RESUMO
Bone fractures or bones subjected to open conduction and internal fixation are easily infected by bacteria; bacterial lipopolysaccharide (LPS) has been recognized as an important pathogenic factor affecting bone fracture healing. Therefore, the effect of LPS on bone metabolism is relevant for bone healing. In this study, we investigated the effect of LPS on the expression of Toll-like receptor (TLR)-4 (an LPS receptor) by using real-time quantitative PCR and western blotting. We also examined the regulatory role of LPS in osteoblast differentiation by measuring the ALP activity, matrix mineralization, and ALP, OCN, and Runx2 mRNA (essential factors affecting osteoblast differentiation) expression in LPS-treated mouse osteoblast MC3T3-E1 cells. We also evaluated the effect of TLR-4 on LPS-mediated inhibition of osteoblast differentiation using RNA interference. LPS promotes TLR-4 mRNA and protein expression in MC3T3-E1 cells (P < 0.05, P < 0.01 or P < 0.001), and inhibits osteoblast differentiation by downregulating matrix mineralization and ALP activity (P < 0.05, P < 0.01 or P < 0.001), and suppressing the expression ALP, OCN, and Runx2 mRNA in MC3T3-E1 cells (P < 0.05 or P < 0.01). Conversely, RNAi-mediated TLR-4 knockdown abrogates the LPS-mediated inhibition of osteoblast differentiation (P < 0.05 or P < 0.01). In summary, LPS was shown to inhibit osteoblast differentiation by suppressing the expression of ALP, OCN, and Runx2 in a TLR-4-dependent manner. The results of this study may provide insights into the signal pathway of LPS-induced bone loss or delayed bone fracture healing.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Fraturas Ósseas/genética , Osteoblastos/metabolismo , Receptor 4 Toll-Like/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Fraturas Ósseas/tratamento farmacológico , Fraturas Ósseas/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/administração & dosagem , Proteínas de Membrana/biossíntese , Camundongos , Osteoblastos/patologia , Osteogênese/efeitos dos fármacos , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/genéticaRESUMO
The US2 protein has been reported to contribute to duck enteritis virus (DEV) infection; however, its kinetics and localization during infection, and whether it is a component of virion, have not been previously reported. To elucidate the function of DEV US2, US2 was amplified by polymerase chain reaction (PCR) and inserted into pET-32a(+); this was expressed, the recombinant US2 protein was purified, and a polyclonal antibody generated. In addition, the kinetics and localization of the US2 gene and protein were determined by quantitative real-time fluorescent PCR, ganciclovir (GCV), and cycloheximide (CHX) treatment, western-blot, and indirect immunofluorescence assay. The packaging of US2 into DEV virions was revealed by a protease protection assay. US2 was found to be transcribed 24 h post-infection (pi) and peaked at 72 h pi; the US2 protein was detected 48 h pi, except in the presence of GCV or CHX. US2 was packed into virions and also localized to the plasma membrane and cytoplasm in infected cells. The results showed that the DEV US2 is a late gene, and that its encoding protein could be a tegument component localized mainly in the cytoplasm. This study provides useful data for the further analysis of DEV US2, including an explanation for the genetic conservation among alphaherpesviruses.
Assuntos
Mardivirus/genética , Proteínas do Envelope Viral/genética , Animais , Linhagem Celular , Patos , Fibroblastos , Expressão Gênica , Mardivirus/efeitos dos fármacos , Transporte Proteico , Proteínas Recombinantes , Proteínas do Envelope Viral/isolamento & purificação , Proteínas do Envelope Viral/metabolismo , VírionRESUMO
MicroRNAs (miRNAs) are thought to play a role in cancer development. We conducted a case-control study to investigate the association between polymorphisms in miR-149C>T and hepatocellular carcinoma (HCC) risk. Duplex polymerase chain reaction with the confronting 2-pair primers were taken to genotype miR-149C>T. The association between genotype frequencies of miR-149C>T and risk of HCC was estimated as odds ratios (ORs) and 95% confidence intervals (95%CIs) using conditional regression analysis. Logistical regression analysis showed that the miR-149 CC genotype and C allele were associated with risk of HCC, with adjusted ORs (95%CI) of 2.07 (1.32-3.26) and 1.42 (1.06-2.12), respectively. Using the TT+TC genotype as a reference, individuals carrying the CC genotype were associated with non-significant increased risk of HCC, adjusted OR (95%CI) of 1.37 (0.91-2.07). Subgroup analysis showed that HBV-infected subjects carrying the miR-149 TC+CC genotype (OR=5.85, 95%CI=2.49-13.77) had an increased risk of HCC. In summary, our study found that miRNA-149C>T polymorphism is associated with risk of HCC, especially in HBV-infected patients.