Toxicogenomics Analysis Of Okadaic Acid

Objective: Okadaic acid(OA)is a common marine algal toxin that can cause severe harm to mammals,including neurotoxicity,liver toxicity,and genotoxicity.Although OA is not a typical neurotoxin,more and more studies have reported its neurotoxic effects.However,most of the existing researches focus on OA-induced serine / threonine protein phosphatase inhibition,and the molecular mechanism of OA-induced neurotoxicity is unclear.In this study,human-derived neuroblastoma SH-SY5 Y cell line was used to study the gene expression level of neurons affected by OA using toxicomics technology.Methods: 1.The effects of different concentrations of OA(0,20,40,60,80,100 n M)on the proliferation and viability of SH-SY5 Y cells were detected by MTT method after 12 h and 24 h exposure.2.SH-SY5 Y cells were treated with OA for 12 h,then total RNA was extracted,and the effect of OA on the whole genome was analyzed by Affymetrix expression profiling chip.3.Use SAM software to screen the differentially expressed genes between the control group and the OA exposure group,and analyze the overlapping of the differential genes in different concentration groups using the Wayne plot.4.Using the GO database for gene annotation and biological interpretation,and KEGG for gene function cluster analysis.5.Flow cytometry was used to analyze the effect of OA on the cell cycle of SH-SY5 Y.6.Real-time fluorescent quantitative PCR(q PCR)was performed to verify the S-phase related differentially expressed genes in 13 cell cycles as shown by the chip results.7.Western-Blot analysis of 5 proteins related to the S phase.8.Comet assay was used to analyze the DNA damage produced by OA on SH-SY5 Y cells.Results: 1.According to the MTT results,select 0,20,40,60,80,100 n M OA poisoning for subsequent experiments,and use 0,20,60 n M as the control group,low concentration and high concentration,respectively.2.According to microarray experiments,cells exposed to low and high concentrations of OA showed 9 and 889 differentially expressed genes,respectively.3.Signal pathways show that cell cycle block is the main reason for OA-induced neurotoxicity.3.Flow cytometry results showed that OA-induced SH-SY5 Y cells mainly developed S phase arrest.4.Perform q PRC verification on 13 S-phase related differential genes obtained from the chip results.The expression levels of MCM2,MCM3,MCM4,MCM5,MCM6,MCM7,CDC45,GINS1,and GINS2 in the high-concentration group were significantly lower than those in the control group,and SKP1 gene expression was significantly reduced.The levels were significantly higher than those in the control group,but the expression levels of MCM10,GIN3 and GINS4 genes were not significantly changed.5.Five S-phase replication initiation-related proteins were verified by Western-Blot technology.Among them,MCM2,MCM3,and MCM4 showed a downward trend in protein expression after the OA concentration reached 40 n M;p-MCM4 protein expression level was obvious at 40 n M Increased,then gradually decreased;p-MCM2 protein expression level did not change significantly.6.Comet experiment results show that high concentration group OA has a significant effect on TDNA% of SH-SY5 Y cellsConclusion: Genomic results show that cell cycle arrest is the main cause of neurotoxicity in OA.OA induces cell cycle arrest in S phase by inhibiting the expression of genes related to DNA replication helicase,and OA is also genotoxic to nerve cells.