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  

Effect of Myod1 on proliferation and apoptosis of oxygenglucose-deprived SHSY5Y cells by regulating lncRNA SNHG15 and miR-24-3p

作　　者：冀方超 张晨昕 任占军 潘云志 逯琦 孙兴元 JI Fangchao;ZHANG Chenxin;REN Zhanjun;PAN Yunzhi;LU Qi;SUN Xingyuan(Department of Neurology,Third Affiliated Hospital,Qiqihar Medical University,Qiqihar 161000,China)

机构地区：[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

出　　处：《吉林大学学报（医学版）》2024年第4期989-999,共11页Journal of Jilin University：Medicine Edition

基　　金：黑龙江省卫健委基金项目(20210303070198)。

摘　　要：目的:探讨肌源性分化蛋白1(Myod1)对氧糖剥夺(OGD)诱导的SH-SY5Y细胞增殖抑制和凋亡的影响,并阐明其作用机制。方法:采用实时荧光定量PCR(RT-qPCR)法检测正常对照组研究对象和缺血性脑梗死组患者外周血及正常培养的SH-SY5Y细胞(对照组)和OGD细胞模型(OGD组)细胞中Myod1和长链非编码RNA(lncRNA)小核仁RNA宿主基因15(SNHG15)mRNA表达水平。分别采用si-Myod1、pcDNA3.0-Myod1、si-SNHG15、pcDNA3.0-SNHG15、si-NC、空载质粒(Vector)、miR-NC和miR-24-3p模拟物(miR-mimics)质粒转染SH-SY5Y细胞后,进行OGD处理,将SH-SY5Y细胞分为对照组、OGD组、OGD+Vector组、OGD+Myod1组、OGD+si-NC组、OGD+si-Myod1组、OGD+si-SNHG15组、OGD+si-SNHG15+Vector组、OGD+si-SNHG15+Myod1组、OGD+miR-NC组、OGD+miR-mimics组、OGD+miR-mimics+Vector组和OGD+miR-mimics+SNHG15组。采用CCK-8法检测各组细胞活性,采用5-乙炔基-2'-脱氧尿苷(EdU)染色法检测各组EdU阳性细胞率,采用原位末端转移酶标记(TUNEL)法检测各组TUNEL阳性细胞率,采用Western blotting法检测各组细胞中裂解的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved caspase-3)、裂解的含半胱氨酸的天冬氨酸蛋白水解酶9(cleaved caspase-9)、B细胞淋巴瘤2(Bcl-2)和Bcl-2相关X蛋白(Bax)蛋白表达水平。染色质免疫共沉淀(CHIP)法评估Myod1和SNHG15之间的关联。双荧光素酶报告基因实验评估Myod1与SNHG15及SNHG15与miR-24-3p的靶向关系。结果:与正常对照组比较,缺血性脑梗死组患者外周血中Myod1和SNHG15 mRNA表达水平均升高(P<0.05)。与对照组比较,OGD组细胞中Myod1和SNHG15 mRNA表达水平均明显升高(P<0.05)。与OGD组比较,48和72 h时OGD+Myod1组细胞活性和EdU阳性细胞率均降低(P<0.01),TUNEL阳性细胞率升高(P<0.01);OGD+si-Myod1组细胞活性和EdU阳性细胞率均升高(P<0.01),TUNEL阳性细胞率降低(P<0.01)。Myod1可与SNHG15的启动子序列结合。SNHG15可吸附miR-24-3p,Myod1与SNHG15及SNHG15与miR-24-3Objective: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 TdTmediated 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 ex

关 键 词：肌源性分化蛋白1 小核仁RNA宿主基因15 微小RNA-24-3p SH-SY5Y细胞 脑梗死 

分 类 号：R743[医药卫生—神经病学与精神病学] R363.2[医药卫生—临床医学]