STC1 competitively binding ßPIX enhances melanoma progression via YAP nuclear translocation and M2 macrophage recruitment through the YAP/CCL2/VEGFA/AKT feedback loop.

This study investigates the role of Stanniocalcin-1 (STC1) in melanoma progression, with a focus on its impact on metastasis, angiogenesis, and immune evasion. Systematic bioinformatics analysis revealed the potential influence of STC1 dysregulation on prognosis, immune cell infiltration, response to immune therapy, and cellular functions. In vitro assays were conducted to assess the proliferation, invasion, migration, and angiogenesis capabilities of A375 cells. In vivo experiments utilizing C57BL/6 J mice established a lung metastasis model using B16-F10 cells to evaluate macrophage infiltration and M2 polarization. A Transwell co-culture system was employed to explore the crosstalk between melanoma and macrophages. Molecular interactions among STC1, YAP, ßPIX, and CCL2 are investigated using mass spectrometry, Co-Immunoprecipitation, Dual-Luciferase Reporter Assay, and Chromatin Immunoprecipitation experiments. STC1 was found to enhance lung metastasis by promoting the recruitment and polarization of M2 macrophages, thereby fostering an immunosuppressive microenvironment. Mechanistically, STC1 competes with YAP for binding to ßPIX within the KER domain in melanoma cells, leading to YAP activation and subsequent CCL2 upregulation. CCL2-induced M2 macrophages secrete VEGFA, which enhances tumor vascularization and increases STC1 expression via the AKT signaling pathway in melanoma cells, establishing a pro-metastatic feedback loop. Notably, STC1-induced YAP activation increases PD-L1 expression, promoting immune evasion. Silencing STC1 enhances the efficacy of PD-1 immune checkpoint therapy in mice. This research elucidates STC1's role in melanoma metastasis and its complex interactions with tumor-associated macrophages, proposing STC1 as a potential therapeutic target for countering melanoma metastasis and augmenting the efficacy of PD-1 immunotherapy.

Knockout of formyl peptide receptor 1 reduces osteogenesis and bone healing.

AIMS: Formyl peptide receptor 1 (FPR1), from a G-protein coupled receptor family, was previously well-characterized in immune cells. But the function of FPR1 in osteogenesis and fracture healing was rarely reported. This study, using the FPR1 knockout (KO) mouse, is one of the first studies that try to investigate FPR1 function to osteogenic differentiation of bone marrow-derived stem cells (BMSCs) in vitro and bone fracture healing in vivo. MATERIALS AND METHODS: Primary BMSCs were isolated from both FPR1 KO and wild type (WT) mice. Cloned mouse BMSCs (D1 cells) were used to examine role of FoxO1 in FPR1 regulation of osteogenesis. A closed, transverse fracture at the femoral midshaft was created to compare bone healing between KO and WT mice. Biomechanical and structural properties of femur were compared between healthy WT and KO mice. KEY FINDINGS: FPR1 expression increased significantly during osteogenesis of both primary and cloned BMSCs. Compared to BMSCs from FPR1 KO mice, WT BMSCs displayed considerably higher levels of osteogenic markers as well as mineralization. Osteogenesis by D1 cells was inhibited by either an FPR1 antagonist cFLFLF or a specific inhibitor of FoxO1, AS1842856. In addition, the femur from WT mice had better biomechanical properties than FPR1 KO mice. Furthermore, bone healing in WT mice was remarkably improved compared to FPR1 KO mice analyzed by X-ray and micro-CT. SIGNIFICANCE: These findings indicated that FPR1 played a vital role in osteogenic differentiation and regenerative capacity of fractured bone, probably through the activation of FoxO1 related signaling pathways.

Role of the tumor microenvironment in malignant melanoma organoids during the development and metastasis of tumors.

Malignant melanoma (MM) is the most metastatic and aggressive form of skin cancer, and carries a high risk of death. Immune-checkpoint inhibitor therapy and molecular-targeted therapy can prolong the survival of patients with advanced MM significantly. However, the low response rate and inevitable drug resistance prevent further improvements in efficacy, which is closely related to the tumor microenvironment (TME). The TME refers to the tumor stroma, including fibroblasts, keratinocytes, immune cells, soluble molecules, and extracellular matrix (ECM). The dynamic interaction between the TME and tumor cells is very important for the growth, local invasion, and metastatic spread of tumor cells. A patient-derived organoid (PDO) model involves isolation of tumor tissue from patients with MM and culturing it in vitro in a three-dimensional pattern. Compared with traditional cultivation methods, the PDO model preserves the heterogeneity of the tissue structure of MM and demonstrates the interaction between MM cells and the TME. It can reproduce the characteristics of proliferation, migration, and invasion of MM cells, and better simulate the structural function of MM in vivo. This review explores the role of each TME component in development of the PDO model. This review will provide a reference for research on the drug screening and targeted treatment using PDOs, particularly for the immunotherapy of MM.

Chitosan targets PI3K/Akt/FoxO3a axis to up-regulate FAM172A and suppress MAPK/ERK pathway to exert anti-tumor effect in osteosarcoma.

Osteosarcoma (OS) is a serve and the most frequent primary malignant tumor of bone. Chitosan was reported to have anti-tumor effect on human cancers including OS. However, the molecular mechanism by which chitosan suppresses tumor growth is not fully illustrated. In this study, human OS cell lines, including both Saos-2 and U2OS cells, were used to dissect the underlying mechanisms. RNA sequencing results show that a candidate biomarker family with sequence similarity 172 member A (FAM172A) was up-regulated in both of the two cell lines treated with chitosan. We observed that the mitogen-activated protein kinase (MAPK) signaling pathway could be inactivated by chitosan, and the MAPK inhibition caused by chitosan was reversed by FAM172A knockdown. Moreover, we uncovered a direct interaction between C-terminal domain of FAM172A (311-415) and mitogen-activated protein kinase kinase 1 (MEK1) (270-307) by immunoprecipitation assay. Finally, we also found that chitosan could bind with subunit p85 of PI3K to further inactivate the PI3K/Akt pathway. Taken together, our study demonstrates that chitosan binds with PI3K p85 subunit to suppress the activity of PI3K/Akt pathway to up-regulate the expression of FAM172A, and which exerts its function by suppressing phosphorylation of MEK1/2 and blocking the activity of MAPK/ERK signaling pathway. Taken together, our study deepens the understanding of the molecular mechanism of MAPK/ERK pathway inhibition induced by chitosan, and provides insights into the development of new targets to enhance the pharmacological effect of chitosan against OS.

Association of Fpr1 gene expression with osteogenesis and adipogenesis of adipose derived stem cells.

Formyl peptide receptors (Fprs) play fundamental roles in multiple cell functions including promotion of osteogenesis and bone fracture healing. In this study, the role of Fpr1 gene in osteogenic and adipogenic differentiation of adipose derived stem cells (ADSCs) was investigated. Primary ADSCs (mADSCs) from either Fpr1 knockout (KO) or wild type (WT) mice and human ADSCs (hADSCs) were treated by osteogenic (OM) or adipogenic (AM) medium, with basal medium as control. Osteogenesis and adipogenesis were measured by histological and biochemical methods. In both hADSCs and mADSCs, Fpr1 gene expression, osteogenic gene expression, as well as mineralization were increased after osteogenic induction. The osteogenic capacity of KO ADSCs was remarkably reduced compared to WT ADSCs, with decreased levels of expression of osteogenic markers, alkaline phosphatase activity, and mineralization. In contrast, the adipogenesis of KO ADSCs was remarkably enhanced compared with WT ADSCs, forming more lipid droplets, and increasing expression of adipogenic markers PPARγ and aP2. Expression of the nuclear transcription factor Forkhead box protein O1 (FoxO1) was decreased in KO ADSCs, while OM and AM caused increase and decrease in FoxO1 expression, respectively. The current study revealed a correlation of Fpr1 gene expression with osteogenesis and adipogenesis of mADSCs, underlying a mechanism involving FoxO1. Our present research suggests that targeting Fpr1 might be a novel strategy to enhance osteogenesis of ADSCs.

Knockdown of NLE1 inhibits development of malignant melanoma in vitro and in vivo NLE1 promotes development of malignant melanoma.

Melanoma, which originates from neural crest derived melanocytes, causes severe pain and even death to numerous patients. Previous studies reported that Notchless Homolog 1 (NLE1) plays an important role in cell proliferation, transcription and signal transduction. However, the clinical significance and biological behavior of NLE1 in melanoma remain a mystery. Thus, the role of NLE1 in melanoma was investigated in vitro and in vivo. The expression of NLE1 in melanoma was elevated and the expression level was positively correlated with lymphatic metastasis and tumor stage. In addition, NLE1 knockdown by shRNA specifically inhibited proliferation, enhanced the apoptotic sensitivity and hindered migration of melanoma cells in vitro. Mice xenograft model further showed that NLE1 knockdown could inhibit the tumor formation of melanoma in vivo. Additionally, the induction of apoptosis of melanoma cells by NLE1 knockdown required the participation of a series of apoptosis-related proteins. Besides, NLE1 can activate the PI3K/AKT signaling pathway. In summary, NLE1 was involved in the development and progression of melanoma, which may be a novel potential target for molecular therapy of melanoma.

IL-18-Mediated SLC7A5 Overexpression Enhances Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells via the c-MYC Pathway.

Objective: To investigate the role of IL-18 in the regulation of osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs). Methods: To assess whether IL-18 affects the osteogenic differentiation of hBMSCs through the c-MYC/SLC7A5 axis, IL-18 dose-response and time-course experiments were performed to evaluate its impact on osteogenic differentiation. To confirm osteogenic differentiation, alizarin red staining calcium measurement were performed. RT-qPCR and western blotting were used to determine the expression levels of bone-specific markers ALP, RUNX2, and BMP2, as well as those of SLC7A5 and c-MYC. Furthermore, SLC7A5 and c-MYC expression was evaluated via immunofluorescence. To elucidate the roles of SLC7A5 and c-MYC in osteoblast differentiation, cells were transfected with SLC7A5 or c-MYC siRNAs, or treated with the SLC7A5-specific inhibitor JPH203 and c-MYC-specific inhibitor 10058-F4, and the expression of SLC7A5, c-MYC, and bone-specific markers ALP, RUNX2, and BMP2 was assessed. Results: Our results demonstrated that IL-18 increased calcium deposition in hBMSCs, and upregulated the expression of SLC7A5, c-MYC, ALP, RUNX2, and BMP2. Silencing of SLC7A5 or c-MYC using siRNA reduced the expression of ALP, RUNX2, and BMP2, while IL-18 treatment partially reversed the inhibitory effect of siRNA. Similar results were obtained by treating hBMSCs with SLC7A5 and c-MYC specific inhibitors, leading to significant reduction of the osteogenesis effect of IL-18 on hBMSCs. Conclusion: In conclusion, our results indicate that IL-18 promotes the osteogenic differentiation of hBMSCs via the SLC7A5/c-MYC pathway and, therefore, may play an important role in fracture healing. These findings will provide new treatment strategies for delayed fracture healing after splenectomy.

Antioxidative nanofullerol inhibits macrophage activation and development of osteoarthritis in rats.

Background: There is emerging evidence which suggests that cellular ROS including nitric oxide (NO) are important mediators for inflammation and osteoarthritis (OA). Water-soluble polyhydroxylated fullerene C60 (fullerol) nanoparticle has been demonstrated to have an outstanding ability to scavenge ROS. Purpose: The objective of this study is to assess the effects of fullerol on inflammation and OA by in vitro and in vivo studies. Methods: For in vitro experiments, primary mouse peritoneal macrophages and a macrophage cell line RAW264.7 were stimulated to inflammatory phenotypes by lipopolysaccharide (LPS) in the presence of fullerol. For the animal study, OA model was created by intra-articular injection of monoiodoacetate into the knee joints of rats and fullerol was intravenously injected immediately after OA induction. Results: NO production and pro-inflammatory gene expression induced by LPS was significantly diminished by fullerol in both macrophage cell types. Meanwhile, fullerol could remarkably reduce phosphorylation of p38 mitogen-activated protein kinase, and protein level of transcription factors nuclear factor-kappaB and forkhead box transcription factor 1 within the nucleus. The animal study delineated that systematic administration of fullerol prevented OA, inhibiting inflammation of synovial membranes and the damage toward the cartilage chondrocytes in the OA joints. Conclusion: Antioxidative fullerol may have a potential therapeutic application for OA.

A very rare presentation of reoperation combined both old Monteggia fracture and secondary distal radioulnar joint dislocation in adult: A case report.

RATIONALE: The old Monteggia fracture is an uncommon lesion pattern in adult, which may lead to the potential complications such as recurrent dislocation of the radial head, heterotopic ossification of the elbow, nerve palsy, malunion of the ulna, and residual forearm deformity. However, the secondary distal radioulnar joint (DRUJ) dislocation is rarely reported in the similar lesion. Here we present a unique reoperation of old Monteggia fracture combined with secondary DRUJ disruption after the initial operation failure. PATIENT CONCERNS: A 38-year-old male presented to our hospital outpatient office complaining of left elbow stiffness and ongoing wrist dysfunction with a history of injury to the left forearm caused by a forklift accident that occurred 5 months previously. DIAGNOSIS AND INTERVENTIONS: At the local hospital, the patient underwent successively fasciotomy and decompression, ulnar open reduction, and internal fixation due to osteofascial compartment syndrome and a misdiagnosed ulnar fracture. Upon examination, the secondary dorsal dislocation of the DRUJ was obvious both clinically and radiographically. We performed a revision surgery called ulnar osteotomy, radioulnar ligament repair, and temporary fixation of the DRUJ with a Kirschner wire. OUTCOMES: The patient received a satisfactory result without observed redislocation and relapse according to the 1-year follow-up. LESSONS: Considering the notoriously poor outcomes, the importance of early recognition and accurate treatment should be emphasized repeatedly in similar lesions. Paying close and continuous attention to the clinical and radiographic examinations of the elbow and wrist joint is necessary to avoid misdiagnosis and missed diagnosis.

Fasciocutaneous flap with perforating branches of peroneal artery repairing soft tissue loss in anterior and middle parts of children's feet: A STROBE-compliant article.

Repairing soft tissue loss in feet's anterior and middle parts has become a problem, especially for children. We observed the feasibility and clinical effects of superficial peroneal fasciocutaneous flap pedicled with terminal perforating branches of peroneal artery for repairing children's feet.Between January 2015 and December 2016, soft tissue loss in anterior and middle regions of feet were repaired using superficial peroneal fasciocutaneous flap pedicled with terminal perforating branches of peroneal artery in 8 children with a median age of 6.5 [4-9, interquartile range (IQR) = 3] years. The skin of lower leg was intact, and the soft tissue loss area was located in the anterior and middle regions of feet with a size of 5 cm × 4 cm to 11 cm × 7 cm combined with the exposure of tendons and joints in all the 8 children. On the basis of the conditions above, there were no indications of free skin grafting. Foot wounds were repaired all with the superficial peroneal fasciocutaneous flap pedicled with terminal perforating branches of peroneal artery (6 cm × 5 cm to 12 cm × 8 cm), and then the donor area was sutured to narrow the donor area followed by intermediate split thickness skin graft. The perforating branch trunk of peroneal artery was used as a rotation point (4 cm above the lateral malleolus) in 5 children and descending branch of perforating branch of peroneal artery as a rotation point (2 cm under the lateral malleolus) in 3 children.All flaps survived with primary healing in the 8 children. Postoperative median 7.5-month (3-12, IQR = 4.5) follow-up indicated that flap color and texture were fine, the appearances of donor and recipient areas were satisfactory, wearing shoes was not affected, and walking function and foot blood circulation were normal.For intractable soft tissue loss in the anterior and middle regions of children's feet, superficial peroneal fasciocutaneous flap pedicled with terminal perforating branches of peroneal artery can improve recipient area appearance and walking function because it has the characteristics of reliable blood supply and convenient rotation. It is worth using this method widely in clinics.

Splenectomy delays fracture healing by affecting the level of tumor necrosis factor alpha, interleukin 6 and bone morphogenetic protein.

BACKGROUND: Abdominal injuries combined with bone fractures are increasing. Splenectomies are often required, but have prolonged healing time for bone fracture. OBJECTIVES: The aim of the study was to explore the molecular mechanism for splenectomy delaying fracture healing. MATERIAL AND METHODS: Eighty-four patients (42 received splenectomy) who received hip fractures operations were recruited in our hospital. One-year follow-up analysis was performed. To ensure the results, an animal model was established. Sprague-Dawley (SD) rats were randomly divided into 5 groups: group A: experimental group, femoral fractures + splenectomy; group B: femoral fractures; group C: splenectomy; group D: femoral fracture + sham splenectomy; group E: sham fracture. After the femoral fracture surgery, the callus status was evaluated by X-ray. RESULTS: After 1-year follow-up, the healing index and bone quality was higher in the fracture-operatedonly group than in the splenectomy group. In contrast, the rate of healing complications was lower in the fracture-operated-only group than in the splenectomy group. Biomarker analysis showed that the serum levels of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and bone morphogenetic protein (BMP) were higher in the fracture-operated-only group than in the splenectomy group. No difference of the callus status was found among the rats in groups B, D and E (p > 0.05), while there were significant differences of the callus status of the rats in groups A and C at different stages (p < 0.05). On the other hand, the levels of TNF-α, IL-6 and BMP increased, reached peak after 7-day splenectomy surgery, and then decreased significantly in groups A and C (p > 0.05). CONCLUSIONS: Splenectomy delays fracture healing by affecting the levels of TNF-α, IL-6 and BMP.

miR-337 can be a key negative regulator in melanoma.

Incidence of melanoma is increasing annually worldwide. There remains a lack of suitable treatment methods which can significantly improve the 5-year survival rates of patients. It is established that micro RNAs (miRNAs) have important roles in the diagnosis and treatment of cancer. MiR-337 had been reported to regulate the development of variety of cancers, as a cancer suppressive factor. In our research we found that miR-337 had a lower expression in melanoma than adjacent tissues. The patients who had a lower miR-337 also got a worse survival. MiR-337 could target STAT3 to regulate the occurrence and development of melanoma.  In summary, our findings suggest that the miR-337/STAT3 axis may serve as a potential target for the treatment of melanoma.

Bone fracture healing is delayed in splenectomic rats.

AIMS: Splenectomy is sometimes required in bone fracture patients. The present study aims to investigate the effect of splenectomy on fracture healing in rats. MAIN METHODS: Rats underwent osteotomy were subjected to splenectomy. The effects of splenectomy were evaluated at day 0, 3, 15 and 30 post-fracture. Double immunofluorescence staining was used to examine the expression of macrophages marker F4/80 and CD11b. H&E staining was used to examine the histological changes in fracture sites. Western blotting was used to detect protein expression of osteoprotegerin (OPG), the ligand for receptor activator of NF-κB (RANKL) and collagens in the fracture site. Activity of alkaline phosphatase (ALP) in the serum was determined using a biochemical kit. Serum levels of osteocalcin and inflammatory cytokines were determined using ELISA kits. Real-time PCR was used to detect mRNA expression of ALP and osteocalcin in the fracture site. KEY FINDINGS: Results showed that the recruitment of macrophages and the production of inflammatory cytokines were inhibited in the fracture rats underwent splenectomy. Importantly, histological examination showed that fracture healing was delayed in splenectomized rats. In addition, the protein expression of OPG and RANKL in the fracture site was diminished, the activity of ALP and the level of osteocalcin in the serum and their mRNA expression in the fracture site were reduced, and the protein expression of type I collage a1 and type II collage a1 was inhibited in fracture rats underwent splenectomy compared with that in rats without splenectomy. SIGNIFICANCE: Our findings indicate that splenectomy delays fracture healing.

CXCL13 Promotes Osteogenic Differentiation of Mesenchymal Stem Cells by Inhibiting miR-23a Expression.

CXC chemokines are essential for osteogenic differentiation of bone mesenchymal stem cells (BMSCs) for use in bone tissue engineering and regenerative medicine in clinical settings. However, an accurate understanding of the underlying mechanisms is still needed. In this study, we analyzed the effects of CXC chemokine ligand-13 (CXCL13) on osteogenic differentiation of rat BMSCs and initiated a preliminary discussion on possible mechanisms. BMSCs were isolated from bone marrow of rat and incubated with CXCL13 recombinant protein in differentiation medium. The main osteogenesis indexes were alkaline phosphatase (ALP) activity and calcium nodes. Expression of Runx2 and CXCR5 was determined using western blot, while miRNAs were determined with quantitative-RT-PCR. Si-CXCR5 was transfected into MSCs to silence CXCR5. A miRNA-23a mimic was transfected into BMSCs for overexpression of miRNA-23a. Recombinant CXCL13 induced ALP activity, deposition of calcium salts, and formation of calcium nodes, and it also increased expression of Runx2. The expression of recombinant CXCL13 suppressed expression of miRNA-23a. Overexpression of miR-23a reversed CXCL13 induced-osteogenic differentiation of BMSCs and expression of Runx2. Recombinant CXCL13 attenuated the interaction of miRNA-23a with the Runx2 3'UTR. Silencing of CXCR5 abrogated recombinant CXCL13-induced downregulation of miRNA-23a expression. In summary, CXCL13 promotes osteogenic differentiation of BMSCs by inhibiting miR-23a expression.

CXCL13 promotes the effect of bone marrow mesenchymal stem cells (MSCs) on tendon-bone healing in rats and in C3HIOT1/2 cells.

OBJECTIVES: Mesenchymal stem cells (MSCs) are potential effective therapy for tissue repair and bone regeneration. In present study, the effects of CXC chemokine ligand-13 (CXCL13) were evaluated on tendon-bone healing of rats. METHODS: Tendon bone healing of the rat model was established and biomechanical testing was performed at 2, 4, 8 weeks after surgery. Murine mesenchymal cell line (C3HIOT1/2 cells) was cultured. The expression of miRNA-23a was detected by real-time PCR. The protein expression of ERK1/2, JNK and p38 was detected by western blotting. MiR-23a mimic and inhibitor were used to overexpress or silence the expression of miR-23a. RESULTS: MSCs significantly elevated the levels of ultimate load to failure, stiffness and stress in specimens of rats, the effects of which were enhanced by CXCL13. The expression of miR-23a was down-regulated and the protein of ERK1/2 level was up-regulated by CXCL13 treatment in both in vivo and in vitro experiments. ERK1/2 expression was elevated by overexpression of miR-23a and reduced by miR-23a inhibitor. CONCLUSIONS: These findings revealed that CXCL13 promoted the tendon-bone healing in rats with MSCs treatment, and implied that the activation of ERK1/2 via miR-23a was involved in the process of MSCs treated bone regeneration.

Venous arterialization for the treatment of large-area foot skin retrograde avulsion.

Between 2009 and 2011, three patients with large-area foot skin retrograde avulsion (more than 1% of the body surface area) underwent venous arterialization. Anastomosis of the artery in the wound surface with the vein in the skin flap and an appropriate number of venous end-to-end anastomoses were performed. The skin flaps survived in all 3 patients. Six months postoperatively, the flap elasticity and appearance were close to that of normal skin, and foot function was better without scar contracture. When venous arterialization is used to treat foot avulsion, the following points should be noted. Surgical indications include no fresh bleeding from the wound edge of the avulsed skin after debridement, more complete avulsed skin, and superficial veins that do not completely separate from the avulsed skin. Venous arterialization is not suitable to avulsion with fresh bleeding, avulsed skin in small fragments, and avulsion with a subcutaneous venous network embolism. During debridement, the subcutaneous venous network should be protected to avoid exposing the vein stems outside the fat layer. If the avulsion is less than 1% of the body surface area, arterial-venous anastomosis can provide adequate blood supply. Venous-venous anastomosis is performed as much as possible to enhance venous return and decrease microcirculatory pressure, which is conducive to the establishment of effective blood circulation.