极低频电磁场辐射干扰后小鼠成纤维细胞转录组测序的生物信息学分析

Bioinformatics analysis of transcriptome sequencing after the interference of extremely low frequency electromagnetic field in mouse fibroblasts

目的通过高通量测序技术分析极低频电磁场(ELF-EMFs)辐射干扰小鼠成纤维细胞后转录组的变化情况,并筛选出可能参与ELF-EMFs调控成纤维细胞生长的相关通路及基因。方法将小鼠NIH/3T3细胞分为电磁辐射组和正常对照组,电磁辐射组细胞置于0.2 mT、50 Hz的电磁辐射系统中,正常对照组置于同等条件未通电的相同线圈系统中,于细胞培养箱中培养24 h后收集细胞,提取RNA。采用第2代高通量测序技术对2个组进行转录组测序,对筛选的差异基因进行基因功能注释及信号通路数据库分析。筛选出部分高表达基因进行实时荧光定量PCR验证。结果本次转录组测序共鉴定出17980个基因,筛选出140个有显著差异的基因,其中上调120个,下调20个。差异基因富集在酶的催化活性、细胞代谢过程、生物调控、生物合成等方面。京都基因与基因组百科全书分析结果显示,差异基因主要涉及55条通路,富集显著的10条通路集中于氨酰基-tRNA的生物合成、血小板活化、神经营养蛋白信号通路、肾素-血管紧张素系统等,与细胞的生物合成密切相关,进一步从中筛选出可能参与细胞辐射后应激的差异基因,包括有丝分裂原激活的蛋白激酶12(MAPK12)、神经营养性酪氨酸激酶受体3型(NTRK3)、2型血管紧张素Ⅱ受体(AGTR2)、血管内皮生长因子(VEGF)等。实时荧光定量PCR结果显示,电磁辐射组MAPK12、NTRK3、AGTR2、VEGF mRNA相对表达量分别为2.389±0.003、2.481±0.350、2.354±0.081和1.559±0.110,明显高于正常对照组的1.011±0.190、1.011±0.180、1.007±0.150、1.008±0.153,差异均有统计学意义(t=12.540、6.309、13.710、3.078,均P<0.05)。结论ELF-EMFs干扰小鼠成纤维细胞后,MAPK12、NTRK3、AGTR2、VEGF等基因表达明显上调,主要涉及神经营养蛋白信号通路、肾素-血管紧张素系统等通路,这部分基因及通路可能是ELF-EMFs影响成纤维细胞的主要途径。

Objective To analyze the changes in the transcriptome of mouse fibroblasts after exposure to extremely low frequency electromagnetic fields(ELF-EMFs)using next-generation sequencing technology,and to screen out related pathways and genes that might be involved in the regulation of fibroblast growth by ELF-EMFs.Methods The mouse NIH/3T3 cells were divided into the radiation group and the normal control group.The cells in the radiation group were placed in a 0.2 mT,50 Hz electromagnetic radiation system,and the cells in the normal control group was placed in the same coil system under the same conditions without power.After 24-hour culture in a cell incubator,RNA was extracted.The next-generation high-throughput sequencing technology was used to sequence the transcriptomes of the two groups,and perform gene function annotation and signal pathway database analysis on the selected differential genes.Some highly expressed genes were screened out and verified by real-time fluorescent quantitative PCR.Results A total of 17980 genes were identified in the transcriptome sequencing,and there were 140 significantly differentially expressed genes(DEGs),of which 120 were up-regulated and 20 were down-regulated.DEGs were enriched in enzyme catalytic activity,cell metabolism process,biological regulation,biosynthesis and so on.According to the Kyoto Encyclopedia of Genes and Genomes analysis,the DEGs mainly involved 55 pathways,among which the most enriched 10 pathways were aminoacyl-tRNA biosynthesis,platelet activation,neurotrophin signaling pathway,renin-angiotensin system,etc.,closely related to cell biosynthesis.The DEGs that might be involved in the post-irradiation stress of cells were further screened out,including mitogen activated protein kinase 12(MAPK12),neurotrophic tyrosine kinase receptor type 3(NTRK3),angiotensinⅡreceptor type 2(AGTR2),vascular endothelial growth factor(VEGF),etc.Real-time fluorescent quantitative PCR results showed that the relative expression levels of MAPK12,NTRK3,AGTR2,VEGF mRNA in the radiation group were 2.389±0.003,2.481±0.350,2.354±0.081,1.559±0.110,respectively,which were significantly higher than 1.011±0.190,1.011±0.180,1.007±0.150,1.008±0.153,respectively in the normal control group(t=12.540,6.309,13.710,3.078;all at P<0.05).Conclusions After the mouse fibroblasts were interfered with ELF-EMFs,the expression levels of MAPK12,NTRK3,AGTR2,VEGF and other genes are significantly up-regulated,which mainly involve neurotrophin signaling pathway,renin-angiotensin system and other pathways.These genes and pathways may be the main way that ELF-EMFs affect fibroblasts.