Effects of paraquat on the proliferation of human neural stem cells via DNA methylation
10.3760/cma.j.issn.1001-9391.2019.03.001
- VernacularTitle: 百草枯通过DNA甲基化途径对人神经干细胞增殖的影响
- Author:
Min HUANG
1
;
Nannan HUANG
;
Yingying LI
;
Kexin WU
;
Muzhen GUO
;
Chendi ZHU
Author Information
1. School of Public Health, Ningxia Medical University, Yinchuan 750004, China
- Publication Type:Journal Article
- Keywords:
Paraquat;
Human neural stem cells;
Proliferation;
DNA methylation;
DNA Methyltransferases
- From:
Chinese Journal of Industrial Hygiene and Occupational Diseases
2019;37(3):161-168
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effects of Paraquat on neural stem cell proliferation in vitro and explore the its mechanism based on DNA methylation pathway.
Methods:Nestin, β-tubulin III, and glial fibrillary acidic protein (GFAP) were detected by indirect immunofluorescence assay to evaluate self renewal and differentiation potentia of ReNcell CX human neural stem. The cells were treated with terminal concentrations of 0, 5, 25, 50, and 100μmol/L PQ for 24 hours, and the cells were induced by 50 μmol/L PQ for different time (6, 12, 24, 48 h). Cell viability was determined by MTT assay. The proliferation of neural stem cells was evaluated using Sox2/Brdu and Nestin/Brdu double immunofluorescence staining. The global DNA methylation level was assayed by MethyflashTM methylated DNA Quantification kit. The expression levels of Dnmts mRNA and protein were analyzed by quantitative reverse transcription polymerase chain reaction(qRT-PCR) and Western blot, respectively.
Results:Immunofluorescence showed that nestin was primarily expressed in proliferative neural stem cell and peotein biomarkers (β-tubulin III, GFAP) for neuron and astrocyte were expressed in differentiated cells. MTT assay showed PQ induced cell survival rate decrease in a time and dose dependent manner. Double immunfluorescence staining of cells showed colocalization of Sox2 and Brdu. The percentage of Brdu/Sox2 positive cells was significantly lower in the PQ-exposed (25, 50, 100μmol/L PQ treatment) groups compared to control (P<0.05); Meanwhile, The percentage of Brdu/Nestin positive cells was also significantly lower in the PQ-exposed(50,100μmol/L PQ treatment) groups compared to control (P<0.05). The results of global DNA methylation revealed a significant decrease in PQ-exposed groups (P<0.05). Western blot showed that compared with control group, the protein and mRNA levels of Dnmt1, Dnmt3a in PQ-exposed group were significantly decreased (P<0.05), but there was a significant increase in expression level of Dnmt3b in 50, 100 μmol/L PQ-treated group(P<0.05).
Conclusion:Paraquat could inhibite the proliferation of human neural stem cells through reducing the level of DNA methylation reaction by suppressing the protein expression and transcription of DNA methylated transferase(Dnmts).
