Role of repressor element 1-silencing transcription factor/repressor element 1-silencing transcription factor coinhibitory factor in reparation of cortical neural axons after traumatic brain injury: an in vitro study

Abstract

Objective To explore the effects of repressor element 1-silencing transcription factor (REST)/REST coinhibitory factor (CoREST) on axonal regeneration and repairmen of mouse cortical neurons after traumatic brain injury (TBI). Methods (1) The primary cortical neurons were obtained from fetal C57BL/6 mice; one, three, 5, 7, 9, and 11 d after cultivation, miR-124 expression was detected by quantitative- (q-) PCR. (2) Neurons cultured for 5 d were divided into miR-124 mimics group, blank control group, and miR-124 inhibitor group, and miR-124 mimics, nonsense control sequences and miR-124 inhibitor were transfected, respectively; 0, 6, 12, 24, 48, and 72 h after transfection, miR-124 expression was detected by q-PCR. (3) Neurons cultured for 7 d were divided into blank control group I, oxygen glucose deprivation (OGD) model group, up-regulated miR-124+OGD model group, and down-regulated miR-124+OGD model group, and neurons in the later two groups were transfected with miR-124 mimics and miR-124 inhibitor; 48 h after transfection, OGD models in the later three groups were prepared; 0, 6, 12, 24, 48, and 72 h after OGD, miR-124 expression was detected by q-PCR; GAP-43, REST and CoREST expressions were detected by Western blotting 48 h after OGD; the REST and CoREST expressions were measured by immunofluorescent staining 48 h after OGD. Results (1) One, three, 5, and 7 d after cultivation, miR-124 expression gradually increased, and 7, 9, and 11 d after cultivation, miR-124 expression gradually decreased, with significant differences (P<0.05). (2) Twenty-four and 48 h after transfection, miR-124 expression in the miR-124 inhibitor group was significantly lower than that in the blank control group (P<0.05); 12, 24, 48 and 72 h after transfection, miR-124 expression in the miR-124 mimics group was significantly higher than that in the blank control group (P<0.05), and peak level was noted at 48 h. (3) The miR-124 expression in the OGD model group was significantly higher than that in the blank control group I at 12, 24, 48 and 72 h after OGD (P<0.05), and peak level was noted at 48 h; 0, 6, 12, 24, 48, and 72 h after OGD, the miR-124 expression in the up-regulated miR-124+OGD model group was significantly higher than that in the blank control group I (P<0.05), and peak level was noted at 48 h; Western blotting indicated that GAP-43 and CoREST gradually increased, and REST gradually decreased in blank control group I, OGD model group and down-regulated miR-124+OGD model group, with significant differences (P<0.05); neurons in the up-regulated miR-124+OGD model group had significantly lower GAP-43 and CoREST expressions, and significantly higher REST expression than those in the OGD model group (P<0.05); the results of immunofluorescence staining were consistent with those of Western blotting. Conclusion REST/CoREST, as a pair regulator, may play a key role in the repairment and regeneration of neuron axons after TBI. Key words: Traumatic brain injury; Oxygen glucose deprivation; Repressor element 1-silencing transcription factor; Repressor element 1-silencing transcription factor coinhibitory factor