BMP9 attenuates occurrence of venous malformation by maintaining endothelial quiescence and strengthening vessel walls via SMAD1/5/ID1/α-SMA pathway.

Venous malformation (VM) is a type of vascular morphogenic defect in humans with an incidence of 1%. Although gene mutation is considered as the most common cause of VM, the pathogenesis of those without gene mutation remains to be elucidated. Here, we aimed to explore the relation of bone morphogenetic protein 9 (BMP9) and development of VM. At first, we found serum and tissue BMP9 expression in VM patients was significantly lower than that in healthy subjects, detected via enzyme-linked immunosorbent assay. Next, with wound healing assay, transwell assay and tube formation assay, we discovered BMP9 could inhibit migration and enhance tube formation activity of human umbilical vein endothelial cells (HUVECs) via receptor activin receptor-like kinase 1 (ALK1). Besides, BMP9 improved the expression of structural proteins alpha-smooth muscle actin (α-SMA) and Desmin in human umbilical vein smooth muscle cells (HUVSMCs) via activation of the SMAD1/5-ID1 pathway, determined by RNA-based next-generation sequencing, qPCR, immunofluorescence and western blotting. Intriguingly, this effect could be blocked by receptor ALK1 inhibitor, SMAD1/5 inhibitor and siRNAs targeting ID1, verifying the BMP9/ALK1/SMAD1/5/ID1/α-SMA pathway. Meanwhile, knocking out BMP9 in C57BL/6 mice embryo led to α-SMA scarcity in walls of lung and mesenteric vessels, as well as walls of small trachea. BMP9-/- zebrafish also exhibited abnormal vascular maturity, indicating a critical role of BMP9 in vascular maturity and remodeling. Finally, a VM mice model revealed that BMP9 might have therapeutic effect in VM progression. Our study discovered that BMP9 might inhibit the occurrence of VM by strengthening the vessel wall and maintaining endothelium quiescence. These findings provide promising evidences of new therapeutic targets that might be used for the management of VM.

The oncolytic virus H101 combined with GNAQ siRNA-mediated knockdown reduces uveal melanoma cell viability.

BACKGROUND: Uveal melanoma (UM) is a severe human malignancy with a high mortality rate, as well as high metastasis and recurrence potential. The active mutation of G protein subunit alpha q (GNAQ) or G protein subunit alpha 11 (GNA11) is a major trigger for UM. Oncolytic adenovirus H101 (H101) is the first oncolytic virus approved for clinical applications in cancer therapy by the China Food and Drug Administration. We investigated whether combining H101 with the downregulation of GNAQ expression would act synergistically in UM therapy. METHODS: Three UM cell lines OMM2.3 and 92.1, harboring GNAQ mutation, and OCM1, harboring B-Raf proto-oncogene mutation, were chosen for our research. The cellular toxicity of adenoviral infection and the cell growth rate were measured with a Cell Counting Kit-8. Western blot analysis was used to detect GNAQ, p-MEK1/2, YAP, and p-YAP expression. The apoptosis and cell-cycle distribution of cells were evaluated with annexin-V and propidium iodide staining. RESULTS: Our results revealed that OMM2.3 and 92.1 cells were more sensitive to H101 infection than OCM1 cells. GNAQ expression was markedly reduced by small interfering RNA, siGNAQ. Combined treatment of siGNAQ and H101 inhibited the proliferation and activated the apoptosis of OMM2.3 and 92.1 cells by blocking the phosphorylation of MEK1/2 and increasing the phosphorylation of YAP. CONCLUSIONS: In summary, a therapy combining H101 and siGNAQ is feasible, with potential utility as a novel targeted molecular therapy for UM, especially those carrying a GNAQ mutation.

The novel roles of circRNAs in human cancer.

Covalently closed single-stranded circular RNAs (circRNAs) consist of introns or exons and are widely present in eukaryotic cells. CircRNAs generally have low expression levels and relatively stable structures compared with messenger RNAs (mRNAs), most of which are located in the cytoplasm and often act in cell type and tissue-specific manners, indicating that they may serve as novel biomarkers. In recent years, circRNAs have gradually become a hotspot in the field of RNA and cancer research, but the functions of most circRNAs have not yet been discovered. Known circRNAs can affect the biogenesis of cancers in diverse ways, such as functioning as a microRNA (miRNA) sponges, combining with RNA binding proteins (RBPs), working as a transcription factor and translation of proteins. In this review, we summarize the characteristics and types of circRNAs, introduce the biogenesis of circRNAs, discuss the emerging functions and databases on circRNAs and present the current challenges of circRNAs studies.

Targeting secreted cytokine BMP9 gates the attenuation of hepatic fibrosis.

Liver fibrosis is overly exuberant wound healing that leads to portal hypertension or liver cirrhosis. Recent studies have demonstrated the functions of bone morphogenetic protein 9 (BMP9) in liver fibrosis, and thus, targeting liver-specific BMP9 abnormalities will become an attractive approach for developing therapeutics to treat liver fibrosis. Here, we reveal that BMP9 serves as a valuable serum diagnostic indicator and efficient therapeutic target to attenuate liver fibrogenesis. Our analysis of biopsies from liver fibrotic patients revealed that higher BMP9 levels accompanied advanced stages of liver fibrosis. In mouse models, recombinant Bmp9 overexpression accelerated liver fibrosis, and adenovirus-mediated Bmp9 knockdown attenuated liver fibrogenesis. Intriguingly, BMP9 directly stimulated hepatic stellate cell activation via the SMAD signaling pathway to enhance hepatic fibrosis. Moreover, an inhibitory monoclonal antibody targeting Bmp9 was efficacious in treatment of mice with liver fibrosis. These observations delineate a novel model in which BMP9 directly drives SMAD/ID1 signaling in hepatic stellate cells, which modulates liver fibrogenesis development. Moreover, the findings unveil a promising surrogate biomarker for the diagnosis of hepatic fibrosis, thereby representing an efficient "BMP9 neutralization" approach in alleviating hepatic fibrosis.

Characterization of a conjunctival melanoma cell line CM-AS16, newly-established from a metastatic Han Chinese patient.

Conjunctival melanoma (CM) is associated with metastases formation, can be fatal, and occurs in all different races. While cell lines are essential for experimental research, all available CM cell lines are derived from Caucasian patients. Furthermore, they are not derived from metastases. We aimed to establish a new CM cell line from a parotid metastasis in a Han Chinese patient and to depict its characteristics. The novel cell line, CM-AS16, was obtained from a surgical parotid sample and determined as a unique one with short tandem repeat (STR) analysis. It has been successively sub-cultured in vitro for more than 100 passages and exhibits rapid proliferation and migration. Chromosome analysis shows abundant chromosome aberrations, while whole exome sequencing (WES) reveals a typical NRAS mutation (Q61R). In vivo tumor growth was successfully established in a NOD/SCID mice model, and the immunophenotypes, such as HMB45, Melan A, S100, SOX10 and Ki67, manifested similar between the original tumor and the xenograft by immunohistochemistry. A MEK inhibitor binimetinib prominently suppressed in vitro cell growth by inhibiting ERK1/2 phosphorylation. In addition, monoclonal cells were used to demonstrate the drug sensitivity of different cells. In conclusion, the first cell line, CM-AS16, that is derived from a CM in a Han Chinese patient has highly malignant characteristics and a typical NRAS mutation. It may be used as a tool for further exploration of the molecular mechanisms of CM.

FMRP ligand circZNF609 destabilizes RAC1 mRNA to reduce metastasis in acral melanoma and cutaneous melanoma.

BACKGROUND: Melanoma is a type of malignant tumor with high aggressiveness and poor prognosis. At present, metastasis of melanoma is still an important cause of death in melanoma patients. However, the potential functions and molecular mechanisms of most circular RNAs (circRNAs) in melanoma metastasis remain unknown. METHODS: circRNAs dysregulated in melanoma cell subgroups with different metastatic abilities according to a screening model based on repeated Transwell assays were identified with a circRNA array. The expression and prognostic significance of circZNF609 in skin cutaneous melanoma and acral melanoma cells and tissues were determined by qRT-PCR, nucleoplasmic separation assays and fluorescence in situ hybridization. In vitro wound healing, Transwell and 3D invasion assays were used to analyse melanoma cell metastasis ability. Tail vein injection and intrasplenic injection were used to study in vivo lung metastasis and liver metastasis, respectively. The mechanism of circZNF609 was further evaluated via RNA immunoprecipitation, RNA pull-down, silver staining, and immunofluorescence colocalization assays. RESULTS: circZNF609 was stably expressed at low levels in melanoma tissues and cells and was negatively correlated with Breslow depth, clinical stage and prognosis of melanoma patients. circZNF609 inhibited metastasis of acral and cutaneous melanoma in vivo and in vitro. Mechanistically, circZNF609 promoted the binding of FMRP protein and RAC1 mRNA, thereby enhancing the inhibitory effect of FMRP protein on the stability of RAC1 mRNA and ultimately inhibiting melanoma metastasis. CONCLUSIONS: Our findings revealed that circZNF609 plays a vital role in the metastasis of acral and cutaneous melanoma through the circRNF609-FMRP-RAC1 axis and indicated that circZNF609 regulates the stability of RAC1 mRNA by combining with FMRP, which might provide insight into melanoma pathogenesis and a new potential target for treatment of melanoma.

Altered expression profile of circular RNAs in conjunctival melanoma.

Aim: We aimed to explore the roles of circular RNA (circRNA) in conjunctival melanoma. Materials & methods: The altered circRNAs were identified by RNA sequencing. The function of one circRNA, circMTUS1, was explored by several experiments in conjunctival melanoma. Bioinformatic analyses and an RNA immunoprecipitation assay were performed to further study the downstream mechanism of circMTUS1. Results: We identified 9300 different circRNAs in conjunctival melanoma tissues compared with adjacent normal tissues. CircMTUS1 was upregulated in conjunctival melanoma. Silencing of circMTUS1 inhibited conjunctival melanoma proliferation in vitro and in vivo. CircMTUS1 may serve as an oncogene by binding to hsa-miR-622 and hsa-miR-1208 to regulate several tumor-related pathways. Conclusion: We demonstrated that circMTUS1 may serve as an oncogene to promote tumor proliferation.