Effects Of Homocysteine On The Genome Stability And Cell Cycle Of Human Neural Cell Line

Homocysteine(Hcy),a sulfur-containing non-essential amino acid,is generated in the pathway of one carbon metabolism.Clinically,plasma concentration of Hcy > 15?mol/L is defined as hyperhomocysteinemia.High Hcy induces genomic damage of human peripheral blood lymphocytes and is also an independent risk factor for cardiovascular disease.Both genetic and dietary factors can affect plasma Hcy levels.Previous studies have suggested that high levels of Hcy and genomic damage are related to the risk of Alzheimer's disease(AD),but its precise underlying mechanism is still elusive.In this study,a cell model of neurobiology research was used to explore the effect of Hcy on genome stability and cell cycle and the underlying molecular mechanisms.According to the physiological concentration of Hcy in human plasma and the influence on the growth of SH-SY5 Y cells,SH-SY5 Y cells were treated with 100 and 1000 ?mol/L Hcy for 14 days,and a negative control was set at the same time.The cytokinesis blocked micronucleus cytome(CBMN-cyt)assay was used to evaluate the genomic instability(GIN)of the cells in each test group;The RNA-seq was used to compare transcription profile difference between Hcy groups and control groups;The bioinformatic analysis was used to enrich and screen the differentially expressed genes in these two transcription profiles;GO and KEGG analysis were used to analyze the cell cycle-related genes that are affected by Hcy treatment,and confirm by the RT-q PCR;On this basis,Impacts of different concentrations of Hcy on cell cycle progression was accessed by flow cytometry.The results showed that:(1)Compared with the control group,both of 100 and 1000?mol/L Hcy caused a significant increase of GIN in SH-SY5 Y cells(P < 0.05).Among the CIN events,micronuclei(MN)and nucleoplasmic bridges(NPB)were significantly increased by Hcy(P < 0.01).(2)The RNA-seq showed that,there were 2,999 significantly differentially expressed genes in 100 ?mol/L Hcy group,of which 2,882 genes were up-regulated and 117 genes are down-regulated.Differentially expressed genes have a strong correlation with cell cycle pathways.(3)confirm by the RT-q PCR,100 ?mol/L Hcy significantly increased the transcription of G1–S transition related genes,including ORC5,ORC6,CCND1,E2F6,CHEK1,CHEK2,and GSPT1(P < 0.01);In contrast,1000 ?mol/L Hcy decreased the transcription of G1–S transition related genes,including ORC4,ORC5,ORC6,CCND1,E2F6,CHEK1,and GSPT1(P < 0.05).These data suggested that different concentrations of Hcy may have different effects on the G1–S transition in SH-SY5 Y cells.(4)Compared with the control group,1000 ?mol/L Hcy significantly increased the percentage of cells in G0/G1 phase(P < 0.01)and significantly decreased the percentage of cells in S phase(P < 0.01),suggesting that 1000 ?mol/L Hcy induces SH-SY5 Y cells to arrest in the G0/G1 phase.To sum up,in vitro,high levels of Hcy can significantly induce neuronal genome damage,interfere with G1/S phase checkpoints,and cause cell arrest in G0/G1 phase.The results of this study provide new evidence for evaluating the AD risk associated with high Hcy in human population.