基于转录组测序探讨EGFR-TKI致HaCaT细胞损伤机制

Transcriptome sequencing to explore the mechanism of EGFR-TKI damage to HaCaT cells

目的:探讨表皮生长因子受体-酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitors,EGFR-TKIs)致人永生化角质形成细胞(HaCaT)损伤机制,从而初步探索该药物诱导表皮不良反应机制。方法:利用MTS及Hoechst染色建立TKI药物对HaCaT细胞损伤模型;通过转录组测序筛选差异基因;利用数据库收集表皮不良反应靶点;将差异基因与表皮不良反应靶点取交集,得到潜在作用靶点;利用DAVID及R软件对潜在靶点进行京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)及基因本体论(gene ontology,GO)分析;应用STRING数据库进行蛋白互作(protein protein interaction,PPI)分析,并采用Cytoscape软件进行可视化分析和拓扑参数分析;通过Autodock软件对关键基因与EGFR-TKI药物进行分子对接;运用实时荧光定量PCR检测分子对接中结合能最低的靶点;利用免疫印迹实验(Wesrtern Blot)法检测通路相关蛋白表达。结果:MTS及Hoechst染色表明,奥希替尼与阿法替尼对细胞存活率有明显抑制作用;取交集后得到176个潜在作用靶点;PPI显示IL6、HSPA5、FN1、VEGFA、XBP1、TLR4、MMP1、CCL2、PTGS2和VWF可能是EGFR-TKI表皮毒性关键靶点;KEGG信号通路包括IL-17、PI3K-Akt通路;GO功能涉及内质网应激、氧化应激、脂多糖反应;分子对接表明,奥西替尼、阿法替尼与PTGS2、HSPA5、MMP1、VEGFA和FN1有较好的结合活性。实时荧光定量PCR显示,奥西替尼与阿法替尼处理HaCaT细胞后FN1、HSPA5和PTGS2表达上调,VEGFA和MMP1表达下调;免疫印迹实验表明FN1蛋白表达增加,VEGFA蛋白表达减少。结论:EGFR-TKI药物可能通过抑制PI3K-Akt、IL-17信号通路中的MMP1、VEGFA产生,促进PTGS2、FN1、HSPA5表达,损伤HaCaT细胞,从而产生表皮毒性。

Objective:To investigate the mechanism of epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs)causing damage to human immortalized keratinocytes(HaCaT)cells,and thus to explore the preliminary mechanism of epidermal adverse reactions induced by this drug.Methods:MTS and Hoechst staining were used to model TKI drug damage to HaCaT cells.Transcriptome sequencing was used to screen for differential genes.The database was used to collect targets for epidermal adverse reactions.The intersection of differential genes and targets for epidermal adverse reactions was taken to obtain potential targets.The potential targets were analyzed by the Kyoto encyclopedia of genes and genomes(KEGG)and gene ontology(GO)using DAVID and R software.Protein protein interaction(PPI)analysis was performed using the STRING database,and Cytoscape software was used to visualisation and topological parameters were analysed using Cytoscape software.Molecular docking of key genes to EGFR-TKI drugs was performed using Autodock software.Real-time fluorescence PCR was used to detect the lowest binding energy targets in molecular docking and Wesrtern Blot was used to detect pathway-related protein expression.Results:MTS and Hoechst staining showed that ositinib and afatinib had a significant inhibitory effect on cell survival.176 potential targets of action were obtained after taking intersection.PPI showed that IL6,HSPA5,FN1,VEGFA,XBP1,TLR4,MMP1,CCL2,PTGS2 and VWF may be key targets of EGFR-TKI epidermal toxicity.KEGG signaling pathway includes IL-17,PI3K-Akt pathway.GO function involves endoplasmic reticulum stress,oxidative stress,lipopolysaccharide response.Molecular docking showed that oxitinib and afatinib have good binding activity with PTGS2,HSPA5,MMP1,VEGFA and FN1.Real-time fluorescence quantitative PCR showed that FN1,HSPA5 and PTGS2 expression were up-regulated and VEGFA and MMP1 expression were down-regulated after treatment of HaCaT cells with axitinib and afatinib.Immunoblotting experiments showed that FN1 protein expression increased and VEGFA protein expression decreased.Conclusion:EGFR-TKI drugs may produce epidermal toxicity by inhibiting MMP1 and VEGFA production in PI3K-Akt and IL-17 signaling pathways,promoting PTGS2,FN1 and HSPA5 expression and damaging HaCaT cells.