基于代谢组学和转录组学筛选丙泊酚致神经毒性关键基因的研究

SCREENING FOR KEY GENES INVOLVED IN PROPOFOL-INDUCED NEUROTOXICITY BASED ON METABOLOMICS AND TRANSCRIPTOMICS

目的通过代谢组学和转录组学联合分析方法筛选丙泊酚致神经毒性的关键基因。方法以0、25、50、75、100 mg/L的丙泊酚培养HT22细胞(分别为Ctrl组、P25组、P50组、P75组、P100组)24 h,采用CCK-8实验检测各组细胞的活性。收集各组处理24 h后HT22细胞提取RNA和代谢物,进行代谢物检测和转录组分析。对提取的各组HT22细胞代谢物进行液相色谱-质谱(LC-MS)分析、差异代谢物分析、趋势分析,筛选出关键代谢物。对提取的各组HT22细胞RNA进行测序序列比对分析、差异基因分析,筛选出差异基因。对上述筛选出的关键代谢物和差异基因进行加权基因共表达分析(WGCNA)和蛋白质相互作用网络(PPI)分析,获得丙泊酚致神经毒性的关键基因。结果各组HT22细胞的细胞活性随丙泊酚浓度的升高而下降(t=11.40~97.25,P<0.05)。对提取的各组HT22细胞代谢物进行LC-MS分析,共检测到2701种代谢物,进一步经差异分析和趋势分析,得到49种关键代谢物。对提取的各组HT22细胞RNA进行序列比对分析,各组HT22细胞的测序序列数量能够满足数据分析需要,Ctrl组与P25组、P50组、P75组、P100组间的差异基因分别为1254、4695、4923和5031个。对关键代谢物以及差异基因进行WGCNA分析和PPI分析,共筛选出了15种关键基因,分别为NTNG2、ENG、SEMA4G、JAG2、FGF11、SERPINE1、GDF15、GADD45G、F3、NGF、FGF21、PGF、EGFL7、SEMA6D、LRFN1。结论丙泊酚所导致的神经毒性的关键基因可能包含NTNG2、ENG、SEMA4G、JAG2、FGF11、SERPINE1、GDF15、GADD45G、F3、NGF、FGF21、PGF、EGFL7、SEMA6D以及LRFN1。筛选出的这些基因为后续丙泊酚致神经毒性的分子机制研究提供了理论基础,同时也为丙泊酚的神经毒性预防用药和治疗用药的开发提供了研究方向。

Objective To screen for the key genes involved in propofol-induced neurotoxicity based on metabolomics and transcriptomics.Methods HT22 cells were cultured with propofol at concentrations of 0,25,50,75,and 100 mg/L for 24 h and were established as Ctrl,P25,P50,P75,and P100 groups,respectively,and CCK-9 assay was used to measure cell viability in each group.HT22 cells were collected after 24 h of treatment to extract RNA and metabolites for metabolite detection and transcriptome analysis.The extracted HT22 cell metabolites were analyzed by liquid chromatography-mass spectrometry(LC-MS),differentially expressed metabolites,and trend analysis to obtain the key metabolites.The RNA of HT22 cells was analyzed by sequence alignment and differentially expressed genes to obtain the differentially expressed genes.Weighted gene co-expression network analysis(WGCNA)and protein-protein interaction(PPI)analysis were performed for the above key metabolites and differentially expressed genes to obtain the key genes involved in propofol-induced neurotoxicity.Results The viability of HT22 cells decreased with the increase in propofol concentration(t=11.40-97.25,P<0.05).LC-MS was performed for the metabolites of HT22 cells and a total of 2701 metabolites were identified,and 49 key metabolites were obtained through further differential ana-lysis and trend analysis.A sequence alignment analysis of the extracted HT22 cell RNA showed that the number of sequences in each group of HT22 cells could meet the requirements for data analysis,and there were 1254 differentially expressed genes between the Ctrl group and the P25 group,4695 differentially expressed genes between the Ctrl group and the P50 group,4923 differentially expressed genes between the Ctrl group and the P75 group,and 5031 differentially expressed genes between the Ctrl group and the P100 group.WGCNA and PPI analyses were performed for the key metabolites and the differentially expressed genes,and a total of 15 key genes were obtained,namely NTNG2,ENG,SEMA4G,JAG2,FGF11,SERPINE1,GDF15,GADD45G,F3,NGF,FGF21,PGF,EGFL7,SEMA6D,and LRFN1.Conclusion The key genes involved in propofol-induced neurotoxicity may include NTNG2,ENG,SEMA4G,JAG2,FGF11,SERPINE1,GDF15,GADD45G,F3,NGF,FGF21,PGF,EGFL7,SEMA6D,and LRFN1.These genes provide a theoretical basis for the subsequent research on the molecular mechanism of propofol-induced neurotoxicity and research directions for the development of preventive and therapeutic drugs for propofol-induced neurotoxicity.