CircZNF367 promotes osteoclast differentiation and osteoporosis by interacting with FUS to maintain CRY2 mRNA stability.

BACKGROUND: Osteoporosis, characterized by reduced bone mass and deterioration of bone quality, is a significant health concern for postmenopausal women. Considering that the specific role of circRNAs in osteoporosis and osteoclast differentiation remains poorly understood, this study aims to shed light on their involvement in these processes to enhance our understanding and potentially contribute to improved treatment strategies for osteoporosis. METHODS: An osteoporotic model was constructed in vivo in ovariectomized mouse. In vitro, we induced osteoclast formation in bone marrow-derived macrophages (BMDMs) using M-CSF + RANKL. To assess osteoporosis in mice, we conducted HE staining. We used MTT and TRAP staining to measure cell viability and osteoclast formation, respectively, and also evaluated their mRNA and protein expression levels. In addition, RNA pull-down, RIP and luciferase reporter assays were performed to investigate interactions, and ChIP assay was used to examine the impact of circZNF367 knockdown on the binding between FUS and CRY2. RESULTS: We observed increased expression of CircZNF367, FUS and CRY2 in osteoporotic mice and M-CSF + RANKL-induced BMDMs. Functionally, knocking down circZNF367 inhibited osteoporosis in vivo. Furthermore, interference with circZNF367 suppressed osteoclast proliferation and the expression of TRAP, NFATc1, and c-FOS. Mechanistically, circZNF367 interacted with FUS to maintain CRY2 mRNA stability. Additionally, knocking down CRY2 rescued M-CSF + RANKL-induced osteoclast differentiation in BMDMs promoted by circZNF367 and FUS. CONCLUSION: This study reveals that the circZNF367/FUS axis may accelerate osteoclasts differentiation by upregulating CRY2 in osteoporosis and suggests that targeting circZNF367 may have potential therapeutic effects on osteoporosis.

METTL14 represses osteoclast formation to ameliorate osteoporosis via enhancing GPX4 mRNA stability.

Excessive bone resorption by osteoclasts results in the development of multiple bone disorders including osteoporosis. This study aimed to explore the biological function of methyltransferase-like14 (METTL14) in osteoclast formation, as well as its related mechanisms. Expression levels of METTL14, GPX4 and osteoclast-related proteins TRAP, NFATc1, c-Fos were detected by qRT-PCR and Western blotting. The osteoporosis model was established in mice by bilateral ovariectomy (OVX). Bone histomorphology was determined by micro-CT and H&E staining. NFATc1 expression in bone tissues was determined by immunohistochemical staining. Proliferation of primary bone marrow macrophages cells (BMMs) was assessed by MTT assay. Osteoclast formation was observed by TRAP staining. The regulatory mechanism was evaluated by RNA methylation quantification assay, MeRIP-qPCR, dual luciferase reporter assay, and RIP, respectively. METTL14 was down-regulated in the serum samples of postmenopausal osteoporotic women, which was positively associated with bone mineral density (BMD). Osteoclast formation was promoted in OVX-treated METTL14+/- mice as compared with wild-type littermates. Conversely, METTL14 overexpression repressed RANKL-induced osteoclast differentiation of BMMs. Mechanistically, METTL14-mediated m6A modification post-transcriptionally stabilized glutathione peroxidase 4 (GPX4), with the assistance of Hu-Antigen R (HuR). Finally, GPX4 depletion-mediated osteoclast formation in BMMs could be counteracted by METTL14 or HuR overexpression. Collectively, METTL14 inhibits osteoclastogenesis and bone resorption via enhancing GPX4 stability through an m6A-HuR dependent mechanism. Therefore, targeting METTL14 might be a novel promising treatment strategy for osteoporosis.

CircZNF367 suppresses osteogenic differentiation of human bone marrow mesenchymal stromal/stem cells via reducing HuR-mediated mRNA stability of LRP5.

Osteoporosis is a highly prevalent disease characterized by bone mass loss and structural deterioration. There are evidences that altered differentiation of human bone marrow mesenchymal stromal/stem cells (hBMSCs) is a major cause for osteoporosis. Recent studies suggest that circular RNAs (circRNAs) are dysregulated in osteoporosis patients and involved in the pathogenesis of osteoporosis. In the present study, we are aimed to analyze the circRNA expression profiles in osteoporosis patients and identify potential circRNAs that involved in the differentiation of hBMSCs during osteoporosis. Transcriptome RNA-sequencing was conducted to search for differentially expressed circRNAs. Transwell assay, ARS and ALP staining, and ectopic bone formation model were performed to evaluate osteogenic differentiation of hBMSCs. RNA pull-down assay, RNA immunoprecipitation, western blot, and in vitro binding assay were conducted to evaluate the interaction of circRNAs and RNA-binding protein HuR. We found that hsa_circ_0008842 (designated as circZNF367) was upregulated in osteoporosis patients and decreased in hBMSCs during osteogenic differentiation. CircZNF367 overexpression suppressed migration, invasion and osteogenic differentiation of hBMSCs in vitro and in vivo. In comparison, knockdown of circZNF367 promoted migration, invasion and osteogenic differentiation of hBMSCs. CircZNF367 could interact with the RNA-binding protein HuR, thus reduced the mRNA stability of LRP5. Furthermore, HuR overexpression or LRP5 restoration abrogated the effects of circZNF367 overexpression on osteogenic differentiation of hBMSCs. Our results indicated that circZNF367 played a role in osteogenic differentiation of hBMSCs via reducing HuR-mediated mRNA stability of LRP5.

Overexpression of circ PTK2 suppresses the progression of nonalcoholic fatty liver disease via the miR-200c/SIK2/PI3K/Akt axis.

Excessive hepatic lipid accumulation is involved in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). A previous study showed that the circular RNA (circRNA) PTK2 was significantly downregulated in NAFLD mice. However, the detailed function of circ PTK2 in NAFLD remains unclear. A high-fat diet (HFD) was used to establish a mouse model of NAFLD, and free fatty acid (FFA) treatment was used to establish an in vitro model of NAFLD. Oil red O staining was used to evaluate lipid accumulation. The pathological changes in mice were observed by HE staining. Western blotting and RT-qPCR were applied to assess protein and mRNA levels, respectively. A dual luciferase reporter assay and RIP were used to explore the relationship among circ PTK2, miR-200c and SIK2. Circ PTK2 and SIK2 were downregulated and miR-200c was upregulated in NAFLD. Upregulation of circ PTK2 reversed lipid accumulation in FFA-treated HepG2 cells. Moreover, circ PTK2 bound to miR-200c, and SIK2 was identified as the direct target of miR-200c. Moreover, the miR-200c inhibitor-induced decrease in lipid accumulation was reversed by SIK2 knockdown. Furthermore, the impact of circ PTK2 overexpression on PI3K/Akt signaling was partially reversed by SIK2 silencing. Circ PTK2 overexpression alleviates NAFLD development via the miR-200c/SIK2/PI3K/Akt axis. Thus, our work might provide new methods for NAFLD treatment.

Circular RNA Circ-03955 Promotes Epithelial-Mesenchymal Transition in Osteosarcoma by Regulating miR-3662/Metadherin Pathway.

Osteosarcoma is the most common primary malignant tumor, especially in children and adolescents. Circular RNAs (circRNAs) are found to play roles in the progression of osteosarcoma. However, the exact functions of circRNAs in osteosarcoma development still need to be clarified. We obtained differentially expressed circRNAs and miRNAs from a GSE99671 data set (GEO database). The gene co-expression network of ceRNAs and osteosarcoma-related genes was analyzed using the STRING database. qRT-PCR was used to detect the expression of circ-03955 and miR-3662. Transwell assays and flow cytometry were performed to detect phenotypic changes in cell function. A xenograft tumor model was established using BALB/c nude mice. Dual luciferase activity and RNA immunoprecipitation assays were performed to assess the relationship between circ-03955, miR-3662, and metadherin (MTDH). Immunohistochemistry, immunofluorescence, and Western blotting were used to assess protein expression levels. Circ-03955 was significantly upregulated, and miR-3662 was downregulated in osteosarcoma. Circ-03955 silencing inhibited the growth and metastasis of osteosarcoma. Mechanism analysis revealed that circ-03955 could bind to miR-3662, and the latter could target MTDH, leading to its suppressed expression and facilitating epithelial-mesenchymal transition (EMT). All these findings demonstrate that the presence of circ-03955 promotes EMT in osteosarcoma by acting as miR-3662 sponge-mediated MTDH expression.

Reconstruction of Large-scale Defects with a Novel Hybrid Scaffold Made from Poly(L-lactic acid)/Nanohydroxyapatite/Alendronate-loaded Chitosan Microsphere: in vitro and in vivo Studies.

A chitosan-based microsphere delivery system has been fabricated for controlled release of alendronate (AL). The present study aimed to incorporate the chitosan/hydroxyapatite microspheres-loaded with AL (CH/nHA-AL) into poly(L-lactic acid)/nanohydroxyapatite (PLLA/nHA) matrix to prepare a novel microspheres-scaffold hybrid system (CM-ALs) for drug delivery and bone tissue engineering application. The characteristics of CM-ALs scaffolds containing 10% and 20% CH/nHA-AL were evaluated in vitro, including surface morphology and porosity, mechanical properties, drug release, degradation, and osteogenic differentiation. The in vivo bone repair for large segmental radius defects (1.5 cm) in a rabbit model was evaluated by radiography and histology. In vitro study showed more sustained drug release of CM-AL-containing scaffolds than these of CM/nHA-AL and PLLA/nHA/AL scaffolds, and the mechanical and degradation properties of CM-ALs (10%) scaffolds were comparable to that of PLLA/nHA control. The osteogenic differentiation of adipose-derived stem cells (ASCs) was significantly enhanced as indicated by increased alkaline phosphates (ALP) activity and calcium deposition. In vivo study further showed better performance of CM-ALs (10%) scaffolds with complete repair of large-sized bone defects within 8 weeks. A microspheres-scaffold-based release system containing AL-encapsulated chitosan microspheres was successfully fabricated in this study. Our results suggested the promising application of CM-ALs (10%) scaffolds for drug delivery and bone tissue engineering.

Dissecting the roles of Ephrin-A3 in malignant peripheral nerve sheath tumor by TALENs.

Malignant peripheral nerve sheath tumor (MPNST) is a rare and aggressive soft tissue sarcoma for which effective treatments have not yet been established due to poor understanding of its pathogenesis. Our previous study indicated that miR-210-mediated Ephrin-A3 (EFNA3) promotion of proliferation and invasion of MPNST cells plays an important role in MPNST tumorigenesis and progression. The purpose of the present study was to further investigate the roles of EFNA3 in MPNST. Constructed transcription activator-like effector nucleases (TALENs) and lentiviral vectors were transfected into MPNST ST88-14 (NF1 wild-type) and sNF96.2 (NF1 mutant type) cell lines to obtain gain- and loss-of-function cell lines for the EFNA3 function study. The results showed that the knockout of ENFA3 increased cellular viability and invasiveness of the MPNST cells. However, the adhesion ability of MPNST cells was enhanced or inhibited when EFNA3 was overexpressed or knocked out, respectively. It was also observed that knockout of EFNA3 significantly decreased the expression of phosphorylated FAK (p-FAK) and the tumor necrosis factor α (TNF-α) compared to that in the control cells, yet the expression of phosphatidylinositol 3-kinase (PI3K), GTPase, integrins, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-α) increased significantly. Inversely, overexpression of EFNA3 significantly increased the expression of p-FAK and TNF-α compared to that in the control cells, yet the expression of PI3K, GTPase, integrins, VEGF and HIF-α decreased significantly. The results indicated that EFNA3 serves as a tumor suppressor in MPNST cells and it may play a critical role in the focal adhesion kinase (FAK) signaling and VEGF-associated tumor angiogenesis pathway. These findings may not only facilitate the better understanding of MPNST pathogenesis, but also suggest EFNA3 as a promising target for MPNST treatment.

Emulsion cross-linked chitosan/nanohydroxyapatite microspheres for controlled release of alendronate.

Sustained delivery of growth factors has emerged as an essential requirement for bone tissue engineering applications for the treatment of various kinds of bone defects. Chitosan (CH) has attracted particular attention for drug delivery and bone tissue engineering because of its favorable biocompatibility and biodegradability. In this study, a composite microsphere system containing CH and nanohydroxyapatite (nHA)-alendronate (AL) particles was fabricated by employing both emulsification and cross-linking strategies. The microspheres were characterized for their surface morphology, composition, size distribution, drug loading efficiency and release properties. The results showed that loading efficiency and sustained release of hydrophilic AL were significantly improved, which is ideal for locally sustained release in the bone microenvironment. In vitro osteogenic studies showed that the microspheres could enhance the osteogenic activity of rabbit adipose-derived stem cells. In conclusion, the CH/nHA-AL composite microspheres exhibit promising properties as a candidate for local treatment for bone defects.

Comparison of initial nonoperative and operative management of radial nerve palsy associated with acute humeral shaft fractures.

The optimal treatment approach for the initial management of radial nerve palsy associated with humeral shaft fractures has yet to be conclusively determined. The authors performed a systematic review of the literature to identify studies that compared the outcomes after initial nonoperative and operative management for radial nerve palsy associated with acute humeral shaft fractures. A meta-analysis of the data from these studies was also performed to determine whether recovery from radial nerve palsy was more favorable in one approach compared with the other. The primary outcome was recovery from radial nerve palsy and the secondary outcome was complaints after treatment. Nine articles (1 prospective observational and 8 retrospective) were included in the meta-analyses. Operative management showed no improved recovery from radial nerve palsy compared with nonoperative management. Nonoperative management was associated with a decreased risk of complaints relative to operative management. Recovery from radial nerve palsy associated with acute humeral shaft fractures is not influenced by the initial management approach.