Regulatory mechanism of TCOF1 gene expression in human neural crest cells
10.3760/cma.j.cn114453-20220425-00128
- VernacularTitle:人神经嵴细胞中 TCOF1基因表达调控机制研究
- Author:
Rong HAN
1
;
Xin FU
;
Ran XIAO
Author Information
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
- Keywords:
Craniofacial abnormalities;
Neural crest;
Human neural crest cells;
TCOF1;
Treacher Collins syndrome
- From:
Chinese Journal of Plastic Surgery
2022;38(6):671-679
- CountryChina
- Language:Chinese
-
Abstract:
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 0.1 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.
