背景:脊髓发育失调可导致严重的神经元损伤和功能障碍,对新生儿造成严重的健康问题。miRNA-138似乎对增殖至关重要,分化,和细胞凋亡。然而,miRNA-138和下游分子在胚胎脊髓发育中的调控仍然难以捉摸。本实验的目的是确定miRNA-138或RNA干扰(RNAi)过表达是否可以调节胎鼠脊髓的发育。
方法:构建pLenti-III-mico-GFP(miRNA-138开放阅读框[ORF])和pLenti-III-miR-Off(miRNA-138短发夹)两种质粒载体,并在妊娠第14天注射入大鼠尾静脉。苏木精-伊红(HE)染色观察细胞形态。QRT-PCR,蛋白质印迹,和免疫染色证实了miRNA-138与下游分子Sonichedgehog(Shh)之间的调节关系。
结果:与对照组相比,miRNA-138的过表达显着增加了神经元的再生并减少了神经元的凋亡。miRNA-138的沉默显著增加神经元凋亡和脊髓萎缩。此外,miRNA-138ORF处理有效地增加了miRNA-138的表达水平并且还上调了Shh的水平。相对而言,miRNA-138的敲减下调骨髓增生异常区域的Shh水平。
结论:这些发现表明miRNA-138过表达可以保护胎鼠的脊髓发育。潜在机制与Shh表达有关。本研究为促进胚胎脊髓发育的分子机制提供了新的策略。
BACKGROUND: Dysregulation of spinal cord development can lead to serious neuronal damage and dysfunction, causing significant health problems in newborns. MiRNA-138 appears to be crucial for proliferation, differentiation, and apoptosis of cells. However, the regulation of miRNA-138 and downstream molecules in embryonic spinal cord development remain elusive. The aim of this experiment is to determine whether overexpression of miRNA-138 or RNA interference (RNAi) can regulate the development of spinal cord in fetal rats.
METHODS: Two plasmid vectors including pLenti-III-mico-GFP (miRNA-138 open reading frame [ORF]) and pLenti-III-miR-Off (miRNA-138 short hairpin) were constructed and injected into the tail vein of rats on the 14th day of pregnancy. Hematoxylin-eosin (HE) staining was used to observe the cell morphology. QRT-PCR, Western blot, and immunostaining confirmed the regulatory relationship between miRNA-138 and downstream molecules sonic hedgehog (Shh).
RESULTS: Overexpression of miRNA-138 increased neuron regeneration significantly and decreased neuronal apoptosis when compared with the control. Silencing of miRNA-138 increased neuronal apoptosis and spinal cord atrophy significantly. Furthermore, miRNA-138 ORF treatment effectively increased the expression level of miRNA-138 and also upregulated the level of Shh. Comparatively, knockdown of miRNA-138 downregulated Shh levels in myelodysplastic regions.
CONCLUSIONS: These findings indicated that miRNA-138 overexpression could protect the spinal cord development of fetal rats, and the underlying mechanisms were associated with Shh expression. The present study provides a novel strategy to promote the molecular mechanism of embryonic spinal cord development.