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0.1 μmol/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。

abstractsObjective: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 0.1 μmol/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). (4) Compared with the negative control group, the expression of TCOF1 mRNA in H9-NCC was up-regulated after RA treatment (RA vs. Ctrl, P=0.041); HOXA3 knockdown inhibited the RA induced TCOF1 mRNA up-regulation in H9-NCC (siHOXA3 vs. siCtrl, P=0.008). Conclusions:The decreased expression of TCOF1 induces H9-NCC apoptosis, miR-654-5p down-regulates TCOF1 mRNA through post-transcriptional regulation, and RA promotes TCOF1 expression through HOXA3 mediated pathway.