Zhang Fangfang, Cui Mingyu, Zhang Lijuan, Ma Bangzhen, Guo Feng, Wang Gang
Department of Pediatric Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, 250021, China.
Pediatr Res. 2024 Apr 24. doi: 10.1038/s41390-024-03158-8.
Hirschsprung disease (HSCR) is a congenital intestinal disease characterised by functional obstruction of the colon. Herein, we investigated the role and mechanism of the gene GFRA4 in HSCR.
GFRA4 expression in the ganglionic and aganglionic segment tissues in patients with HSCR and healthy colon tissues were detected using qRT-PCR, western blot, and immunohistochemistry. Cell proliferation, cycle distribution, apoptosis, changes in mitochondrial membrane potential, and differentiation were assessed in mouse enteric neural crest stem cells (ENCSCs) using the CCK-8 assay, EdU staining, flow cytometry, JC-1 probe, and immunofluorescence, respectively. GSEA analysis was performed to screen the signaling pathways regulated by GFRA4.
GFRA4 was downregulated in aganglionic segment tissues compared to control and ganglionic segment tissues. GFRA4 overexpression promoted proliferation and differentiation, and inhibited apoptosis in ENCSCs, while GFRA4 down-regulation had the opposite result. GFRA4 activated the hedgehog pathway. GFRA4 overexpression enhanced the expression of key factors of the hedgehog pathway, including SMO, SHH, and GLI1. However, GFRA4 down-regulation reduced their expression. An antagonist of hedgehog pathway, cyclopamine, attenuated the effect of GFRA4 overexpression on proliferation, differentiation, and apoptosis of ENCSCs.
GFRA4 promotes proliferation and differentiation but inhibits apoptosis of ENCSCs via the hedgehog pathway in HSCR.
This study confirms that GFRA4 improves the proliferation and differentiation of ENCSCs via modulation of the hedgehog pathway. This study for the first time revealed the role and the mechanism of the action of GFRA4 in HSCR, which indicates that GFRA4 may play a role in the pathological development of HSCR. Our findings may lay the foundation for further investigation of the mechanisms underlying HSCR development and into targets of HSCR treatment.
先天性巨结肠（HSCR）是一种以结肠功能性梗阻为特征的先天性肠道疾病。在此，我们研究了基因GFRA4在HSCR中的作用及机制。
采用qRT-PCR、蛋白质免疫印迹法和免疫组织化学法检测HSCR患者神经节段和无神经节段组织以及健康结肠组织中GFRA4的表达。分别使用CCK-8法、EdU染色、流式细胞术、JC-1探针和免疫荧光法评估小鼠肠神经嵴干细胞（ENCSCs）的细胞增殖、细胞周期分布、细胞凋亡、线粒体膜电位变化和分化情况。进行基因集富集分析（GSEA）以筛选受GFRA4调控的信号通路。
与对照组织和神经节段组织相比，无神经节段组织中GFRA4表达下调。GFRA4过表达促进ENCSCs增殖和分化，并抑制其凋亡，而GFRA4表达下调则产生相反结果。GFRA4激活了刺猬信号通路。GFRA4过表达增强了刺猬信号通路关键因子SMO、SHH和GLI1的表达。然而，GFRA4表达下调则降低了它们的表达。刺猬信号通路拮抗剂环杷明减弱了GFRA4过表达对ENCSCs增殖、分化和凋亡的影响。
在HSCR中，GFRA4通过刺猬信号通路促进ENCSCs增殖和分化，但抑制其凋亡。
本研究证实GFRA4通过调节刺猬信号通路改善ENCSCs的增殖和分化。本研究首次揭示了GFRA在HSCR中的作用及作用机制，表明GFRA4可能在HSCR的病理发展中发挥作用。我们的研究结果可能为进一步研究HSCR发生发展机制及治疗靶点奠定基础。
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