摘要
目的探讨缺糖缺氧/再灌注(OGD/R)对星形胶质细胞外泌体中微小RNA(miRNAs)表达的影响。方法对新生原代培养的SD大鼠大脑皮层星形胶质细胞进行缺糖缺氧(OGD)或OGD/R处理,采用超高速离心法分别收集培养基上清液和细胞内的外泌体。通过透射电镜和流式细胞术鉴定外泌体。采用Illumina Hiseq2500测序平台检测外泌体miRNAs的表达谱,从中选取表达受OGD及OGD/R处理影响的miRNAs。并用实时荧光定量聚合酶链反应(qPCR)对生物信息学分析中差异有统计学意义的miRNAs进行实验验证。使用metascape和miRWalk预测高表达miRNAs的靶基因,再将挑选出的共同靶基因在Webgestalt中进行KEGG富集分析。采用Western blot(WB)检测OGD/R及其星形胶质细胞外泌体对全反式维甲酸(ATRA)诱导分化的SH-SY5Y细胞Erk磷酸化水平的影响。结果与对照组相比,OGD 4 h处理改变了星形胶质细胞外泌体miRNAs的表达谱;而再灌注24 h后改变的miRNAs大部分恢复到对照组水平。qPCR验证显示:3种miRNAs在外泌体中的分布水平远高于细胞,而且这种富集作用可能不受OGD刺激的影响。KEGG富集分析显示:表达上调miRNAs的靶基因信号通路主要为代谢通路,包括PI3K-AKT、MAPK和mTOR信号通路等。OGD、OGD/R及正常星形胶质细胞外泌体处理均能上调相应培养条件下SH-SY5Y细胞的p-Erk1和p-Erk2水平。结论OGD处理能显著影响星形胶质细胞外泌体miRNAs的表达,其外泌体中富集分布的miRNAs均靶向作用于代谢通路,如PI3K-AKT、MAPK和mTOR等信号通路。
Objective To investigate the effect of oxygen-glucose deprivation/reperfusion(OGD/R)on the expression of miRNAs in astrocyte exosomes.Methods Primary cultured neonatal cortical astrocytes of SD rats were subjected to oxygen-glucose deprivation(OGD)or OGD/R treatment,and the culture medium supernatant and intracellular exosomes were collected by ultracentrifugation.Exosomes were identified by transmission electron microscopy and flow cytometry.The Illumina Hiseq2500 sequencing platform was used to detect the expression profile of exosomal miRNAs,and the miRNAs whose expression was affected by OGD and OGD/R were selected.Real-time fluorescence quantitative polymerase chain reaction(qPCR)was used to verify the differentially expressed miRNAs in bioinformatics analysis.Metascape and miRWalk were used to predict the target genes of the highly expressed miRNAs,and then the selected common target genes were analyzed by KEGG enrichment in Webgestalt.Western blot(WB)was used to detect the effect of exosomes derived from OGD/R and astrocytes on the phosphorylation of Erk in SH-SY5Y cells induced by all-trans retinoic acid(ATRA).Results Compared with the control group,OGD 4 h treatment changed the expression profile of exosomal miRNAs in astrocytes.However,most of the altered miRNAs after 24 h of reperfusion returned to the control level.qPCR validation showed that the distribution level of the three miRNAs in exosomes was much higher than that in cells,and this enrichment effect may not be affected by OGD stimulation.KEGG enrichment analysis showed that the target gene signaling pathways of up-regulated miRNAs were mainly metabolic pathways,including PI3K-AKT,MAPK and mTOR signaling pathways.The levels of p-Erk1 and p-Erk2 in SH-SY5Y cells treated with exosomes from OGD,OGD/R and normal astrocytes were significantly up-regulated under corresponding culture conditions.Conclusion OGD treatment can significantly affect the expression of exosome miRNAs in astrocytes.The enriched and distributed miRNAs in exosome of astrocytes target metabolic pathways,such as PI3K-AKT,MAPK,and mTOR signaling pathways.
作者
韩韦华
黄庭睿
文田甜
沈耀
HAN Weihua;HUANG Tingrui;WEN Tiantian;SHEN Yao(School of Laboratory Medicine(School of Life Sciences)/Key Laboratory of Ministry of Education for Laboratory Medicine,Wenzhou Medical University,Wenzhou,Zhejiang 325035,China)
出处
《国际检验医学杂志》
CAS
2023年第4期430-435,共6页
International Journal of Laboratory Medicine
基金
浙江省大学生科技创新活动计划(新苗人才计划)项目(2020R413038)
浙江省自然科学基金项目(LY19H090010)。
