Role Of RUNX1T1 In Neural Differentiation Of Hippocampal Radial Glial Cells

RUNX1T1, also named MTG8 and ETO, involved in the development of various systems as an important transcription factor. Currently, more researches about RUNX1T1 focuse on the pathogenesis of leukemia, while on the nervous system is still few. RUNX1T1 mainly participates in proliferation of cells and neuronal differentiation during the development of nervous system as a co-repressor. This research will study the function of RUNX1T1 on hippocampal neural regeneration.Objective:The changes and effects of astrocytes, activated by transected hippocampal extracts, on hippocampal neural regeneration were observed. We investigated and detected dynamic change and location of RUNX1T1 expression in hippocampus after fimbria-fornix transaction, the expression of RUNX1T1 and neural differentiation of RGCs in vitro. And then we studied the influence of up-regulation and down-regulation of RUNX1T1 on the differentiation of hippocampal radial glial cells to make clear that if there was connection between RUNX1T1 and hippocampal neural regeneration in vivo and in vitro.Methods:(1) The rats’ hippocampal fimbria-fornix was transected and the normal and transected hippocampal extracts were prepared successfully;(2) The astrocytes were cultured in vitro, normal and transected hippocampal extracts were added into the culture mediums to activate the astrocytes, and then investigated the co-expression of BLBP, Vimentin, Nestin, Sox2 and GFAP in cells.(3) The astrocyte-conditioned medium was prepared and added into the cultured NSCs, then investigated the migration and differentiation of cells.(4) Using ELISA to detect the density of Clusterin in astrocyte-conditioned medium, we investigated the function of Clusterin in the neuronal differentiation of NSCs;(5) RUNX1T1 m RNA and protein expression were detected at the 3rd, 7th, 14 th, 21 st, and 28 th day after transecting the hippocampal fimbria-fornix of rats.(6) The RGCs were cultured in vitro, normal and transected hippocampal extracts were added into the culture mediums, then investigated the expression of RUNX1T1 and the function of hippocampal extracts in the neuronal differentiation of RGCs;(7) Lentivirus si RNA-RUNX1T1 and LV-RUNX1T1-overexpression were transfected into the cultured RGCs in vitro, respectively. The differentiation of hippocampal RGCs into neurons were detected;(8) The lentivirus si RNA-RUNX1T1 were injected into the fimbria-fornix transected hippocampal dentate gyrus, then the DCX positive neurons together with the gene and protein expression of RUNX1T1 were investigated;(9) The lentivirus LV-RUNX1T1-overexpression were injected into the normal hippocampal dentate gyrus, then the DCX positive neurons and expression of RUNX1T1 were investigated in hippocampal dentate gyrus.Results:(1) The astrocytes, activated by transected hippocampal extracts expressed the glial precursor markers BLBP and Vimentin, and neural stem cell markers Nestin and Sox2.(2) The activation astrocyte-conditioned medium induces the NSCs neurospheres lively. Transwell migration assay showed that more cells had migrated through the PET membrane in activation astrocyte-conditioned medium than other groups. More neurons were detected in activation astrocyte-conditioned medium.(3) The density of Clusterin in astrocytes-conditioned medium of the cutting group, was higher than in control and normal groups. Among them, it is highest in the group 24 h. And Clusterin could induce the NSCs differentiation into neurons.(4) The gene and protein expression of RUNX1T1 were increased gradually and reached the peak on 3rd day after transaction. The RUNX1T1 positive cells located in the hippocampal subgranular zone. The number of RUNX1T1 positive cells in the transected side was obviously higher than those in the normal side, and most on 3rd day after transaction;(5) The transected hippocampal extracts could promote the gene and protein expression of RUNX1T1, and induce the neuronal differentiation of RGCs;(6) After si RNA-RUNX1T1 transfected into hippocampal RGCs, the gene and protein expression of RUNX1T1 were decreased significantly, and the number of MAP-2 positive neurons was fewer, with shorter, fewer and less obvious processes, compared with the mock and LV-NC group. The results indicated that down-regulation of RUNX1T1 could inhibit the neuronal differentiation of hippocampal RGCs in vitro. The expression of RUNX1T1 was increased significantly, and more MAP-2 positive cells were detected, with more and longer processes after LV-RUNX1T1-overexpression transfected into hippocampal RGCs. It indicated that up-regulation of RUNX1T1 could induce the hippocampal RGCs differentiating into neurons.(7) The hippocampal fimbria-fornix transaction combined with si RNA-RUNX1T1 injection of dentate gyrus, the number of DCX positive neurons was decreased significantly, the gene and protein expression of RUNX1T1 were also decreased. This result indicated that down-regulation of RUNX1T1 could inhibit the level of neural regeneration in hippocampus. While the LV-RUNX1T1-overexpression was injected into normal hippocampal dentate gyrus, the number of DCX positive neurons and the expression of RUNX1T1 were increased significantly. It indicated that up-regulation of RUNX1T1 could promote the neural regeneration in hippocampus.Conclusions:(1) The astrocytes, activated by transected hippocampal extracts, highly expressed the glial precursor markers and neural stem cell markers, reversed to RGCs and appeared embryonic origin.(2) The activated astrocytes could promote hippocampal NSCs survival, migration and neuronal differentiation.(3) The Clusterin secreted by activated astrocytes could induce the neuronal differentiation of hippocampal NSCs.(4) The expression of RUNX1T1 was increased briefly after transaction and located in the hippocampal subgranular zone.(5) The transected hippocampal extracts could promote the expression of RUNX1T1 and the neuronal differentiation of RGCs.(6) Down-regulation of RUNX1T1 could inhibit the neuronal differentiation of cultured hippocampal RGCs, while up-regulation of RUNX1T1 could induce the hippocampal RGCs differentiating into neurons in vivo.(7) Down-regulation of RUNX1T1 could inhibit the level of neural regeneration in hippocampus, while up-regulation of RUNX1T1 could promote the neural regeneration in hippocampus in vitro.