Involvement of the lncRNA AFAP1-AS1/microRNA-195/E2F3 axis in proliferation and migration of enteric neural crest stem cells of Hirschsprung's disease
Involvement of the lncRNA AFAP1-AS1/microRNA-195/E2F3 axis in proliferation and migration of enteric neural crest stem cells of Hirschsprung's disease
复制标题
lncRNA AFAP1-AS1/microRNA-195/E2F3轴参与先天性巨结肠肠神经嵴干细胞的增殖和迁移
DOI：
10.1113/ep088780
复制
发表时间：
2020-10-07
影响因子：
2.7
通讯作者：
Gao, Ya
中科院分区：
文献类型：
--
作者：
Pan, Weikang;Wu, Ali;Gao, Ya
新发现这项研究的中心问题是什么？长非编码RNA（lncRNA）广泛参与先天性巨结肠（HSCR）的进展，但肌动蛋白丝相关蛋白1反义RNA1（AFAP1-AS1）这种lncRNA在HSCR中的作用此前尚未被探索过。主要发现及其重要性是什么？下调AFAP1-AS1可阻断肠神经嵴干细胞的增殖、分化、迁移和侵袭，并通过miR-195/E2F3轴促进HSCR的发生，表明AFAP1-AS可能是HSCR患者的潜在生物标志物。长链非编码 RNA (lncRNA) 与多种人类疾病有关。然而，目前尚不清楚它们是否与先天性巨结肠症 (HSCR) 中肠神经嵴干细胞 (ENCSC) 的表型有关。因此，我们设计本研究来探讨AFAP1-AS1对HSCR的致病性。利用微阵列分析和生物信息学工具筛选出 HSCR 患者中差异的 lncRNA 和 microRNA (miRNA)。应用小干扰RNA转染在ENCSC中进行功能实验。通过细胞计数试剂盒-8、5-乙炔基-2'-脱氧尿苷、Transwell实验和流式细胞术检测细胞活性。最后，进行挽救实验来检查AFAP1-AS1和miR-195以及miR-195和E2F转录因子3（E2F3）的协同功能。HSCR患者中AFAP1-AS1减少。同时，敲低AFAP1-AS1会降低细胞迁移和增殖能力，促进细胞凋亡并导致G0/G1期停滞。当miR-195上调时，E2F3减弱，抑制AFAP1-AS1会降低其与miR-195结合的能力。总而言之，AFAP1-AS1 沉默作为内源性 RNA，通过与 miR-195 相互作用改变 E2F3 表达，从而对 ENCSC 活性产生抑制作用并促进 HSCR 进展。
New FindingsWhat is the central question of this study?Long non-coding RNAs (lncRNAs) are widely involved in the progression of Hirschsprung's disease (HSCR), but the role of actin filament associated protein 1 antisense RNA1 (AFAP1-AS1), an lncRNA, in HSCR has not been explored before. What is the main finding and its importance?Downregulation ofAFAP1-AS1blocks enteric neural crest stem cell proliferation, differentiation, migration and invasion and promotes the occurrence of HSCR via themiR-195/E2F3axis, indicating thatAFAP1-ASmight be a potential biomarker for HSCR patients. Long non-coding RNAs (lncRNAs) are involved in several human disorders. Nevertheless, it remains unclear whether they are implicated in the phenotypes of enteric neural crest stem cells (ENCSCs) in Hirschsprung's disease (HSCR). Therefore, we designed this study to explore the pathogenicity ofAFAP1-AS1for HSCR. Microarray analysis and bioinformatic tools were used to screen out the differentially lncRNAs and microRNAs (miRNAs) in patients with HSCR. Small interference RNA transfection was applied to carry out functional experiments in ENCSCs. Cellular activities were detected by cell counting kit-8, 5-ethynyl-2 '-deoxyuridine, Transwell assays and flow cytometry. Finally, rescue experiments were performed to examine the cofunction ofAFAP1-AS1andmiR-195and ofmiR-195and E2F transcription factor 3 (E2F3).AFAP1-AS1was reduced in HSCR patients. Meanwhile, knockdown ofAFAP1-AS1reduced the cell migratory and proliferative capacities and facilitated cell apoptosis along with G0/G1 phase arrest.E2F3was diminished whenmiR-195was upregulated, andAFAP1-AS1inhibition reduced its ability to bind tomiR-195. Altogether,AFAP1-AS1silencing acts as an endogenous RNA by interacting withmiR-195to alterE2F3expression, thus conferring repressive effects on ENCSC activity and promoting HSCR progression.