The serum of healthy controls and SCI patients at the acute stage were collected for exosomes isolation, and the two different exosomes were used to treat human astrocytes (HA). The cell viability, apoptosis, and cycle were determined, and the expression of the related proteins were detected by western blot. Then, the two different exosomes were sent for tsRNA sequencing, and four significant known differentially expressed tsRNAs (DE-tsRNAs) were selected for RT-qPCR validation. Finally, tRT-41 was chosen to further explore its roles and related mechanisms in SCI.
After sequencing, 21 DE-tsRNAs were identified, which were significantly enriched in pathways of Apelin, AMPK, Hippo, MAPK, Ras, calcium, PI3K-Akt, and Rap1. RT-qPCR showed that tRF-41 had higher levels in the SCI-associated exosomes. Compared with the control HA, healthy exosomes did not significantly affect the growth of HA cells, but SCI-associated exosomes inhibited viability of HA cells, while promoted their apoptosis and increased the HA cells in G2/M phase; but tRF-41 inhibitor reversed the actions of SCI-associated exosomes. Additionally, SCI-associated exosomes, similar with tRF-41 mimics, down-regulated IGF-1, NGF, Wnt3a, and β-catenin, while up-regulated IL-1β and IL-6; but tRF-41 inhibitor had the opposite actions, and reversed the effects induced by SCI-associated exosomes.
SCI-associated exosomes delivered tRF-41 may inhibit the growth of HA through regulating Wnt/ β-catenin pathway and inflammation response, thereby facilitating the progression of SCI.
方法:收集健康对照组和急性期SCI患者的血清进行外泌体分离,两种不同的外泌体用于治疗人星形胶质细胞(HA)。细胞活力,凋亡,并确定了周期,用westernblot检测相关蛋白的表达。然后,两个不同的外泌体被送去进行tsRNA测序,选择四种显著的已知差异表达的tsRNA(DE-tsRNA)用于RT-qPCR验证。最后,选择tRT-41进一步探讨其在SCI中的作用及相关机制。
结果:测序后,鉴定了21个DE-tsRNAs,它们在Apelin途径中显著富集,AMPK,河马,MAPK,拉斯,钙,PI3K-Akt,Rap1RT-qPCR显示tRF-41在SCI相关的外泌体中具有较高的水平。与对照HA相比,健康的外泌体没有显著影响HA细胞的生长,但SCI相关的外泌体抑制HA细胞的活力,同时促进其凋亡并增加处于G2/M期的HA细胞;但tRF-41抑制剂逆转了SCI相关外泌体的作用。此外,SCI相关外泌体,类似于TRF-41模仿,下调IGF-1,NGF,WNT3a,和β-连环蛋白,而上调IL-1β和IL-6;但tRF-41抑制剂具有相反的作用,并逆转SCI相关外泌体诱导的效应。
结论:SCI相关外泌体递送tRF-41可能通过调节Wnt/β-catenin通路和炎症反应来抑制HA的生长,从而促进SCI的进展。