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1.
Cell Mol Biol Lett ; 26(1): 37, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399682

RESUMO

BACKGROUND: Iron overload can promote the development of osteoporosis by inducing apoptosis in osteoblasts. However, the mechanism by which miRNAs regulate apoptosis in osteoblasts under iron overload has not been elucidated. METHOD: The miRNA expression profile in MC3T3-E1 cells under iron overload was detected by next generation sequencing. qRT-PCR was used to determine the expression of miR-3074-5p in MC3T3-E1 cells under iron overload. The proliferation of MC3T3-E1 cells was tested using CCK-8 assays, and apoptosis was measured using flow cytometry. The miRanda and TargetScan databases were used to predict the target genes of miR-3074-5p. Interaction between miR-3074-5p and the potential target gene was validated by qRT-PCR, luciferase reporter assay and western blotting. RESULTS: We found that iron overload decreased the cell viability and induced apoptosis of MC3T3-E1 cells. The results of next generation sequencing analysis showed that miR-3074-5p expression was significantly increased in MC3T3-E1 cells under iron overload conditions, which was confirmed by further experiments. The inhibition of miR-3074-5p attenuated the apoptosis of iron-overloaded MC3T3-E1 cells. Furthermore, the expression of Smad4 was decreased and was inversely correlated with miR-3074-5p expression, and overexpression of Smad4 partially reversed the viability inhibition of iron-overloaded MC3T3-E1 cells by relieving the suppression of ERK, AKT, and Stat3 phosphorylation, suggesting its regulatory role in the viability inhibition of iron-overloaded MC3T3-E1 cells. The luciferase reporter assay results showed that Smad4 was the target gene of miR-3074-5p. CONCLUSION: miR-3074-5p functions as an apoptosis promoter in iron-overloaded MC3T3-E1 cells by directly targeting Smad4.


Assuntos
Sobrecarga de Ferro/metabolismo , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/patologia , Camundongos , MicroRNAs/genética , Osteoblastos/patologia , Transdução de Sinais , Proteína Smad4/metabolismo
2.
Oncotarget ; 8(57): 96913-96923, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29228581

RESUMO

We utilized one-step multiplex reverse transcription-PCR (RT-PCR) and Luminex xMAP technology to develop a respiratory multiplex liquid-chip assay (rMLA) for simultaneous detection of 6 common respiratory viruses, including influenza virus type A (FluA) and type B (FluB), para-influenza virus type 3 (PIV-3), respiratory syncytial virus (RSV), human metapneumovirus (MPV) and a threatening virus to China, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Performance of rMLA was evaluated by comparing with real-time RT-PCR. Detection data from clinical specimens showed that the rMLA had diagnostic sensitivities of 97.10% for FluA, 94.59% for FluB, 98.68% for PIV-3, 94.87% for RSV and 95.92% for MPV (No Data for MERS-CoV due to the lack of positive specimens). Data of analytical sensitivities showed that the detection limits of the rMLA assay were 5-25 viral RNA copies per µl for FluA, FluB, PIV-3 and MERS-CoV, approximate to the real-time RT-PCR assay; while the values were 8 and 22copies/µl for MPV and RSV, lower than the real-time RT-PCR(78 and 114 copies/µl respectively). The results indicated that the rMLA is a sensitive, specific detection tool and comparable to real-time RT-PCR, especially suitable for high-throughput detection of respiratory specimens.

3.
J Virol Methods ; 234: 22-7, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27058642

RESUMO

This study involved a human infection with avian influenza H7N9(A) virus in Zhejiang province, the first one after implementing the closure measures of living poultry markets in China. The clinical symptoms, epidemiological and virological characteristics of the case were described briefly, and as the emphasis, H7N9 virus was detected quantitatively and continuously from the collected samples in 10 different periods of the patient's treatment in order to reveal changes of viral load in patient's body during the treatment. This study first used reverse-transcription droplet digital PCR (RT-ddPCR) assays to monitor viral load dynamically for human H7N9 infection, synchronously performing real-time RT-PCR as a reference technology to obtain more comprehensive data for comparison. Our results indicated that RT-ddPCR compared to real-time RT-PCR is more sensitive and accurate for quantifying H7N9 viral load without the use of standard curves. Furthermore it can provide reference data for clinical policies including infectivity judgement, ward transferring and therapy adjustment for the patient during treatment.


Assuntos
Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Influenza Humana/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral , Feminino , Genoma Viral , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Humana/diagnóstico , Influenza Humana/tratamento farmacológico , Pessoa de Meia-Idade , Filogenia , Sensibilidade e Especificidade
4.
J Virol Methods ; 189(2): 277-82, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23454645

RESUMO

A one-step multiplex real-time reverse transcription-PCR (RT-PCR) assay was developed for one-tube and simultaneous detection of three genogroups of human norovirus, genogroup I, II and IV (GI, GII and GIV). The specificity and sensitivity of the assay were evaluated and 50 samples were tested by using this assay. The results showed that the multiplex assay had high sensitivity and specificity. The amplification efficiencies of the assay were 91.3%, 90.1%, 88.9% and the detection limits were up to 16.9, 6.3, 43.0 copies/reaction respectively for norovirus GI, GII and GIV detection. No cross-reaction with the other examined RNA viruses was observed, and the qualitative analysis of samples showed that the multiplex assay had a good consistency with its corresponding monoplex assays for the detection of norovirus GI, GII and GIV (Kappa values were 0.848, 0.876 and 0.812 respectively).


Assuntos
Infecções por Caliciviridae/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Norovirus/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Virologia/métodos , Infecções por Caliciviridae/virologia , Genótipo , Humanos , Norovirus/classificação , Norovirus/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo
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