SCUBE3 promotes osteogenic differentiation and mitophagy in human bone marrow mesenchymal stem cells through the BMP2/TGF-ß signaling pathway.

In clinical settings, addressing large bone defects remains a significant challenge for orthopedic surgeons. The use of genetically modified bone marrow mesenchymal stem cells (BMSCs) has emerged as a highly promising approach for these treatments. Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a multifunctional secreted glycoprotein, the role of which remains unclear in human hBMSCs. This study used various experimental methods to elucidate the potential mechanism by which SCUBE3 influences osteogenic differentiation of hBMSCs in vitro. Additionally, the therapeutic efficacy of SCUBE3, in conjunction with porous GeLMA microspheres, was evaluated in vivo using a mouse bone defect model. Our findings indicate that SCUBE3 levels increase significantly during early osteogenic differentiation of hBMSCs, and that reducing SCUBE3 levels can hinder this differentiation. Overexpressing SCUBE3 elevated osteogenesis gene and protein levels and enhanced calcium deposition. Furthermore, treatment with recombinant human SCUBE3 (rhSCUBE3) protein boosted BMP2 and TGF-ß expression, activated mitophagy in hBMSCs, ameliorated oxidative stress, and restored osteogenic function through SMAD phosphorylation. In vivo, GELMA/OE treatment effectively accelerated bone healing in mice. In conclusion, SCUBE3 fosters osteogenic differentiation and mitophagy in hBMSCs by activating the BMP2/TGF-ß signaling pathway. When combined with engineered hydrogel cell therapy, it could offer valuable guidance for the clinical management of extensive bone defects.

Clinical features associated with maternal uniparental disomy for chromosome 6.

BACKGROUND: Maternal uniparental disomy for chromosome 6 (upd(Cajaiba MM, Witchel S, Madan-Khetarpal S, Hoover J, Hoffner L, Macpherson T, et al. Prenatal diagnosis of trisomy 6 rescue resulting in paternal UPD6 with novel placental findings. Am J Med Genet Part A. 2011;155 A(8):1996-2002.)mat) has been previously reported to cause intrauterine growth restriction (IUGR), but the specific clinical phenotype has not been defined. Based on clinical data from two new cases and patients from the literature, specific phenotypes and mechanisms will be discussed further. CASE PRESENTATION: In case 1, a maternal isodisomy mixed with a heterodisomy was found on chromosome 6, including a regional absence of heterozygosity between 6q23.3 and 6q27. In case 2, a homozygous SCUBE3 mutation and upd(Cajaiba MM, Witchel S, Madan-Khetarpal S, Hoover J, Hoffner L, Macpherson T, et al. Prenatal diagnosis of trisomy 6 rescue resulting in paternal UPD6 with novel placental findings. Am J Med Genet Part A. 2011;155 A(8):1996-2002.)mat, involving the 6p21.1-25.1 region were found. Clinical data related to upd(Cajaiba MM, Witchel S, Madan-Khetarpal S, Hoover J, Hoffner L, Macpherson T, et al. Prenatal diagnosis of trisomy 6 rescue resulting in paternal UPD6 with novel placental findings. Am J Med Genet Part A. 2011;155 A(8):1996-2002.)mat were also reviewed. Of all the 21 reported cases with upd(Cajaiba MM, Witchel S, Madan-Khetarpal S, Hoover J, Hoffner L, Macpherson T, et al. Prenatal diagnosis of trisomy 6 rescue resulting in paternal UPD6 with novel placental findings. Am J Med Genet Part A. 2011;155 A(8):1996-2002.)mat (including our 2 cases), 18 (85.7%) presented IUGR. CONCLUSIONS: The phenotypes of the two newly identified patients with upd(Cajaiba MM, Witchel S, Madan-Khetarpal S, Hoover J, Hoffner L, Macpherson T, et al. Prenatal diagnosis of trisomy 6 rescue resulting in paternal UPD6 with novel placental findings. Am J Med Genet Part A. 2011;155 A(8):1996-2002.)mat further suggest that IUGR is associated with upd(Cajaiba MM, Witchel S, Madan-Khetarpal S, Hoover J, Hoffner L, Macpherson T, et al. Prenatal diagnosis of trisomy 6 rescue resulting in paternal UPD6 with novel placental findings. Am J Med Genet Part A. 2011;155 A(8):1996-2002.)mat and case 2 is the first reported upd(Cajaiba MM, Witchel S, Madan-Khetarpal S, Hoover J, Hoffner L, Macpherson T, et al. Prenatal diagnosis of trisomy 6 rescue resulting in paternal UPD6 with novel placental findings. Am J Med Genet Part A. 2011;155 A(8):1996-2002.)mat patient with a homozygous SCUBE3 gene mutation. However, the specific phenotypes involved in upd(Cajaiba MM, Witchel S, Madan-Khetarpal S, Hoover J, Hoffner L, Macpherson T, et al. Prenatal diagnosis of trisomy 6 rescue resulting in paternal UPD6 with novel placental findings. Am J Med Genet Part A. 2011;155 A(8):1996-2002.)mat and the related mechanisms need to be further studied.

Biochemical study of ZNF76 rs10947540 and SCUBE3 rs1888822 single nucleotide polymorphisms in the Egyptian patients with systemic lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a common autoimmune disease marked by the formation of apoptotic debris and the presence of autoantibodies that target nuclear components. At this moment, the actual cause of SLE is uncertain. Genetic variables have been well proven to have a significant role in the propensity of SLE. This study aimed to investigate the effect of (ZNF76) rs (10947540) and (SCUBE) rs (1888822) gene polymorphism in patients with systemic lupus erythematosus. A case control study has been carried out at Medical Biochemistry & Molecular biology and Rheumatology unit of Internal Medicine Departments, Faculty of Medicine, Menoufia University, Egypt, for 1-year duration between 1 June 2022 and 1 June 2023. Sixty patients were females (75%) and twenty patients were males (25%). Their ages ranged from 19 to 53 years. Their disease durations ranged from 7 months to 20 years. The findings indicated that the TC genotype of the ZNF76 rs10947540 gene increases the risk of SLE by 2.274-fold, while the dominant TC + CC increases the risk by 2.472-fold, and the C allele increases the risk by 2.115-fold. Additionally, the results showed that the TT genotype of the SCUBE3 rs1888822 gene increases the risk of SLE by 3.702-fold, the dominant GT + TT increases the risk by 2.304-fold, and the T allele increases the risk by 2.089-fold, while the GT genotype increases the risk by 1.918-fold. The study revealed significant associations between the genotypes of these polymorphisms and certain clinical parameters in SLE patients. These findings highlight the potential genetic contributions to SLE susceptibility and its clinical manifestations, providing valuable insights for future research and potential personalized approaches to the management of this complex autoimmune disease.

The role of α7nAchR and PD-L1 in neuroimmune regulation of keloid treatment.

Keloid formation, characterized by aberrant fibroproliferation and immune dysregulation, remains a challenging clinical concern. This study aims to elucidate the neuroimmune mechanisms underlying keloid pathogenesis and explores the efficacy of a combined treatment approach involving modulation of the α7 nicotinic acetylcholine receptor (α7nAchR), a key player in neural transmission, and programmed death ligand 1 (PD-L1), an immune checkpoint molecule, for keloid intervention. A key innovation lies in the identification of signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3) as a potential target gene influenced by this dual treatment. We elucidate the underlying mechanism, wherein the hypoxic keloid microenvironment fosters an upsurge in SCUBE3 secretion. Subsequently, SCUBE3 forms complexes with TGF-ß, initiating the activation of the PI3K/AKT/NF-κB signaling pathway. Notably, SCUBE3 is secreted in the form of exosomes, thereby exerting a profound influence on the differentiation of T cells and macrophages within the keloid milieu. This research not only provides a comprehensive understanding of the molecular mechanisms involved but also offers a promising avenue for the development of targeted therapies to address keloid-associated fibrosis and immune dysregulation. In conclusion, the combined inhibition of α7nAchR and PD-L1 represents a promising therapeutic strategy with SCUBE3 as a pivotal molecular target in the complex landscape of keloid pathophysiology.

Two previously undescribed cholestanol saponins from the rhizomes of Paris fargesii var. petiolata.

Two previously undescribed cholestanol saponins, parpetiosides F - G (1-2), and six known analogs (3-8) were isolated from the rhizomes of Paris fargesii var. petiolata. Their structures were elucidated by extensive spectroscopic data analysis and chemical methods. Compound 1 was a rare 6/6/6/5/5 fused-rings cholestanol saponin with disaccharide moiety linked at C-26 of aglycone which was hardly seen in genus Paris. All of these compounds were discovered in this plant for the first time. In addition, the cytotoxicities of saponins (1-8) against three human cancer cell lines (U87, HepG2 and SGC-7901) were evaluated by CCK-8 method, and saponins 5-8 displayed certain cytotoxicities. The strong interactions between saponins 5-8 and SCUBE3, an oncogene for glioma cells, were displayed by molecular docking.

ß-catenin/TCF4-induced SCUBE3 upregulation promotes ovarian cancer development via HIF-1 signaling pathway.

The precise involvement and mechanistic role of the signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3) in ovarian cancer (OV) remain poorly understood. Here, leveraging comprehensive data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, we unveil the selective overexpression of SCUBE3 in ovarian cancer tissues and cells. Intriguingly, elevated SCUBE3 expression levels correlate with an unfavorable prognosis in patients. Through meticulous manipulation of SCUBE3 expression, we elucidate its consequential impact on in vitro proliferation and invasion of ovarian cancer cells, as well as in vivo tumor growth in mice. Our multifaceted investigations, encompassing luciferase reporter assays, chromatin immunoprecipitation (ChIP) experiments, and mining of public databases, successfully identify SCUBE3 as a direct downstream target gene of TCF4-a pivotal positive regulator within the ß-catenin/TCF4 complex. Furthermore, utilizing a recessive mutant mouse line (kta41) harboring a functionally impaired point mutation at position 882 in the SCUBE3 gene, we uncover SCUBE3's involvement in the intricate regulation of angiogenesis and epithelial-mesenchymal transition (EMT). Strikingly, Spearman correlation coefficient analysis unveils a close association between SCUBE3 and HIF1A in OV, with SCUBE3 exerting tight control over HIF1A mRNA expression. Moreover, functional inhibition of HIF1A significantly impedes the pro-proliferative and invasive capabilities of SCUBE3-overexpressing ovarian cancer cells. Collectively, our findings underscore the pivotal role of SCUBE3 in driving ovarian cancer progression, shedding light on its intricate molecular mechanisms and establishing it as a potential therapeutic target for this devastating disease.

Epithelium-derived SCUBE3 promotes polarized odontoblastic differentiation of dental mesenchymal stem cells and pulp regeneration.

BACKGROUND: Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3), a secreted multifunctional glycoprotein whose transcript expression is restricted to the tooth germ epithelium during the development of embryonic mouse teeth, has been demonstrated to play a crucial role in the regulation of tooth development. Based on this, we hypothesized that epithelium-derived SCUBE3 contributes to bio-function in dental mesenchymal cells (Mes) via epithelium-mesenchyme interactions. METHODS: Immunohistochemical staining and a co-culture system were used to reveal the temporospatial expression of the SCUBE3 protein during mouse tooth germ development. In addition, human dental pulp stem cells (hDPSCs) were used as a Mes model to study the proliferation, migration, odontoblastic differentiation capacity, and mechanism of rhSCUBE3. Novel pulp-dentin-like organoid models were constructed to further confirm the odontoblast induction function of SCUBE3. Finally, semi-orthotopic animal experiments were performed to explore the clinical application of rhSCUBE3. Data were analysed using one-way analysis of variance and t-tests. RESULTS: The epithelium-derived SCUBE3 translocated to the mesenchyme via a paracrine pathway during mouse embryonic development, and the differentiating odontoblasts in postnatal tooth germ subsequently secreted the SCUBE3 protein via an autocrine mechanism. In hDPSCs, exogenous SCUBE3 promoted cell proliferation and migration via TGF-ß signalling and accelerated odontoblastic differentiation via BMP2 signalling. In the semi-orthotopic animal experiments, we found that SCUBE3 pre-treatment-induced polarized odontoblast-like cells attached to the dental walls and had better angiogenesis performance. CONCLUSION: SCUBE3 protein expression is transferred from the epithelium to mesenchyme during embryonic development. The function of epithelium-derived SCUBE3 in Mes, including proliferation, migration, and polarized odontoblastic differentiation, and their mechanisms are elaborated for the first time. These findings shed light on exogenous SCUBE3 application in clinic dental pulp regeneration.

Illuminating DEPDC1B in Multi-pronged Regulation of Tumor Progression.

DEPDC1B (aliases BRCC3, XTP8, XTP1) is a DEP (Dishevelled, Egl-1, Pleckstrin) and Rho-GAP-like domains containing predominately membrane-associated protein. Earlier, we and others have reported that DEPDC1B is a downstream effector of Raf-1 and long noncoding RNA lncNB1, and an upstream positive effector of pERK. Consistently, DEPDC1B knockdown is associated with downregulation of ligand-stimulated pERK expression. We demonstrate here that DEPDC1B N-terminus binds to the p85 subunit of PI3K, and DEPDC1B overexpression results in decreased ligand-stimulated tyrosine phosphorylation of p85 and downregulation of pAKT1. Collectively, we propose that DEPDC1B is a novel cross-regulator of AKT1 and ERK, two of the prominent pathways of tumor progression. Our data showing high levels of DEPDC1B mRNA and protein during the G2/M phase have significant implications in cell entry into mitosis. Indeed, DEPDC1B accumulation during the G2/M phase has been associated with disassembly of focal adhesions and cell de-adhesion, referred to as a DEPDC1B-mediated de-adhesion mitotic checkpoint. DEPDC1B is a direct target of transcription factor SOX10, and SOX10-DEPDC1B-SCUBE3 axis has been associated with angiogenesis and metastasis. The Scansite analysis of the DEPDC1B amino acid sequence shows binding motifs for three well-established cancer therapeutic targets CDK1, DNA-PK, and aurora kinase A/B. These interactions and functionalities, if validated, may further implicate DEPDC1B in regulation of DNA damage-repair and cell cycle progression processes. Finally, a survey of the publicly available datasets indicates that high DEPDC1B expression is a viable biomarker in breast, lung, pancreatic and renal cell carcinomas, and melanoma. Currently, the systems and integrative biology of DEPDC1B is far from comprehensive. Future investigations are necessary in order to understand how DEPDC1B might impact AKT, ERK, and other networks, albeit in a context-dependent manner, and influence the actionable molecular, spatial, and temporal vulnerabilities within these networks in cancer cells.

Hedgehog signaling reprograms hair follicle niche fibroblasts to a hyper-activated state.

Hair follicle stem cells are regulated by dermal papilla fibroblasts, their principal signaling niche. Overactivation of Hedgehog signaling in the niche dramatically accelerates hair growth and induces follicle multiplication in mice. On single-cell RNA sequencing, dermal papilla fibroblasts increase heterogeneity to include new Wnt5ahigh states. Transcriptionally, mutant fibroblasts activate regulatory networks for Gli1, Alx3, Ebf1, Hoxc8, Sox18, and Zfp239. These networks jointly upregulate secreted factors for multiple hair morphogenesis and hair-growth-related pathways. Among these is non-conventional TGF-ß ligand Scube3. We show that in normal mouse skin, Scube3 is expressed only in dermal papillae of growing, but not in resting follicles. SCUBE3 protein microinjection is sufficient to induce new hair growth, and pharmacological TGF-ß inhibition rescues mutant hair hyper-activation phenotype. Moreover, dermal-papilla-enriched expression of SCUBE3 and its growth-activating effect are partially conserved in human scalp hair follicles. Thus, Hedgehog regulates mesenchymal niche function in the hair follicle via SCUBE3/TGF-ß mechanism.

SCUBE3 downregulation modulates hepatocellular carcinoma by inhibiting CCNE1 via TGFß/PI3K/AKT/GSK3ß pathway.

OBJECTIVES: We aimed to verify the role of signal peptide-CUB-EGF-like domain-containing protein3 (SCUBE3) in the hepatocellular carcinoma (HCC) progression. METHODS: The role of SCUBE3 in HCC cell proliferation, apoptosis, and cell cycle in vitro were detected using MTT assay, colony formation assay, 5-ethynyl-2´-deoxyuridine assay (EDU), Celigo cell counting assay, Caspase3/7 activity assay, and flow cytometry. The effect of SCUBE3 on HCC cell proliferation in vivo was inspected by a xenograft tumour model in nude mice. The related mechanisms were further studied. RESULTS: The level of SCUBE3 was upregulated in HCC tissues and cell lines. Knockdown of SCUBE3 inhibited proliferation, promoted apoptosis, and induced cell cycle arrest in HCC cell lines in vitro and in vivo. Screening of cell cycle-related proteins revealed that CCNL2, CDK6, CCNE1, and CCND1 exhibited a significantly different expression profile. We found that SCUBE3 may promote the proliferation of HCC cells by regulating CCNE1 expression. The pathway enrichment analysis showed that the TGFß signalling pathway and the PI3K/AKT signalling pathway were significantly altered. Co-immunoprecipitation results showed that SCUBE3 binds to the TGFßRII receptor. SCUBE3 knockdown inhibited the PI3K/AKT signalling pathway and the phosphorylation of GSK3ß to inhibit its kinase activity. CONCLUSIONS: SCUBE3 promotes HCC development by regulating CCNE1 via TGFß/PI3K/AKT/GSK3ß pathway. In addition, SCUBE3 may be a new molecular target for the clinical diagnosis and treatment of HCC.

DEPDC1B Promotes Melanoma Angiogenesis and Metastasis through Sequestration of Ubiquitin Ligase CDC16 to Stabilize Secreted SCUBE3.

The ability of melanoma to acquire metastasis through the induction of angiogenesis is one of the major causes of skin cancer death. Here, it is found that high transcript levels of DEP domain containing 1B (DEPDC1B) in cutaneous melanomas are significantly associated with a poor prognosis. Tissue microarray analysis indicates that DEPDC1B expression is positively correlated with SOX10 in the different stages of melanoma. Consistently, DEPDC1B is both required and sufficient for melanoma growth, metastasis, angiogenesis, and functions as a direct downstream target of SOX10 to partly mediate its oncogenic activity. In contrast to other tumor types, the DEPDC1B-mediated enhancement of melanoma metastatic potential is not dependent on the activities of RHO GTPase signaling and canonical Wnt signaling, but is acquired through secretion of signal peptide, CUB domain and EGF like domain containing 3 (SCUBE3), which is crucial for promoting angiogenesis in vitro and in vivo. Mechanistically, DEPDC1B regulates SCUBE3 protein stability through the competitive association with ubiquitin ligase cell division cycle 16 (CDC16) to prevent SCUBE3 from undergoing degradation via the ubiquitin-proteasome pathway. Importantly, expression of SOX10, DEPDC1B, and SCUBE3 are positively correlated with microvessel density in the advanced stage of melanomas. In conclusion, it is revealed that a SOX10-DEPDC1B-SCUBE3 regulatory axis promotes melanoma angiogenesis and metastasis, which suggests that targeting secreted SCUBE3 can be a therapeutic strategy against metastatic melanoma.

SCUBE3 serves as an independent poor prognostic factor in breast cancer.

BACKGROUND: Accumulating evidences indicate that the signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3) plays a key role in the development and progression of many human cancers. However, the underlying mechanism and prognosis value of SCUBE3 in breast cancer are still unclear. METHODS: The clinical data of 137 patients with breast cancer who underwent surgical resection in Taizhou Hospital of Zhejiang Province were retrospectively analyzed. We first conducted a comprehensive study on the expression pattern of SCUBE3 using the Tumor Immune Estimation Resource (TIMER) and UALCAN databases. In addition, the expression of SCUBE3 in breast tumor tissues was confirmed by immunohistochemistry. The protein-protein interaction analysis and functional enrichment analysis of SCUBE3 were analyzed using the STRING and Enrichr databases. Moreover, tissue microarray (TMA) was used to analyze the relationship between SCUBE3 expression levels and clinical-pathological parameters, such as histological type, grade, the status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2). We further supplemented and identified the above results using the UALCAN and bc-GenExMiner v4.4 databases from TCGA data. The correlation between the expression of SCUBE3 and survival was calculated by multivariate Cox regression analysis to investigate whether SCUBE3 expression may be an independent prognostic factor of breast cancer. RESULTS: We found that the expression level of SCUBE3 was significantly upregulated in breast cancer tissue compared with adjacent normal tissues. The results showed that the distribution of breast cancer patients in the high expression group and the low expression group was significantly different in ER, PR, HER2, E-cadherin, and survival state (p < 0.05), but there was no significant difference in histologic grade, histologic type, tumor size, lymph node metastasis, TMN stage, subtypes, or recurrence (p > 0.05). In addition, the high expression of SCUBE3 was associated with relatively poor prognosis of ER- (p = 0.012), PR- (p = 0.029), HER2 + (p = 0.007). The multivariate Cox regression analysis showed that the hazard ratio (HR) was 2.80 (95% CI 1.20-6.51, p = 0.0168) in individuals with high SCUBE3 expression, and HR was increased by 1.86 (95% CI 1.06-3.25, p = 0.0300) for per 1-point increase of SCUBE3 expression. CONCLUSIONS: These findings demonstrate that the high expression of SCUBE3 indicates poor prognosis in breast cancer. SCUBE3 expression may serve as a potential diagnostic indicator of breast cancer.

Activation of TGF-ß1 Pathway by SCUBE3 Regulates TWIST1 Expression and Promotes Breast Cancer Progression.

Background: Recently, signal peptide-CUB-EGF-like domain containing protein 3 (SCUBE3) has been found to be associated with the development of several cancers. However, the biological role of SCUBE3 in breast cancer progression has not been reported. Materials and Methods: Western blotting and quantitative real-time polymerase chain reaction were used to measure the expressions of SCUBE3, TGF-ß (transforming growth factor-ß) signaling pathway markers, and epithelial-mesenchymal transition markers. The influence of SCUBE3 on the breast cancer cell proliferation, invasion, and migration was detected using methyl thiazolyl tetrazolium, wound healing, colony formation, and transwell assay. The role of SCUBE3 in vivo was confirmed using tumor xenograft experiment. Results: SCUBE3 expression was markedly increased in breast cancer cells and tissues. Knockdown of SCUBE3 suppressed cell growth, invasion, and migration, while SCUBE3 overexpression promoted cell growth, invasion, and migration in breast cancer cells. In addition, TGF-ß1 and its downstream proteins were positively regulated by SCUBE3, and the promotion effect on TWIST1 expression induced by pcDNA3.1-SCUBE3 can be reversed by TGF-ß1 inhibitor in breast cancer cell lines. Moreover, silencing of SCUBE3 suppressed breast cancer cell growth and tumorigenesis through reducing TGF-ß1 in vivo. Conclusion: Knockdown of SCUBE3 downregulated TGF-ß1 and TWIST1 expression, thereby inhibiting breast cancer cell growth and tumorigenesis.

The First Scube3 Mutant Mouse Line with Pleiotropic Phenotypic Alterations.

The vertebrate Scube (Signal peptide, CUB, and EGF-like domain-containing protein) family consists of three independent members, Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3N294K/N294K), which clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to our understanding of SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC). Scube3N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function, and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3 In addition, alterations in energy metabolism, behavior, and neurological functions were detected in Scube3N294K/N294K mice. The Scube3N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function.

The Expression of HSPD1, SCUBE3, CXCL14 and Its Relations with the Prognosis in Osteosarcoma.

The aim of the study was to explore the expression of three genes, HSPD1, SCUBE3, and CXCL14, in osteosarcoma cells and tissue, as well as their association with the prognosis of patients with osteosarcoma. The expression of HSPD1, SCUBE3, and CXCL14 in osteosarcoma cells was detected by using Western blotting method. siRNA was used to knockdown the expression of the three genes. CCK8 cell proliferation assay was used to observe the effect of siRNA interference on U2OS cell proliferation. The expression of the three genes in osteosarcoma tissue was detected employing immunohistochemical method. Kaplan-Meier survival analysis was used to compare the relations between the expression of the three genes and prognosis. The Western blotting results showed that the expression of Hsp70, SCUBE3 protein, and CXCL14 chemotactic factor in osteosarcoma cells was significantly higher than that in normal osteocytes (p < 0.05). After the three genes were interfered by siRNA, the mRNA and protein expression levels of these genes in osteosarcoma cells were significantly decreased (p < 0.05). The growth rate of U2OS cell after the siRNA interference was significantly lower than that before interference and that in the control group transfected with negative control siRNA (p < 0.05). The result of immunohistochemistry demonstrated that the expression of Hsp70, SCUBE3 protein, and CXCL14 chemotactic factor in osteosarcoma tissues was significantly higher than that in adjacent muscle tissue (p < 0.05). Kaplan-Meier survival analysis indicated that the survival rate of the patients with high expression of those three kinds of genes was obviously lower than that of other patients (p < 0.05). There was no significant difference between the survival rates of patients with high or low expression of two genes (p > 0.05). The expression of HSPD1, SCUBE3, and CXCL14 was all high in osteosarcoma tissues and cells; moreover, the three kinds of genes had close correlations with the prognosis of the patients. Targeted inhibition of these three genes could inhibit the proliferation of the tumor, which may become a new therapeutic target.

SCUBE3 (signal peptide-CUB-EGF domain-containing protein 3) modulates fibroblast growth factor signaling during fast muscle development.

SCUBE3 (signal peptide CUB-EGF-like domain-containing protein 3) belongs to a newly identified secreted and cell membrane-associated SCUBE family, which is evolutionarily conserved in vertebrates. Scube3 is predominantly expressed in a variety of developing tissues in mice such as somites, neural tubes, and limb buds. However, its function during development remains unclear. In this study, we first showed that knockdown of SCUBE3 in C2C12 myoblasts inhibited FGF receptor 4 expression and FGF signaling, thus resulting in reduced myogenic differentiation. Furthermore, knockdown of zebrafish scube3 by antisense morpholino oligonucleotides specifically suppressed the expression of the myogenic marker myod1 within the lateral fast muscle precursors, whereas its expression in the adaxial slow muscle precursors was largely unaffected. Consistent with these findings, immunofluorescent staining of fast but not slow muscle myosin was markedly decreased in scube3 morphants. Further genetic studies identified fgf8 as a key regulator in scube3-mediated fast muscle differentiation in zebrafish. Biochemical and molecular analysis showed that SCUBE3 acts as a FGF co-receptor to augment FGF8 signaling. Scube3 may be a critical upstream regulator of fast fiber myogenesis by modulating fgf8 signaling during zebrafish embryogenesis.