functional loss or inactivation of both alleles of the retinoblastoma 1 (RB1) gene during retinal development appears to be the predominant etiology for Rb, genes associated with tumor angiogenesis are also likely to be involved in the development of this condition. Netrin-1 is a factor that regulates pathological angiogenesis, w ...[more]

PMID: 34988192

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Project description:Netrin-1 binds to the high-affinity receptor CD146 to activate downstream signaling and angiogenesis. Here, we examine the role and underlying mechanisms of G protein subunit alpha i1 (Gαi1) and Gαi3 in Netrin-1-induced signaling and pro-angiogenic activity. In mouse embryonic fibroblasts (MEFs) and endothelial cells, Netrin-1-induced Akt-mTOR (mammalian target of rapamycin) and Erk activation was largely inhibited by silencing or knockout of Gαi1/3, whereas signaling was augmented following Gαi1/3 overexpression. Netrin-1 induced Gαi1/3 association with CD146, required for CD146 internalization, Gab1 (Grb2 associated binding protein 1) recruitment and downstream Akt-mTOR and Erk activation. Netrin-1-induced signaling was inhibited by CD146 silencing, Gab1 knockout, or Gαi1/3 dominant negative mutants. Netrin-1-induced human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation were inhibited by Gαi1/3 short hairpin RNA (shRNA), but were potentiated by ectopic Gαi1/3 overexpression. In vivo, intravitreous injection of Netrin-1 shRNA adeno-associated virus (AAV) significantly inhibited Akt-mTOR and Erk activation in murine retinal tissues and reduced retinal angiogenesis. Endothelial knockdown of Gαi1/3 significantly inhibited Netrin1-induced signaling and retinal angiogenesis in mice. Netrin-1 mRNA and protein expression were significantly elevated in retinal tissues of diabetic retinopathy (DR) mice. Importantly, silence of Netrin-1, by intravitreous Netrin-1 shRNA AAV injection, inhibited Akt-Erk activation, pathological retinal angiogenesis and retinal ganglion cells degeneration in DR mice. Lastly, Netrin-1 and CD146 expression is significantly increased in the proliferative retinal tissues of human proliferative diabetic retinopathy patients. Together, Netrin-1 induces CD146-Gαi1/3-Gab1 complex formation to mediate downstream Akt-mTOR and Erk activation, important for angiogenesis in vitro and in vivo.

Project description:ObjectiveDiscogenic low back pain (LBP) is associated with nociceptive nerve fibers that grow into degenerated intervertebral discs (IVD) but the etiopathogenesis of disease is not fully understood. The purpose of this study was to clarify the role of Netrin-1 in causing discogenic LBP.MethodsThe level of nociceptive nerve innervation was examined in disc degenerative patients and rat needle-punctured models by immunohistochemistry. Nucleus pulposus (NP) cells were isolated from IVD tissues of rats and induced degeneration by interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα). The candidate genes related to neuron outgrowth and migration were selected by Next-generation sequencing (NGS). CRISPR/Cas9 was used to knockdown Netrin-1 in NP cells. The impact of Netrin-1 on nerve innervation were evaluated with P2X2、NF200 staining and microfluidics assay. Meanwhile the CD31 staining and transwell assay were used to evaluate the impact of Netrin-1 in angiogenesis. The proteins and RNA extracted from NP cells related to catabolism and anabolism were examined by western blot assay and RT-qPCR experiment. ChIP and luciferase experiments were used to assess the intracellular transcriptional regulation of Netrin-1. Further, a needle-punctured rat model followed by histomorphometry and immunofluorescence histochemistry was used to explore the potential effect of Netrin-1 on LBP in vivo.ResultsThe level of nerve innervation was increased in severe disc degenerative patients while the expression of Netrin-1 was upregulated. The supernatants of NP cells stimulated with IL-1β or TNFα containing more Netrin-1 could promote axon growth and vascular endothelial cells migration. Knocking down Netrin-1 or overexpressing transcription factor TCF3 as a negative regulator of Netrin-1 attenuated this effect. The needle-punctured rat model brought significant spinal hypersensitivity, nerve innervation and angiogenesis, nevertheless knocking down Netrin-1 effectively prevented disc degeneration-induced adverse impacts.ConclusionDiscogenic LBP was induced by Netrin-1, which mediated nerve innervation and angiogenesis in disc degeneration. Knocking down Netrin-1 by CRISPR/Cas9 or negatively regulating Netrin1 by transcription factor TCF3 could alleviate spinal hypersensitivity.The translational potential of this articleThis study on Netrin-1 could provide a new target and theoretical basis for the prevention and treatment for discogenic back pain.

Dataset Information

Netrin-1 promotes retinoblastoma-associated angiogenesis.

Background

Methods

Results

Conclusions

Publications

Netrin-1 promotes retinoblastoma-associated angiogenesis.

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