MiR-199a-3p/5p participated in TGF-β and EGF induced EMT by targeting DUSP5/MAP3K11 in pterygium
MiR-199a-3p/5p participated in TGF-β and EGF induced EMT by targeting DUSP5/MAP3K11 in pterygium
复制标题
MiR-199a-3p/5p 通过靶向 DUSP5/MAP3K11 参与翼状胬肉中 TGF-β 和 EGF 诱导的 EMT
DOI：
10.1186/s12967-020-02499-2
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发表时间：
2020-09-01
影响因子：
7.4
通讯作者：
Yan, Ming
中科院分区：
文献类型：
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作者：
He, Siying;Huang, Yifang;Yan, Ming
研究背景近年来，有报道称miRNA参与了翼状胬肉的发病，但其确切的发病机制尚不清楚，需要进一步研究。方法采用基因芯片技术检测翼状胬肉组织中miRNA的差异表达，并采用实时荧光定量聚合酶链反应（qRT-PCR）进行验证。培养人结膜上皮细胞（HCE）并用转化生长因子β（TGF-β）和表皮生长因子（EGF）处理，并用miR-199 a-3 p/5 p模拟物和抑制剂转染。采用免疫印迹法和免疫组化法检测HCEs中上皮间质转化（EMT）标志物。采用创伤愈合和transwell实验检测细胞迁移能力，流式细胞仪检测细胞凋亡。通过双荧光素酶报告基因分析确认miR-199 a的靶基因。结果TGF-β和EGF可诱导HCEs发生EMT，增加miR-199 a-3 p/5 p，但抑制靶基因DUSP 5和MAP 3 K11。随着EMT的发生，细胞迁移能力增强，凋亡受阻。促进miR-199 a-3 p/5 p表达可在无TGF-β和EGF的HCE中诱导EMT，而抑制miR-199 a-3 p/5 p可在TGF-β和EGF诱导的HCE中抑制EMT。综上所述，TGF-β和EGF诱导的EMT可能与miR-199 a-3 p/5 p-DUSP 5/MAP 3 K11轴有关。组织验证结果显示，与对照结膜组织相比，miR-199 a-3 p/5 p在翼状胬肉组织中的表达更高，而DUSP 5/MAP 3 K11的表达更低。此外，生物信息学分析表明miR-199 a-3 p/5 p-DUSP 5/MAP 3 K11属于MAPK信号通路。结论TGF-β和EGF通过miR-199 a-3 p/5 p-DUSP 5/MAP 3 K11轴诱导HCEs发生EMT，可能为翼状胬肉的防治提供新的靶点。
Background Recently, it has been reported that miRNA is involved in pterygium, however the exact underlying mechanism in pterygium is unrevealed and require further investigation. Methods The differential expression of miRNA in pterygium was profiled using microarray and validated with quantitative real-time polymerase chain reaction (qRT-PCR). Human conjunctival epithelial cells (HCEs) were cultured and treated with transforming growth factor beta (TGF-beta) and epidermal growth factor (EGF) and transfected with miR-199a-3p/5p mimic and inhibitor. Markers of epithelial-mesenchymal transition (EMT) in HCEs were detected using western blot and immunohistochemistry. Cell migration ability was determined using wound healing and transwell assay, while apoptosis was determined by flow cytometry. The target genes of miR-199a were confirmed by the dual-luciferase reporter assay. Results TGF-beta and EGF could induced EMT in HCEs and increase miR-199a-3p/5p but suppress target genes, DUSP5 and MAP3K11. With the occurrence of EMT, cell migration ability was enhanced, and apoptosis was impeded. Promoting miR-199a-3p/5p expression could induce EMT in HCEs without TGF-beta and EGF, while suppressing miR-199a-3p/5p could inhibit EMT in TGF-beta and EGF induced HCEs. In a word, TGF-beta and EGF induced EMT could be regulated with miR-199a-3p/5p-DUSP5/MAP3K11 axes. The validated results in tissues showed that, compared with control conjunctival tissues, miR-199a-3p/5p were more overexpressed in pterygium, while DUSP5/MAP3K11 were lower expressed. In addition, bioinformatics analysis indicated the miR-199a-3p/5p-DUSP5/MAP3K11 was belong to MAPK signalling pathway. Conclusions TGF-beta and EGF induce EMT of HCEs through miR-199a-3p/5p-DUSP5/MAP3K11 axes, which explains the pathogenesis of EMT in pterygium and may provide new targets for pterygium prevention and therapy.