Exosomal long noncoding RNAs MAGI2-AS3 and CCDC144NL-AS1 in oral squamous cell carcinoma development via the PI3K-AKT-mTOR signaling pathway.

BACKGROUND: Long noncoding RNAs (lncRNAs) are essential and critical components of signal and transduction, regulating the intracellular microenvironment. Serum exosomes (SEs) are involved in rearranging the intercellular functional lncRNAs, which may also play a role in oral squamous cell carcinoma (OSCC). The function of lncRNAs at the transcription level in SEs of patients with OSCC is partially understood. MATERIALS AND METHODS: The lncRNA expression profiles were examined derived from SEs from patients with OSCC with lymph node metastasis (OSCC-LNM), OSCC with no LNM (OSCC-NLNM), postoperative metastasis and recurrence OSCC (rOSCC) and healthy controls (HCs). Bioinformatics analysis was used to analyse differentially expressed lncRNAs (DE lncRNAs) and a total of 150 subjects were enrolled for RT-PCR verifications. The correlations of four lncRNAs and clinicopathologic factors, biochemical indexes were evaluated. MAGI2-AS3 and CCDC144NL-AS1 were overexpressed or silenced in oral cancer (OC) cells. The proliferation, invasion, and migration were evaluated to investigate the effect of MAGI2-AS3 and CCDC144NL-AS1 on the development of OSCC. The related proteins of PI3K-AKT-mTOR signal pathway were also detected. RESULTS: The expressions of the lncRNAs, namely MAGI2-AS3 and CCDC144NL-AS1, were significantly upregulated in rOSCC and OSCC-LNM. MAGI2-AS3 was overexpressed in cancer tissue compared to other control groups. AC109587.1 and AC010978.1 were significantly associated with the clinical stage, and CCDC144NL-AS1 was significantly associated with aging. MAGI2-AS3 and CCDC144NL-AS1 might promote cell proliferation, invasion, and migration in OSCC cells by regulating the PI3K-AKT-mTOR pathway. CONCLUSIONS: our results suggest that MAGI2-AS3 an d CCDC144NL-AS1 may have clinical applications in the treatment of OSCC.

ECFC-derived exosomal THBS1 mediates angiogenesis and osteogenesis in distraction osteogenesis via the PI3K/AKT/ERK pathway.

Background: Distraction osteogenesis (DO) is a widely used bone regenerative technique. However, the DO process is slow, and the consolidation phase is long. Therefore, it is of great clinical significance to explore the mechanism of DO, and shorten its duration. Recent studies reported that stem cell exosomes may play an important role in promoting angiogenesis related to DO, but the mechanism remains unclear. Methods: Canine endothelial colony-forming cells (ECFCs) were isolated and cultured, and the expression of THBS1 in canine ECFCs were inhibited using a lentiviral vector. The exosomes secreted by canine ECFCs were isolated and extracted, and the effect of exosomes on the angiogenic activity of Human umbilical vein endothelial cells (HUVECs) was detected by proliferation, migration, and tube formation experiments. WB and qRT-PCR were used to explore the effects and mechanisms of THBS1-mediated ECFC-Exos on HUVECs angiogenesis. Then, a mandibular distraction osteogenesis (MDO) model was established in adult male beagles, and exosomes were injected into the canine peripheral blood. Micro-CT, H&E, Masson, and IHC staining were used to explore the effects and mechanisms of THBS1-mediated ECFC-Exos on angiogenesis and osteogenesis in the DO area. Results: ECFC-Exo accelerated HUVECs proliferation, migration and tube formation, and this ability was enhanced by inhibiting the expression of THBS1 in ECFC-Exo. Using Western blot-mediated detection, we demonstrated that inhibiting THBS1 expression in ECFCs-Exo activated PI3K, AKT, and ERK phosphorylation levels in HUVECs, which promoted VEGF and bFGF expressions. In the DO model of the canine mandible, ECFCs-Exo injected into the peripheral blood aggregated into the DO gap, thus promoting angiogenesis and bone formation in the DO tissue by reducing THBS1 expression in ECFC-Exo. Conclusion: Our findings suggested that ECFC-Exos markedly enhances angiogenesis of endothelial cells, and promotes bone healing in canine MDO. Thus, THBS1 plays a crucial role in the ECFC-Exos-mediated regulation of canine MDO angiogenesis and bone remodeling. The translational potential of this article: This study reveals that the angiogenic promotion via THBS1 suppression in ECFC-Exos may be a promising strategy for shortening the DO duration.

Identification of the key exosomal lncRNAs/mRNAs in the serum during distraction osteogenesis.

BACKGROUND: Distraction osteogenesis (DO), a kind of bone regenerative process, is not only extremely effective, but the osteogenesis rate is far beyond ordinary bone fracture (BF) healing. Exosomes (Exo) are thought to play a part in bone regeneration and healing as key players in cell-to-cell contact. The object of this work was to determine whether exosomes derived from DO and BF serum could stimulate the Osteogenic Differentiation in these two processes, and if so, which genes could be involved. METHODS: The osteogenesis in DO-gap or BF-gap was evaluated using radiographic analysis and histological analysis. On the 14th postoperative day, DO-Exos and BF-Exos were isolated and cocultured with the jaw of bone marrow mesenchymal stem cells (JBMMSCs). Proliferation, migration and osteogenic differentiation of JBMMSCs were ascertained, after which exosomes RNA-seq was performed to identify the relevant gene. RESULTS: Radiographic and histological analyses manifested that osteogenesis was remarkably accelerated in DO-gap in comparison with BF-gap. Both of the two types of Exos were taken up by JBMMSCs, and their migration and osteogenic differentiation were also seen to improve. However, the proliferation showed no significant difference. Finally, exosome RNA-seq revealed that the lncRNA MSTRG.532277.1 and the mRNA F-box and leucine-rich repeat protein 14(FBXL14) may play a key role in DO. CONCLUSIONS: Our findings suggest that exosomes from serum exert a critical effect on the rapid osteogenesis in DO. This promoting effect might have relevance with the co-expression of MSTRG.532277.1 and FBXL14. On the whole, these findings provide new insights into bone regeneration, thereby outlining possible therapeutic targets for clinical intervention.

A Review Into the Insights of the Role of Endothelial Progenitor Cells on Bone Biology.

In addition to its important transport functions, the skeletal system is involved in complex biological activities for the regulation of blood vessels. Endothelial progenitor cells (EPCs), as stem cells of endothelial cells (ECs), possess an effective proliferative capacity and a powerful angiogenic capacity prior to their differentiation. They demonstrate synergistic effects to promote bone regeneration and vascularization more effectively by co-culturing with multiple cells. EPCs demonstrate a significant therapeutic potential for the treatment of various bone diseases by secreting a combination of growth factors, regulating cellular functions, and promoting bone regeneration. In this review, we retrospect the definition and properties of EPCs, their interaction with mesenchymal stem cells, ECs, smooth muscle cells, and immune cells in bone regeneration, vascularization, and immunity, summarizing their mechanism of action and contribution to bone biology. Additionally, we generalized their role and potential mechanisms in the treatment of various bone diseases, possibly indicating their clinical application.

Dietary ellagic acid ameliorated Clostridium perfringens-induced subclinical necrotic enteritis in broilers via regulating inflammation and cecal microbiota.

BACKGROUND: Subclinical necrotic enteritis (SNE), a common intestinal disease of broiler caused by Clostridium perfringens, could reduce production performance of broilers by chronic intestinal damage and poor absorption of nutrients. Ellagic acid (EA) has been reported to present antioxidant and anti-inflammatory properties on human and animals in many aspects. This study was conducted to evaluate the effect and mechanism of EA in relieving SNE in broilers induced by C. perfringens. RESULTS: C. perfringens challenge decreased body weight (BW), average daily gain (ADG), jejunal villi height/crypt depth (V/C) ratio, the activity of catalase (CAT) and the mRNA expression of zonula occludens 1 (ZO-1) in jejunal mucosa of broilers. While feed conversion ratios (FCR), jejunal crypt depth (CD), the activities of myeloperoxidase (MPO) and diamine oxidase (DAO), as well as the concentrations of interleukin 6 (IL-6), C-reactive protein (CRP) and procalcitonin (PCT) in serum, the activities of inducible nitric oxide synthase (iNOS) and lysozyme (LZM), the concentration of malondialdehyde (MDA), and the mRNA expressions of claudin-2, TNF-α, IL-1ß, TLR-4, TLR-2, NF-κB, JAK3, STAT6 and iNOS in jejunal mucosa of broilers were increased by C. perfringens challenge. Dietary EA supplement relieved these adverse effects, and heightened jejunal villi height (VH), the concentration of D-xylose in plasma, activity of superoxide dismutase (SOD), and the mRNA expression of occludin in jejunal mucosa of broilers. The alpha diversity of cecal microbiota indicated that dietary EA supplement increased observed species and Shannon index. C. perfringens challenge increased the relative abundance of Firmicutes and decreased the relative abundance of Desulfobacterota in cecal microbiota. EA increased the relative abundance of Firmicutes in cecal microbiota. LEfSe analysis showed that C. perfringens challenge triggered the imbalance of cecal microbiota in broilers, dietary EA supplementation led to a small beneficial effect on microbiota, while the simultaneous effect of them seemed to stimulate the immune function of broilers by improving the microbiota balance. CONCLUSIONS: Dietary EA ameliorated C. perfringens-induced SNE in broilers via regulating jejunal inflammation signaling pathways TLR/NF-κB and JAK3/STAT6, relieving jejunal oxidative stress and balancing cecal microbiota to inhibit intestinal barrier damage, prevent systemic inflammatory response and improve nutrient absorption capacity, finally protect and enhance growth performance of broilers.

Panax notoginseng Saponin Promotes Bone Regeneration in Distraction Osteogenesis via the TGF-ß1 Signaling Pathway.

Distraction osteogenesis (DO) is an efficient strategy that is employed for the treatment of large bone defects in craniomaxillofacial surgery. Despite its utility, however, DO is associated with a prolonged consolidation phase and a high complication rate that hinder its more widespread utilization. Panax notoginseng saponin (PNS) is a traditional Chinese medicine that is frequently administered for the treatment of a range of conditions. Herein, we explored the ability of PNS treatment to influence osteogenic differentiation using both rabbit bone marrow mesenchymal cells (BMSCs) and a model of mandibular DO. BMSC proliferation was assessed via CCK-8 assay, while osteogenic differentiation was monitored through ALP and alizarin red S staining. A PCR approach was used to evaluate the expression of genes associated with osteogenesis (ALP, Runx2, and OCN) and genes linked to the TGF pathway (TßR-II, SMAD2, SMAD3, and PPM1A). For in vivo experiments, treated BMSCs were locally injected into the DO gap, with PNS being injected into treated rabbits every other day throughout the experimental period. The quality of the regenerative process was assessed via scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray imaging, and hematoxylin and eosin (H&E) staining. These analyses revealed that PNS was able to promote BMSC osteogenesis and mandibular generation, driving the upregulation of osteogenesis-related genes at the mRNA levels through the modulation of the TGF-ß1/Smad pathway. Consistently, the overexpression or silencing of TßR-II in PNS-treated BMSCs was sufficient to modulate their osteogenic potential. Analyses of in vivo mandibular DO outcomes revealed significantly augmented new bone growth in the PNS-treated group relative to control animals, with maximal osteogenesis in the group overexpressing rabbit TßR-II. Together, these results highlight the PNS as a promising and cost-effective therapeutic tool with the potential to enhance bone regeneration in clinical contexts through the modulation of the TGF-ß1/Smad pathway.

The RNA Methyltransferase METTL3 Promotes Endothelial Progenitor Cell Angiogenesis in Mandibular Distraction Osteogenesis via the PI3K/AKT Pathway.

Distraction osteogenesis (DO) is used to treat large bone defects in the field of oral and maxillofacial surgery. Successful DO-mediated bone regeneration is dependent upon angiogenesis, and endothelial progenitor cells (EPCs) are key mediators of angiogenic processes. The N6-methyladenosine (m6A) methyltransferase has been identified as an important regulator of diverse biological processes, but its role in EPC-mediated angiogenesis during DO remains to be clarified. In the present study, we found that the level of m6A modification was significantly elevated during the process of DO and that it was also increased in the context of EPC angiogenesis under hypoxic conditions, which was characterized by increased METTL3 levels. After knocking down METTL3 in EPCs, m6A RNA methylation, proliferation, tube formation, migration, and chicken embryo chorioallantoic membrane (CAM) angiogenic activity were inhibited, whereas the opposite was observed upon the overexpression of METTL3. Mechanistically, METTL3 silencing reduced the levels of VEGF and PI3Kp110 as well as the phosphorylation of AKT, whereas METTL3 overexpression reduced these levels. SC79-mediated AKT phosphorylation was also able to restore the angiogenic capabilities of METTL3-deficient EPCs in vitro and ex vivo. In vivo, METTL3-overexpressing EPCs were additionally transplanted into the DO callus, significantly enhancing bone regeneration as evidenced by improved radiological and histological manifestations in a canine mandibular DO model after consolidation over a 4-week period. Overall, these results indicate that METTL3 accelerates bone regeneration during DO by enhancing EPC angiogenesis via the PI3K/AKT pathway.

MicroRNA-205 mediates endothelial progenitor functions in distraction osteogenesis by targeting the transcription regulator NOTCH2.

BACKGROUND: Distraction osteogenesis (DO) is a highly efficacious form of reconstructive bone regeneration, but its clinical utility is limited by the prolonged period required for bone consolidation to occur. Understanding the mechanistic basis for DO and shortening this consolidation phase thus represent promising approaches to improving the clinical utility of this procedure. METHODS: A mandibular DO (MDO) canine model was established, after which small RNA sequencing was performed to identify relevant molecular targets genes. Putative miRNA target genes were identified through bioinformatics and confirmed through qPCR, Western blotting, and dual-luciferase reporter assays. Peripheral blood samples were collected to isolate serum and endothelial colony-forming cells (ECFCs) in order to measure miR-205, NOTCH2, and angiogenic cytokines expression levels. Lentiviral constructs were then used to inhibit or overexpress miR-205 and NOTCH2 in isolated ECFCs, after which the angiogenic activity of these cells was evaluated in migration, wound healing, proliferation, tube formation, and chick chorioallantoic membrane (CAM) assay. Autologous ECFCs transfected to knockdown miR-205 and were injected directly into the distraction callus. On days 14, 28, 35 and 42 after surgery, bone density was evaluated via CBCT, and callus samples were collected and evaluated via histological staining to analyze bone regeneration and remodeling. RESULTS: MiR-205 was identified as being one of the miRNAs that was most significantly downregulated in MDO callus samples. Downregulation of miR-205 was also observed in DO-ECFCs and serum of animals undergoing MDO. Inhibiting miR-205 markedly enhanced angiogenesis, whereas overexpressing miR-205 had the opposite effect in vitro. Importantly, NOTCH2, which is a unique regulator in bone angiogenesis, was identified as a miR-205 target gene. Consistent with this regulatory relationship, knocking down NOTCH2 suppressed angiogenesis, and transduction with a miR-205 inhibitor lentivirus was sufficient to rescue angiogenic activity. When ECFCs in which miR-205 had been inhibited were transplanted into the MDO callus, this significantly bolstered osteogenesis, and remodeling in vivo. CONCLUSIONS: MiR-205 is a significant regulator of the MDO process, and inhibiting this miRNA can accelerate MDO-related mineralization. Overall, these results offer new insights into the mechanistic basis for this procedure, highlighting potential targets for therapeutic clinical intervention.

Panax notoginseng saponins promote endothelial progenitor cell angiogenesis via the Wnt/ß-catenin pathway.

BACKGROUND: Distraction osteogenesis (DO) is an effective treatment in craniomaxillofacial surgery. However, the issue of sufficient blood supply at the regeneration tissue has limited its wide application. Panax notoginseng saponins (PNS) is a Traditional Chinese Medicine that is commonly used to treat a range of angiogenic diseases. However, the mechanisms whereby PNS alters angiogenesis in endothelial progenitor cells (EPCs) have yet to be clarified. METHODS: EPCs were identified by immunofluorescence, confirmed by their uptake of fluorescently labeled Dil-ac-LDL and FITC-UEA-1. EPCs were treated with different concentrations of PNS, and the effects of PNS on cell proliferation were measured on the optimal concentration of PNS determined. The effects of PNS on angiogenesis and migration, angiogenic cytokines mRNA expression and the proteins of the Wnt pathway were investigated. Then knocked down ß-catenin in EPCs and treated with the optimum concentrational PNS, their angiogenic potential was evaluated in tube formation and migration assays. In addition, the expression of cytokines associated with angiogenesis and Wnt/ß-catenin was then assessed via WB and RT-qPCR. RESULTS: We were able to determine the optimal concentration of PNS in the promotion of cell proliferation, tube formation, and migration to be 6.25 mg/L. PNS treatment increased the mRNA levels of VEGF, bFGF, VE-Cadherin, WNT3a, LRP5, ß-catenin, and TCF4. After knocked down ß-catenin expression, we found that PNS could sufficient to partially reverse the suppression of EPC angiogenesis. CONCLUSIONS: Overall, 6.25 mg/L PNS can promote EPC angiogenesis via Wnt/ß-catenin signaling pathway activation.