Effect of Myod1 on proliferation and apoptosis of oxygen-glucose-deprived SHSY5Y cells by regulating lncRNA SNHG15 and miR-24-3p
10.13481/j.1671-587X.202404013
- VernacularTitle:Myod1通过调节lncRNA SNHG15和miR-24-3p对氧糖剥夺SH-SY5Y细胞增殖及凋亡的影响
- Author:
Fangchao JI
1
;
Chenxin ZHANG
;
Zhanjun REN
;
Yunzhi PAN
;
Qi LU
;
Xingyuan SUN
Author Information
1. 齐齐哈尔医学院附属第三医院神经内科,黑龙江齐齐哈尔 161000
- Keywords:
Myogenic differentiation 1;
Small nuclear RNA host gene 15;
MicroRNA-24-3p;
SH-SY5Y cell;
Cerebral infarction
- From:
Journal of Jilin University(Medicine Edition)
2024;50(4):989-999
- CountryChina
- Language:Chinese
-
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.
