人神经嵴细胞中TCOF1基因表达调控机制研究  

Regulatory mechanism of TCOF1 gene expression in human neural crest cells

作　　者：韩蓉 傅歆 肖苒 Han Rong;Fu Xin;Xiao Ran(Research Center of Plastic Surgery Hospital,Key Laboratory of Body Surface Tissue and Organ Regeneration,Chinese Academy of Medical Sciences,Beijing 100144,China)

机构地区：[1]中国医学科学院整形外科医院研究中心,中国医学科学院体表组织器官再造研究重点实验室,北京100144

出　　处：《中华整形外科杂志》2022年第6期671-679,共9页Chinese Journal of Plastic Surgery

基　　金：北京协和医学院协和青年基金(3332015154);中国医学科学院中央级公益性科研院所基本科研业务费专项资金(2018PT32015)。

摘　　要：目的探讨TCOF1 mRNA表达下降对人胚胎干细胞系H9来源神经嵴细胞(H9-NCC)凋亡的影响,并阐明对TCOF1表达发挥转录后调控作用的微RNA(miRNA)以及致畸因子视黄酸(RA)调控TCOF1表达的作用和机制。方法将人胚胎干细胞系H9诱导为H9-NCC,通过免疫荧光染色、流式细胞分析检测神经嵴细胞(NCCs)表面标志物P75、HNK-1、AP2,通过实时定量PCR(RT-qPCR)检测NCCs标志物基因SOX9、ZIC1、AP2、P75、PAX3和SOX10表达情况。采用针对TCOF1基因的慢病毒RNA干扰载体(sh-TCOF1)在H9-NCC中敲减TCOF1,并设阴性对照组(sh-Scramble),观察细胞凋亡状况。通过生物信息学分析,预测可能与TCOF1 mRNA结合的miRNA为miR-654-5p,利用该miRNA的模拟物,并设阴性对照,采用RT-qPCR方法观察对TCOF1 mRNA水平的影响。用0.1μmol/L终浓度的RA处理H9-NCC,观察对TCOF1 mRNA表达的影响。预测TCOF1上游转录因子(HOXA3)并通过RNA干扰技术敲减该转录因子,采用RT-qPCR方法检测TCOF1 mRNA表达水平,探讨RA调控TCOF1 mRNA表达的机制。结果(1)H9-NCC细胞系诱导成功,免疫荧光染色、流式细胞分析及RT-qPCR检测显示诱导产生的H9-NCC表达NCCs特异性标志物。(2)与阴性对照组相比,sh-TCOF1可以敲减H9-NCC中TCOF1的表达水平(P<0.001),敲减TCOF1后可增加细胞晚期凋亡水平(P=0.029),上调caspase3表达(P<0.001)。(3)与阴性对照组相比,miR-654-5p模拟物可降低H9-NCC中TCOF1 mRNA水平(P=0.019)。(4)与阴性对照组相比,RA处理H9-NCC后TCOF1 mRNA表达上调(P=0.041);敲减HOXA3可抑制RA对H9-NCC中TCOF1 mRNA的上调能力(P=0.008)。结论TCOF1表达降低诱导H9-NCC凋亡;miR-654-5p通过转录后调控下调TCOF1 mRNA表达;RA通过HOXA3促进TCOF1表达。Objective To investigate the effect of decreased TCOF1 mRNA level on apoptosis of human embryonic stem cell(ESC)line H9-derived neural crest cells(H9-NCC),and to elucidate the post-transcriptional regulation of TCOF1 expression by microRNAs(miRNAs),as well as the role and mechanism of the teratogenic factor retinoic acid(RA)in regulating TCOF1 expression.Methods Human H9 ESC was induced into H9-NCC and the surface markers of neural crest cells(NCCs),which included P75,HNK-1 and AP2,were detected by immunofluorescence staining and flow cytometry.The expressions of NCCs marker genes SOX9,ZIC1,AP2,P75,PAX3 and SOX10 were detected by real-time quantitative PCR(RT-qPCR).TCOF1 targeting lentiviral RNA interference vector(sh-TCOF1)was used to knock out TCOF1 in H9-NCC,and apoptosis level was measured.Negative control group was set up to observe apoptosis.MiR-654-5p was predicted as the miRNA that may bind to TCOF1 mRNA with bioinformatics analysis.The effects of miRNA mimics on TCOF1 mRNA levels was compared with negative control by RT-qPCR results.The expression of TCOF1 mRNA was detected by treating H9-NCC with 0.1μmol/L RA,an important metabolite during embryogenesis.The upstream transcription factor of TCOF1 was predicted,which contained HOXA3.HOXA3 knockdown with small interference RNA and TCOF1 mRNA change with RT-qPCR was used to explore the regulatory mechanism of TCOF1 transcription by RA signaling.Results(1)H9-NCC cell line was successfully induced.Immunofluorescence staining,flow cytometry analysis and RT-qPCR result showed that the induced H9-NCC expressed specific markers of NCCs.(2)Compared with the negative control group,sh-TCOF1 could knock down TCOF1 expression level in H9-NCC(sh-TCOF1 vs.sh-Scramble,P=0.029).TCOF1 knockdown could increase the level of late apoptosis(sh-TCOF1 vs.sh-Scramble,P=0.029)and up-regulate the expression of caspase-3(sh-TCOF1 vs.sh-Scramble,P<0.001).(3)Compared with the negative control group,miR-654-5p mimic decreased TCOF1 mRNA level in H9-NCC(miR-654-5p vs.miR-Ctrl,P=0.019).

关 键 词：颅面骨畸形 神经嵴 人神经嵴细胞 TCOF1基因 Treacher Collins综合征 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]