Changes in the small noncoding RNA transcriptome in osteosarcoma cells.

BACKGROUND: Osteosarcoma has the highest incidence among bone malignant tumors and mainly occurs in adolescents and the elderly, but the pathological mechanism is still unclear, which makes early diagnosis and treatment very difficult. Bone marrow mesenchymal stem cells (BMSCs) are considered to be one of the sources of osteosarcoma cells. Therefore, a full understanding of the gene expression differences between BMSCs and osteosarcoma cells is very important to explore the pathogenesis of osteosarcoma and facilitate the early diagnosis and treatment of osteosarcoma. Small noncoding RNAs (sncRNAs) are a class of RNAs that do not encode proteins but directly play biological functions at the RNA level. SncRNAs mainly include Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), repeat RNAs and microRNAs (miRNAs). METHODS: In this study, we compared the expression of sncRNAs in BMSCs and osteosarcoma cells by high-throughput sequencing and qPCR and looked for differentially expressed sncRNAs. CCK-8, clone formation and transwell assay were used to detect the effect of sncRNA in MG63 cells. RESULTS: We found that 66 piRNAs were significantly upregulated and 70 piRNAs were significantly downregulated in MG63 cells. As for snoRNAs, 71 snoRNAs were significantly upregulated and 117 snoRNAs were significantly downregulated in MG63 cells. As for snRNAs, 35 snRNAs were significantly upregulated and 17 snRNAs were significantly downregulated in MG63 cells. As for repeat RNAs, 6 repeat RNAs were significantly upregulated and 7 repeat RNAs were significantly downregulated in MG63 cells. As for miRNAs, 326 miRNAs were significantly upregulated and 281 miRNAs were significantly downregulated in MG63 cells. Overexpression of piRNA DQ596225, snoRNA ENST00000364830.2, snRNA ENST00000410533.1 and miRNA hsa-miR-369-5p inhibited the proliferation and migration of MG63 cells. CONCLUSIONS: Our results provide a theoretical basis for the pathogenesis, early diagnosis and treatment of osteosarcoma.

Study on the Raman spectral characteristics of dynamic and static blood and its application in species identification.

This paper proposes a method to identify the blood of 4 poultry species (chicken, duck, goose and pigeon) based on Raman spectroscopy and its baseline. Samples were prepared by pretreatment methods of freezing, thawing, and dilution. The Raman spectra of dynamic blood and static blood were measured, respectively, and the spectral differences between the two research schemes were analyzed. The four species of poultry blood were identified based on the Raman spectroscopy and its baseline. The results show that the method can realize the identification of four species of poultry blood. In addition, the potential of Raman spectroscopy as a technique for determining carotenoids in blood has been clearly confirmed, which opens up the possibility to quickly determine whether poultry eats feed containing carotenoids without sample preparation.

TRAF3, a Target of MicroRNA-363-3p, Suppresses Senescence and Regulates the Balance Between Osteoblastic and Adipocytic Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells.

Bone marrow-derived mesenchymal stem cells (BMSCs) have the potential to differentiate into osteoblasts or adipocytes, and an imbalance between adipogenesis and osteogenesis causes age-related bone loss. In this study, we determined the influence of tumor necrosis factor receptor-associated factor 3 (TRAF3) on senescence and osteoblastic and adipocytic differentiation of rat BMSCs. TRAF3 expression increased during osteogenic differentiation but decreased during adipocytic differentiation of rat BMSCs, and compared with day 0 cultures, on day 14, the differences were significant. Overexpression of TRAF3 significantly promoted BMSC osteogenic differentiation and suppressed adipogenic differentiation and senescence. Furthermore, Traf3 was determined to be a target gene of miR-363-3p in BMSCs, and TRAF3 expression in BMSCs was reduced by miR-363-3p overexpression. This overexpression attenuated the effects of TRAF3 on BMSC adipogenic differentiation, osteogenic differentiation, and senescence. Taken together, these results uncovered the mechanism by which TRAF3 promotes BMSC osteogenic differentiation and suppresses adipogenic differentiation and senescence, indicating that the miR-363-3p-TRAF3 axis might be a novel therapeutic target for BMSC-based bone tissue engineering in osteoporosis.

Inflammatory and fibrosis infiltration in synovium associated with the progression in developmental dysplasia of the hip.

Developmental dysplasia of the hip (DDH) is a common musculoskeletal disorder characterized by progressive joint soreness and limited mobility. The aim of the present study was to investigate the pathological changes and inflammatory infiltration in the hypertrophic synovium of the hip joint associated with the progression of DDH. Synovial biopsies in the hip joint are obtained from patients with moderate DDH and severe DDH during surgery. These biopsies are processed for histological and immunohistochemical (IHC) analysis and investigation of the pathological processes in a synovium, including types of inflammatory cell infiltration, synovial angiogenesis and fibrosis, neuron endings and neuropeptide invasion. Correlation analysis was performed between the mean optical density (MOD) of each antibody, and Harris hip score (HHS) and visual analogue score (VAS) using the Spearman correlation test. Chronic inflammation in the synovium was observed via the positive IHC staining of inflammatory cells, such as T cells, B cells, macrophages and leukocytes. Excessive staining of vimentin and α smooth muscle actin in the synovium of severe DDH represented significant fibrosis and angiogenesis. These targets were also significantly correlated with HHS in severe DDH. The MOD levels of CD68 (indicators of macrophage) indicated apparent correlations with HHS and VAS in patients with severe DDH. The labels of nerve fibers and pain transmission indicators were as follows: Neurofilament­200 and substance P. Calcitonin gene­related peptide was upregulated in the synovium of severe DDH in contrast to that in the synovium of moderate DDH. The MOD levels of NF­200, SP and CGRP were correlated with VAS in severe DDH. The pathology of DDH includes chronic inflammatory cell infiltration corresponding with nerve fibers and fibroblastic proliferation, which might contribute to arthritis progression and joint soreness in DDH.

Nano-sized Al2O3 particle-induced autophagy reduces osteolysis in aseptic loosening of total hip arthroplasty by negative feedback regulation of RANKL expression in fibroblasts.

Aseptic loosening is mainly caused by wear debris generated by friction that can increase the expression of receptor activation of nuclear factor (NF)-κB (RANKL). RANKL has been shown to support the differentiation and maturation of osteoclasts. Although autophagy is a key metabolic pathway for maintaining the metabolic homeostasis of cells, no study has determined whether autophagy induced by Al2O3 particles is involved in the pathogenesis of aseptic loosening. The aim of this study was to evaluate RANKL levels in patients experiencing aseptic loosening after total hip arthroplasty (THA) and hip osteoarthritis (hOA) and to consequently clarify the relationship between RANKL and LC3II expression. We determined the levels of RANKL and autophagy in fibroblasts treated with Al2O3 particles in vitro while using shBECN-1 interference lentivirus vectors to block the autophagy pathway and BECN-1 overexpression lentivirus vectors to promote autophagy. We established a novel rat model of femoral head replacement and analyzed the effects of Al2O3 particles on autophagy levels and RANKL expression in synovial tissues in vivo. The RANKL levels in the revision total hip arthroplasty (rTHA) group were higher than those in the hOA group. In patients with rTHA with a ceramic interface, LC3II expression was high, whereas RANKL expression was low. The in vitro results showed that Al2O3 particles promoted fibroblast autophagy in a time- and dose-dependent manner and that RANKL expression was negatively correlated with autophagy. The in vivo results further confirmed these findings. Al2O3 particles induced fibroblast autophagy, which reduced RANKL expression. Decreasing the autophagy level promoted osteolysis and aseptic prosthetic loosening, whereas increasing the autophagy level reversed this trend.

Down-regulation of microRNA-216b inhibits IL-1ß-induced chondrocyte injury by up-regulation of Smad3.

Osteoarthritis (OA) is the most common type of joint disease, leading to a major cause of pain and disability. OA is characterized by the continuous degradation of articular cartilage, mainly resulting in an imbalance between synthesis and degradation of articular chondrocyte extracellular matrix (ECM). Aberrant miR-216b expression has been found in multiple cancers. However, the level of miR-216b in OA cartilage and its role in progression of this disease are still unknown. In the present study, the functional roles of miR-216b and its expression in OA tissues and interleukin-1ß (IL-1ß)-induced chondrocytes were examined. We found that the level of miR-216b was significantly higher and Smad3 expression was obviously lower in OA cartilage and IL-1ß-induced chondrocytes than in normal tissues and cells. Furthermore, a bioinformatics analysis and luciferase reporter assay identified Smad3 as a direct target gene of miR-216b, and Smad3 expression was reduced by miR-216b overexpression at both the mRNA and protein levels. A functional analysis demonstrated that miR-216b down-regulation obviously alleviated the IL-1ß-induced inhibition in cell proliferation, type II collagen, and aggrecan down-regulation and matrix metalloproteinase-13 (MMP-13) up-regulation, while miR-216b overexpression had the opposite effects. Knockdown of Smad3 by siRNA reversed the effects of the miR-216b inhibitor on cell proliferation, the expressions of type II collagen, aggrecan, and MMP-13. Our results suggested that miR-216b contributes to progression of OA by directly targeting Smad3, providing a potential therapeutic target for treatment of OA.

Inhibition of substance P signaling aggravates the bone loss in ovariectomy-induced osteoporosis.

INTRODUCTION: Substance P signaling regulates the functions of both osteoblast and osteoclast. Available reports on the effects of substance P on bone mass are contradictory. The objective of this study was to determine the change of substance P expression in the osteoporotic bone of OVX mice. The effects of substance P signaling blockade by using its specific receptor antagonist L-703606 on bone remodeling in sham-operated mice and OVX mice were also investigated. METHODS: Forty-eight nine-week-old female C57BL/6J mice were evenly distributed into three groups with sham surgery, OVX or OVX with estrogen replacement. Substance P expression in the bones of each group of mice was evaluated by immunohistochemistry and enzyme immunoassay. Another thirty-two nine-week-old female C57BL/6J mice were divided into a SHAM group (sham surgery followed by vehicle treatment with DMSO), a SHAM + L group (sham surgery followed by 15 mg/kg/d L-703606 repeated intraperitoneal injections), an OVX group (ovariectomy with the same vehicle treatment) and an OVX + L group (ovariectomy with the same L-703606 injections), with 8 mice in each group. Treatment started 3 weeks after surgery and last for 3 weeks. A 2 × 2 factorial experimental design was used to detect the effects of substance P signaling blockade on bone remodeling in sham-operated mice and OVX mice. Techniques including micro-computed tomography, biomechanical testing, histomorphometric analysis, enzyme immunoassay, and real-time PCR were employed. RESULTS: Immunohistochemistry and enzyme immunoassay revealed that substance P expression significantly decreased in the bones of OVX mice both at 3 weeks and 6 weeks after surgery. Micro-CT tomography demonstrated that application of L-703606 led to bone loss in sham-operated mice, and aggravated the micro-structural deterioration of bones in OVX mice. This was shown by reduced BV/TV (Mean bone volume fraction), Tb.N (Mean trabecular number) and Tb.Th (Mean trabecular thickness), and increased Tb.Sp (Mean trabecular separation). Biomechanical analysis demonstrated that blockade of substance P signaling reduced the maximum stress and maximum load of L3 vertebrae and tibiae. Inhibited recruitment of bone mesenchymal stem cells (BMSCs) to bone remodeling sites, which was evidenced by increased number of osteoclasts, decreased number of osteoblasts and increased osteoid volume in the secondary spongiosa, was observed in the mice treated with L-703606. A significant decrease of OPG/RANKL ratio was also found in the bones of mice treated with L-703606. Body weight, uterine weight and serum estradiol level were not significantly different between the mice treated with L-703606 and those treated with vehicle. CONCLUSION: The results demonstrated that blocking substance P signaling led to bone loss in sham-operated mice, and exacerbated the bone loss in OVX mice. Substance P signaling had an important role in the maintenance of bone mass.

Increasing expression of substance P and calcitonin gene-related peptide in synovial tissue and fluid contribute to the progress of arthritis in developmental dysplasia of the hip.

INTRODUCTION: Developmental dysplasia of the hip (DDH) is a common musculoskeletal disorder that has pain and loss of joint function as major pathological features. In the present study, we explored the mechanisms of possible involvement and regulation of substance P (SP) and calcitonin gene-related peptide (CGRP) in the pathological and inflammatory processes of arthritis in DDH. METHODS: Blood, synovial tissue and fluid samples were collected from patients diagnosed with different severities of DDH and from patients with femoral neck fracture. Levels of SP, CGRP and inflammatory cytokines in synovium and synovial fluid (SF) in the different groups were evaluated by immunohistochemistry, real-time PCR and enzyme-linked immunosorbent assay (ELISA). Correlations between neuropeptides and inflammatory cytokines in SF were evaluated by partial correlation analysis. The proinflammatory effects of SP and CGRP on synoviocytes obtained from patients with moderate DDH were investigated in vitro by real-time PCR and ELISA. The mechanisms of those effects were evaluated by Western blot analysis and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) DNA binding assay. RESULTS: Significantly increased levels of neuropeptides and inflammatory cytokines were observed in synovium and SF from patients in the severe DDH group compared with the moderate DDH and control groups. In moderate DDH samples, SP in SF correlated with tumor necrosis factor (TNF)-α, and CGRP in SF correlated with TNF-α and interleukin (IL)-10. In the severe DDH group, SP in SF correlated with interleukin (IL)-1ß, TNF-α and IL-10. CGRP in SF correlated with TNF-α. Additionally, SP might have had obvious proinflammatory effects on synoviocytes through the activation of NF-κB. CONCLUSIONS: The upregulation of SP and CGRP in synovium and SF might participate in the inflammatory process of arthritis in DDH. The activation of the NF-κB pathway seems indispensable in the proinflammatory effect of SP on synoviocytes. This original discovery may indicate a potential clinical drug target and the development of innovative therapies for DDH.

Sympathetic neuron can promote osteoblast differentiation through BMP signaling pathway.

Communication between sympathetic neurons and osteoblasts through the adrenergic receptor pathway has already been reported. To investigate whether the sympathetic neurons have a direct effect on osteoblast differentiation, an in vitro Transwell coculture system was established in which osteoblasts were cocultured with sympathetic neurons with no cell-to-cell contact. The expression of osteogenesis-related genes was upregulated in osteoblasts cocultured with sympathetic neurons. Meanwhile, bone morphogenetic protein (BMP) mRNA and protein expressions were detected in sympathetic neurons, and BMP secretion from sympathetic neurons was also confirmed. However, transfection with BMP-2 and/or BMP-6 siRNA in sympathetic neurons caused a down-regulation of osteogenesis-related genes in the cocultured osteoblasts. Sympathetic neurons promoted osteoblast differentiation through BMP signaling pathway, implying that the integrity of sympathetic neurons was important for optimal bone formation and remodeling.

The roles of the sympathetic nervous system in osteoporotic diseases: A review of experimental and clinical studies.

With the rapid aging of the world population, the issue of skeletal health is becoming more prominent and urgent. The bone remodeling mechanism has sparked great interest among bone research societies. At the same time, increasing clinical and experimental evidence has driven attention towards the pivotal role of the sympathetic nervous system (SNS) in bone remodeling. Bone remodeling is thought to be partially controlled by the hypothalamus, a process which is mediated by the adrenergic nerves and neurotransmitters. Currently, new knowledge about the role of the SNS in the development and pathophysiology of osteoporosis is being generated. The aim of this review is to summarize the evidence that proves the involvement of the SNS in bone metabolism and to outline some common osteoporotic diseases that occur under different circumstances. The adrenergic signaling pathway and its neurotransmitters are involved to various degrees of importance in the development of osteoporosis in postmenopause, as well as in spinal cord injury, depression, unloading and the complex regional pain syndrome. In addition, clinical and pharmacological studies have helped to increase the comprehension of the adrenergic signaling pathway. We try to individually examine the contributions of the SNS in osteoporotic diseases from a different perspective. It is our hope that a further understanding of the adrenergic signaling by the SNS will pave the way for conceptualizing optimal treatment regimens for osteoporosis in the near future.

Is patellar resurfacing superior than nonresurfacing in total knee arthroplasty? A meta-analysis of randomized trials.

Conflicting results from abundant studies have made it unclear whether the patella should be resurfaced during total knee replacement. A meta-analysis was undertaken to pool the results of randomized controlled studies (RCTs) and to compare the outcomes and postoperative complications after total knee arthroplasty with patellar resurfacing or nonresurfacing. Sixteen RCTs including 3034 knees between 1966 and December 2009 were analyzed. Reoperation for patellofemoral problems was significantly more likely in the nonresurfacing group (P = 0.03). There was no difference between the two groups in terms of anterior knee pain rate, knee pain score, knee society score and knee function score. The results indicate that patellar resurfacing would reduce the risk of reoperation after total knee replacement, but it seems that the benefits are limited on other aspects, and the analysis of high-quality studies shows no advantage of resurfacing over nonresurfacoing group, even in the aspect of reoperation risk. More carefully and scientifically designed RCTs are beneficial and necessary to further prove the results.