细胞光老化模型的构建及多组学关联分析

Construction and multi-omics analysis of a cell photoaging model

目的构建血清饥饿预处理的人永生化角质形成细胞(HaCaT)光老化模型。通过多组学关联分析,探索中波紫外线(UVB)照射对HaCaT细胞光老化过程的生物学影响。方法血清饥饿法预处理HaCaT细胞24 h,对HaCaT细胞进行20 mJ/cm^(2) UVB照射。利用流式细胞术检测凋亡、高内涵细胞成像检测谷胱甘肽及线粒体跨膜电位、划痕实验检测细胞运动能力,判断光老化模型是否构建成功。对光照前后的HaCaT细胞进行高通量测序,分析miRNA及mRNA转录组中表达差异基因,挑选部分基因进行实时荧光定量PCR(qRT-PCR)验证,对差异基因进行通路富集。结果UVB照射后细胞凋亡增加、谷胱甘肽水平降低、线粒体跨膜电位降低、运动迁移能力减弱,表明无血清条件下光老化建模成功。miRNA组学与mRNA组学关联分析,共得到106个交集差异基因。挑选其中4个关键基因:Ras激酶抑制因子1(KSR1)、胞质分裂作用因子1(DOCK1)、血管内皮生长因子A(VEGFA)、肌球蛋白轻链激酶(MYLK)进行qRT-PCR验证,UVB照射前后的变化趋势与转录组趋势一致。对106个基因进行KEGG功能富集,其显著富集于p53及黏着斑信号通路。结论血清饥饿预处理后,20 mJ/cm^(2) UVB照射能成功构建HaCaT细胞光老化模型,p53及黏着斑信号通路是参与光老化过程的关键通路。

Objective To construct a photoaging model of human immortalized keratinocytes(HaCaT)pretreated with serum starvation and explore the biological effects of UVB radiation on the photoaging process of HaCaT cells via multiomics correlation analysis.Methods HaCaT cells were pretreated using serum starvation method for 24 h before being irradiated with 20 mJ/cm^(2) UVB.Apoptosis was detected by flow cytometry,glutathione and mitochondria membrane potential were detected by high-content screening and cell motility was measured by scratch test to find out whether the photoaging model was properly constructed.HaCaT cells were subjected to high-throughput sequencing before and after light exposure to analyze differentially expressed genes in miRNA genomics and mRNA genomics.Some genes were selected for quantitative real-time PCR(qRT-PCR),and pathway enrichment of differential genes was performed.Results After UVB irradiation,the number of apoptotic cells was increased,glutathione levels decreased,mitochondrial membrane potential decreased,and the ability of motility and migration was weakened,indicating that the photoaging model was workable under serum-free conditions.A total of 106 intersecting differential genes were obtained through the association analysis of miRNA genomics and mRNA genomics.Kinase suppressor of Ras 1(KSR1),dedicator of cytokinesis 1(DOCK1),vascular endothelial growth factor A(VEGFA)and myosin light chain kinase(MYLK)-were selected for qRT-PCR verification.The trend before and after UVB irradiation was consistent with the mRNA genomics.KEGG functional enrichment was performed on 106 genes,which were significantly enriched in p53 and focal adhesion signaling pathways.Conclusion After pretreatment with serum starvation,20 mJ/cm^(2) UVB irradiation can help construct the photoaging model of HaCaT cells.The p53 and focal adhesion signaling pathways are critical to the photoaging process.