Research on the effects of NSUN2 gene knockout on the differentiation and apoptosis of PC12 cells
10.3969/j.issn.1002-0152.2024.12.005
- VernacularTitle:NSUN2基因敲除对PC12细胞分化和凋亡的影响研究
- Author:
Lili ZHANG
1
;
Jiufang HE
;
Lin CHEN
;
Xike WANG
Author Information
1. 复旦大学附属闵行医院儿科(上海 201199)
- Publication Type:Journal Article
- Keywords:
NSUN2;
CRISPR/Cas9;
Gene knockout;
Intellectual disability;
PC12 cells;
Cell differentiation;
Cell apoptosis;
Nervous system development
- From:
Chinese Journal of Nervous and Mental Diseases
2024;50(12):735-741
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the role of the NOP2/Sun RNA methyltransferase 2(NSUN2)gene in functions such as differentiation and apoptosis in PC12 cells,with the aim of exploring the function of the NSUN2 gene in nervous system development.Methods PC12 cells were divided into NSUN2-KO group and NSUN2 group.Immunofluorescence was used to detect neuronal characteristics of PC12 cells of each group.Enzyme-linked immunosorbent assay(ELISA),laser confocal and flow cytometry,nitroblue tetrazolium(NBT),thiobarbituric acid(TBA),cell counting kit-8(CCK-8)and flow cytometry were used to detect the content of cell neurotransmitters,intracellular calcium ion influx,superoxide dismutase activity,malondialdehyde content,cell proliferation,cell cycle and apoptosis,respectively.Results NSUN2-KO group showed reduced and shortened synapses(neuron-specific enolase 44.26 vs.63.21,P=0.002;neurofilament protein 10.12 vs.33.63,P<0.001);increased excitatory neurotransmitters(aspartic acid 618.83 nmol/L vs.372.38 nmol/L,P<0.001;glutamic acid 880.45 nmol/L vs.525.17 nmol/L,P<0.001)and decreased inhibitory neurotransmitters(glycine 197.40 nmol/L vs.286.15 nmol/L,P<0.001;γ-aminobutyric acid 134.91 nmol/L vs.208.25 nmol/L,P<0.001).NSUN2-KO group also exhibited increased fluorescence intensity value of intracellular calcium ions(51319.70 vs.24546.06,P<0.001);the prolonged G1 phase(73.38%vs.68.48%,P<0.001),shortened G2 phase(14.69%vs.16.99%,P=0.008)and S phase(11.92%vs.14.52%,P=0.002);increased oxidative stress levels(superoxide dismutase 11.05 U/mgprot vs.21.59 U/mgprot,P=0.002;malondialdehyde 1.42 nmol/mgprot vs.0.80 nmol/mgprot,P=0.008);increased apoptosis(21.55%vs.10.53%,P<0.001).Conclusion Knockout of the NSUN2 gene reduces and shortens synapses in NGF-induced PC12 cells,enhances the ability to release excitatory neurotransmitters,increases cytotoxicity,decreases cell viability,and promotes apoptosis,which indicates that the NSUN2 gene may be an important neuroprotective factor for neuronal proliferation,differentiation,and maintenance of their morphology and function.
