Single-cell profiling of peripheral neuroblastic tumors identifies an aggressive transitional state that bridges an adrenergic-mesenchymal trajectory.

Peripheral neuroblastic tumors (PNTs) represent a spectrum of neural-crest-derived tumors, including neuroblastoma, ganglioneuroblastoma, and ganglioneuroma. Malignant cells in PNTs are theorized to interconvert between adrenergic/noradrenergic and mesenchymal/neural crest cell states. Here, single-cell RNA-sequencing analysis of 10 PNTs demonstrates extensive transcriptomic heterogeneity. Trajectory modeling suggests that malignant neuroblasts move between adrenergic and mesenchymal cell states via an intermediate state that we term "transitional." Transitional cells express programs linked to a sympathoadrenal development and aggressive tumor phenotypes such as rapid proliferation and tumor dissemination. Among primary bulk tumor patient cohorts, high expression of the transitional gene signature is predictive of poor prognosis compared with adrenergic and mesenchymal expression patterns. High transitional gene expression in neuroblastoma cell lines identifies a similar transitional H3K27-acetylation super-enhancer landscape. Collectively, our study supports the concept that PNTs have phenotypic plasticity and uncovers potential biomarkers and therapeutic targets.

Pharmacological modulation of the cAMP signaling of two isoforms of melanocortin-3 receptor by melanocortin receptor accessory proteins in the tetrapod Xenopus laevis.

As a member of the seven-transmembrane rhodopsin-like G protein-coupled receptor superfamily, the melanocortin-3 receptor (MC3R) is vital for the regulation of energy homeostasis and rhythms synchronizing in mammals, and its pharmacological effect could be directly influenced by the presence of melanocortin receptor accessory proteins (MRAPs), MRAP1 and MRAP2. The tetrapod amphibian Xenopus laevis (xl) retains higher duplicated genome than extant teleosts and serves as an ideal model system for embryonic development and physiological studies. However, the melanocortin system of the Xenopus laevis has not yet been thoroughly evaluated. In this work, we performed sequence alignment, phylogenetic tree, and synteny analysis of two xlMC3Rs. Co-immunoprecipitation and immunofluorescence assay further confirmed the co-localization and in vitro interaction of xlMC3Rs with xlMRAPs on the plasma membrane. Our results demonstrated that xlMRAP2.L/S could improve α-MSH-stimulated xlMC3Rs signaling and suppress their surface expression. Moreover, xlMC3R.L showed a similar profile on the ligands and surface expression in the presence of xlMRAP1.L. Overall, the distinct pharmacological modulation of xlMC3R.L and xlMC3R.S by dual MRAP2 proteins elucidated the functional consistency of melanocortin system during genomic duplication of tetrapod vertebrates.

Identification of novel GPCR partners of the central melanocortin signaling.

OBJECTIVE: Homo- or heterodimerization of G protein-coupled receptors (GPCRs) generally alters the normal functioning of these receptors and mediates their responses to a variety of physiological stimuli in vivo. It is well known that melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) are key regulators of appetite and energy homeostasis in the central nervous system (CNS). However, the GPCR partners of MC3R and MC4R are not well understood. Our objective is to analyze single cell RNA-seq datasets of the hypothalamus to explore and identify novel GPCR partners of MC3R and MC4R and examine the pharmacological effect on the downstream signal transduction and membrane translocation of melanocortin receptors. METHODS: We conducted an integrative analysis of multiple single cell RNA-seq datasets to reveal the expression pattern and correlation of GPCR families in the mouse hypothalamus. The emerging GPCRs with important metabolic functions were selected for cloning and co-immunoprecipitation validation. The positive GPCR partners were then tested for the pharmacological activation, competitive binding assay and surface translocation ELISA experiments. RESULTS: Based on the expression pattern of GPCRs and their function enrichment results, we narrowed down the range of potential GPCR interaction with MC3R and MC4R for further confirmation. Co-immunoprecipitation assay verified 23 and 32 novel GPCR partners that interacted with MC3R and MC4R in vitro. The presence of these GPCR partners exhibited different effects in the physiological regulation and signal transduction of MC3R and MC4R. CONCLUSIONS: This work represented the first large-scale screen for the functional GPCR complex of central melanocortin receptors and defined a composite metabolic regulatory GPCR network of the hypothalamic nucleuses.

Elucidation of the dimeric interplay of dual MRAP2 proteins in the zebrafish.

The melanocortin receptor accessory protein 2 (MRAP2) plays an essential role in the regulation of metabolic homeostasis and deletion of which results in severe obesity syndrome in mice and human. Mammalian MRAP2 is recognized as an endogenous physiological mediator through the potentiation of the MC4R signaling in vivo. Two isoforms of MRAP2 are identified in zebrafish genome, zMRAP2a and zMRAP2b. However, the mechanism of assembling dual topology and the regulatory roles of each complex on the melanocortin cascades remains unclear. In this study, we showed the bidirectional homo- and hetero-dimeric topologies of two zebrafish MRAP2 isoforms on the plasma membrane. Orientation fixed chimeric proteins could affect the trafficking and pharmacological properties of zMC4R signaling. Reciprocal replacement of zMRAP2a and zMRAP2b proteins elucidated the major participation of the carboxyl terminal as the functional domain for modulating zMC4R signaling. Our findings revealed the complex and dynamic conformational regulation of dual zebrafish MRAP2 proteins in vitro.

miR-137 inhibits proliferation, invasion and migration of cervical cancer cells by targeting Wnt5a / 中国肿瘤生物治疗杂志

@# Objective： ：To investigate the expression of miR-137 in cervical cancer tissues and cells, and to explore its effect on proliferation, migration and invasion of cervical cancer cells as well as the mechanisms. Methods： ：Thirty-two pairs of cervical cancer tissues and corresponding para-cancerous tissues that surgically resected at the Department of Gynecology and Obstetrics of Dongguan People's Hospital from January 2017 to March 2018 were collected for this study. In addition, cervical cancer cell lines C33A, HeLa, SiHa and cervical epithelial immortalized cell line H8 were also collected. The expression of miR-137 in cervical cancer tissues and cell lines was detected by RT-PCR. miR-137 mimics and miR-137 NC were respectively transfected into C33Aand HeLa cells, and the effects of miR-137 over-expression on proliferation, migration and invasion of cervical cancer cell lines were observed by CCK-8 and Transwell assay. Luciferase reporter gene assay and WB were used to determine the relationship between miR-137 and Wnt5a in cervical cancer. Wnt5a over-expression vector was constructed, and the effects of simultaneous over-expression of Wnt5a and miR-137 on proliferation, migration and invasion of C33Aand HeLa cells were observed. Results： ：The expression level of miR-137 was significantly down-regulated in cervical cancer tissues and cell lines, as compared to para-cancerous tissues and H8 cells (all P<0.05). The over-expression of miR-137 significantly inhibited cell proliferation, migration and invasion of C33A and HeLa cells (all P<0.05). Moreover, Wnt5a was identified as a target of miR-137 by luciferase reporter gene assay. Furthermore, Wnt5a over-expression, to a certain degree, attenuated the suppressive effects of miR-137 on the proliferation, migration and invasion of C33A and HeLa cells. Conclusion： ：miR-137 can inhibit the proliferation, migration and invasion of cervical cancer cells via targeting Wnt5a, which may be an effective target for the treatment of cervical cancer.