The transforming growth factor-beta (TGF-β) signaling pathway plays critical roles in the development of various diseases. The current study investigated whether TGF-β was involved in the pathogenesis of osteosarcoma from the genetic polymorphism perspective and serum level perspective. We first examined two TGF-β1 polymorphisms, rs1800469C/T and rs1800470T/C, in 202 osteosarcoma patients and 216 healthy controls. Data revealed that the prevalence of rs1800470TT genotype and T allele was significantly elevated in patients than in controls (odds ratio [OR]=2.28, 95% confidence interval [CI]: 1.30-3.98, p<0.001, and OR=1.49, 95% CI: 1.14-1.96, p<0.001). Function analyses showed that healthy controls carrying rs1800470TT genotype had a significantly higher serum level of TGF-β than those carrying the rs1800470CC genotype (191.1±15.7 pg/mL vs. 129.4±10.9 pg/mL, p=0.003). We then compared the serum level of TGF-β between osteosarcoma patients and healthy controls. Results demonstrated a significantly increased serum level of TGF-β in patients than in controls. Further analyses identified that patients with metastasis had augmented levels of serum TGF-β than those without metastasis. These data indicate that TGF-β may be closely involved in the pathogenesis of osteosarcoma.
Various of microRNAs (miRNAs) have been reported to regulate the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), however, whether miR-134 plays a role in this biological process remains undetermined. In this present study, we firstly evaluated the chondrogenic differentiation of BMSCs by Alcian blue staining, and examined the miR-134 expression by qRT-PCR during this process. And miR-134 inhibitor was used to investigate the functions of miR-134 in chondrogenic differentiation of BMSCs by Alcian blue staining, qRT-PCR and western blot. Subsequently, the correlation between miR-134 and SMAD6 was assessed via bioinformatics analysis and dual luciferase reporter assay. Finally, the roles of SMAD6 in chondrogenic differentiation of BMSCs was also determined through Alcian blue staining, qRT-PCR and western blot. As results showed that miR-134 expression was significantly downregulated during chondrogenic differentiation, and inhibition of miR-134 obviously promoted chondrogenic differentiation. Dual luciferase reporter assay indicated that miR-134 could directly targeted the 3'-UTR of SMAD6, inhibited miR-134 expression in BMSCs could upregulate SMAD6 expression. Moreover, we found that overexpression of SMAD6 significantly promoted chondrogenic differentiation, and this SMAD6 induced promotion of chondrogenic differentiation could be reversed by miR-134 mimics. In conclusion, our findings suggested that miR-134 may act as a negative regulator during chondrogenic differentiation of BMSCs by interacting with SMAD6.
ObjectiveWear‐induced aseptic loosening has been accepted as one of the main reasons for failure of total hip arthroplasty. Ceramic wear debris is generated following prosthesis implantation and plays an important part in the upregulation of inflammatory factors in total hip arthroplasty. The present study investigates the influence of ceramic debris on osteoblasts and inflammatory factors.MethodsCeramic debris was prepared by mechanical grinding of an aluminum femoral head and added to cultures of MC3T3‐E subclone 14 cells at different concentrations (i.e. 0, 5, 10, and 15 μg/mL). Cell proliferation was evaluated using a Cell Counting Kit (CCK‐8), and cell differentiation was assessed by mRNA expression of alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In addition, cell bio‐mineralization was evaluated through alizarin red S staining, and release of tumor necrosis factor alpha (TNF‐α), interleukin‐1 beta (IL‐1β), and interleukin‐6 (IL‐6) was measured through enzyme‐linked immunosorbent assays (ELISA). Furthermore, mRNA expression of Smad1, Smad4, and Smad5 and protein expression of phosphorylated Smad1, Smad4, and Smad5 were measured by reverse transcriptase polymerase chain reaction (RT‐PCR) and western blotting.ResultsThe ceramic debris had irregular shapes and sizes, and analysis of the size distribution using a particle size analyzer indicated that approximately 90% of the ceramic debris was smaller than 3.2 μm (2.0 ± 0.4 μm), which is considered clinically relevant. The results for mRNA expression of ALP, OCN, and OPN and alizarin red S staining indicated that cell differentiation and bio‐mineralization were significantly inhibited by the presence of ceramic debris at all tested concentrations (P < 0.05, and the values decreased gradually with the increase of ceramic debris concentration), but the results of the CCK‐8 assay showed that cell proliferation was not significantly affected (P > 0.05; there was no significant difference between the groups at 1, 3, and 5 days). In addition, the results of ELISA, RT‐PCR, and western blotting demonstrated that ceramic debris significantly promoted the release of inflammatory factors, including TNF‐α, IL‐β, and IL‐6 (P < 0.05, and the values increased gradually with the increase of ceramic debris concentration), and also greatly reduced the mRNA expression of Smad1, Smad4, and Smad5 (the values decreased gradually with the increase of ceramic debris concentration) as well as protein expression of phosphorylated Smad1, Smad4, and Smad5.ConclusionCeramic debris may affect differentiation and bio‐mineralization of MC3T3‐E subclone 14 cells through the bone morphogenetic protein/Smad signaling pathway.
The sarcolemmal ATP-sensitive K+ (sarcKATP) channel plays a cardioprotective role during stress. However, the role of the sarcKATP channel in the apoptosis of cardiomyocytes and association with mitochondrial calcium remains unclear. For this purpose, we developed a model of LPS-induced sepsis in neonatal rat cardiomyocytes (NRCs). The TUNEL assay was performed in order to detect the apoptosis of cardiac myocytes and the MTT assay was performed to determine cellular viability. Exposure to LPS significantly decreased the viability of the NRCs as well as the expression of Bcl-2, whereas it enhanced the activity and expression of the apoptosis-related proteins caspase-3 and Bax, respectively. The sarcKATP channel blocker, HMR-1098, increased the apoptosis of NRCs, whereas the specific sarcKATP channel opener, P-1075, reduced the apoptosis of NRCs. The mitochondrial calcium uniporter inhibitor ruthenium red (RR) partially inhibited the pro-apoptotic effect of HMR-1098. In order to confirm the role of the sarcKATP channel, we constructed a recombinant adenovirus vector carrying the sarcKATP channel mutant subunit Kir6.2AAA to inhibit the channel activity. Kir6.2AAA adenovirus infection in NRCs significantly aggravated the apoptosis of myocytes induced by LPS. Elucidating the regulatory mechanisms of the sarcKATP channel in apoptosis may facilitate the development of novel therapeutic targets and strategies for the management of sepsis and cardiac dysfunction.
Although the pathogenesis of age-related macular degeneration (AMD) is unclear, genetic screening has revealed that polymorphisms in the complement system may be associated with AMD development. Production of autoantibodies was also found in AMD patients. In this study, we analyzed the antibody response in AMD patients. We found that purified B cells from AMD patients tended to respond to lower concentrations of bacterial antigen stimulation, and produced higher amounts of antibodies, especially in IgM and IgA secretions. When examining clinical symptoms, patients with more severe wet-form AMD tended to exhibit higher sensitivity to bacterial antigens and secreted more IgM and IgA antibodies than those with less severe dry-form cases. In conclusion, our study discovered an altered B-cell antibody production in response to bacterial antigens in AMD patients, which potentially contributes to AMD pathogenesis.
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