Abstract:

Objective To investigate the effect of myogenic differentiation protein 1 （Myod1） on the proliferation inhibition and apoptosis of the SH-SY5Y cells induced by oxygen-glucose deprivation （OGD）， and to elucidate its mechanism. Methods Real-time quantitative fluorescence PCR （RT-qPCR） method was used to detect the mRNA levels of Myod1 and long non-coding RNA （lncRNA） small nucleolar RNA host gene 15 （SNHG15） in peripheral blood of the subjects in normal group and the patients in ischemic cerebral infarction group as well as the normal cultured SH-SY5Y cells（control group） and the cells in OGD model （OGD group）. After transfecting SH-SY5Y cells with si-Myod1， pcDNA3.0-Myod1， si-SNHG15， pcDNA3.0-SNHG15、si-NC， Vector， miR-NC， and miR-24-3p mimics， the cells were treated with OGD， and then the SH-SY5Y cells were divided into control group， OGD group， OGD+Vector group， OGD+Myod1 group， OGD+si-NC group， OGD+si-Myod1 group， OGD+si-SNHG15 group， OGD+si-SNHG15+Vector group， OGD+si-SNHG15+Myod1 group， OGD+miR-NC group， OGD+miR-mimics group， OGD+miR-mimics+Vector group， and OGD+miR-mimics+SNHG15 group. CCK-8 method was used to detect the cell activities in various groups； 5-ethynyl-2'-deoxyuridine （EdU） staining was used to detect the rates of EDU positive cells in various groups； the rates of TdT-mediated dUTP nick end labeling （TUNEL） positive cells in various groups were detected by TUNEL staining； Western blotting method was used to detect the expression levels of cleaved caspase-3， cleaved caspase-9， B-cell lymphoma 2 （Bcl-2） and Bcl-2 associated X protein （Bax） proteins in the cells in various groups； the association between Myod1 and SNHG15 was evaluated by chromatin immunoprecipitate （CHIP）； dual luciferase reporter gene experiment was used to evaluate the targeting relationships between Myod1 and SNHG15 as well as SNHG15 and miR-24-3p. Results Compared with normal control group， the expression levels of Myod1 and SNHG15 mRNA in peripheral blood of the patients in ischemic cerebral infarction group were significantly increased （P<0.05）. Compared with control group， the expression levels of Myod1 and SNHG15 mRNA in the SH-SY5Y cells in OGD group were significantly increased （P<0.05）. Compared with OGD group， the cell activities and rates of EdU positive cells in OGD+Myod1 group at 48 and 72 h were decreased （P<0.01）， and the rates of TUNEL positive cells were increased （P<0.05）； the cell activities and rates of EdU positive cells in OGD+si-Myod1 group were increased （P<0.05）， while the rates of TUNEL positive cells were decreased （P<0.01）. Myod1 binded to the promoter sequence of SNHG15. SNHG15 could absorb miR-24-3p， and there were target relatronships between Myod1 and SNHG15 as well as SNHG15 and miR-24-3p. After SNHG15 knockdown， compared with OGD group， the cell activities and rates of EdU positive cells in OGD+si-SNHG15 group at 48 and 72 h were increased （P<0.01）， and the rates of TUNEL positive cells were decreased （P<0.01）， the expression levels of Bax， cleaved caspase-3 and cleaved caspase-9 proteins were decreased （P<0.01）， and the expression levels of Bcl-2 protein were increased （P<0.01）. Compared with OGD+si-SNHG15 group， the cell activities and rates of EdU positive cells in OGD+si-SNHG15+Myod1 group at 48 and 72 h were decreased （P<0.05）， the rates of TUNEL positive cells were （P<0.05）， the expression levels of Bax， cleaved caspase-3， and cleaved caspase-9 proteins were increased （P<0.05）， and the expression levels of Bcl-2 were decreased （P<0.05）. After over-expression of miR-24-3p and SNHG15， compared with OGD group， the cell activities and rates of EdU positive cells in OGD+miR-mimics group at 48 and 72 h were increased （P<0.01）， the rates of TUNEL positive cells were significantly decreased （P<0.01）， the protein expression levels of Bax， cleaved caspase-3 and cleaved caspase-9 were decreased （P<0.05）， and the expression levels of Bcl-2 were increased （P<0.01）. Compared with OGD+miR-mimics group， the cell activities and rates of EdU positive cells in OGD+miR-mimics+SNHG15 group at 48 and 72 h were decreased （P<0.05）， and the rates of TUNEL positive cells were increased （P<0.05）， the expression levels of Bax， cleaved caspase-3 and cleaved caspase-9 proteins were increased （P<0.05）， and the expression levels of Bcl-2 protein were decreased （P<0.05）. Conclusion Myod1 can promote the proliferation inhibition and apoptosis of OGD-induced SH-SY5Y cells by binding to the SNHG15 promoter region and then absorbing miRNA-24.

Key words: Myogenic differentiation 1, Small nuclear RNA host gene 15, MicroRNA-24-3p, SH-SY5Y cell, Cerebral infarction