被α-synuclein激活的小胶质细胞通过外泌体转运miR-19a-3p抑制神经细胞自噬

Microglia Activated byα-Synuclein Inhibits Autophagy in SH-SY5Y Cells by Transporting miR-19a-3p Via Exosomes

【目的】研究被过表达α-synuclein的SH-SY5Y细胞外泌体刺激后的小胶质细胞重新分泌的外泌体(SNCA-HM Exo)的miRNAs成分变化,及其对神经细胞自噬的影响。【方法】收集小胶质细胞外泌体进行miRNAs芯片分析和PCR检测,筛选出差异表达的miRNAs,对其靶基因进行KEGG及GO分析,筛选出与PI3K/Akt/信号通路相关的miRNAs。将SH-SY5Y细胞分为四组,分别为Con-HM Exo组,Con-HM Exo+miR-19a mimic组,SNCA-HM Exo组以及SNCA-HM Exo+miR-19a-3p inhibitor组,采用Western Blot进行验证试验。【结果】芯片分析和PCR共筛选出15个差异表达的miRNAs。KEGG及GO分析发现,其靶基因富集分值最高的是PI3K/Akt信号通路,其中miR-19a-3p调控PI3K/Akt信号通路的靶基因包括PTEN等。验证试验发现,miR-19a模拟物组及SNCA-HM Exo组与对照组比较PTEN、LC3Ⅱ/Ⅰ下降,p-Akt/Akt、p-mTOR/mTOR、P62升高(P<0.05),而miR-19a抑制剂可逆转这一作用(P<0.05)。【结论】SNCA-HM Exo可通过miR-19a-3p调控PTEN,激活PI3K/Akt/mTOR信号通路,进而抑制神经细胞自噬。

【Objective】To study the changes of miRNAs in exosomes secreted from microglia after being activated by exosomes of SH-SY5Y cells overexpressing α-synuclein(SNCA-HM Exo)and their effects on autophagy of SH-SY5Y cells.【Methods】Microglia exosomes were collected for miRNAs microarray analysis and PCR detection,and the differentially expressed miRNAs were screened out.Their target genes were analyzed by Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO).MiRNAs and their target genes related to PI3K/Akt/signaling pathway were screened out and verified by Western blot.The SH-SY5Y cells were divided into four groups:Con-HM Exo,Con-HM Exo+miR-19a mimic,SNCA-HM Exo and SNCA-HM Exo+miR-19a-3p inhibitor.【Results】Fifteen differentially expressed miRNAs were screened out by microarray analysis and PCR.KEGG and GO analysis showed that PI3K/Akt signaling pathway had the highest enrichment score of target genes,and PTEN was one of the target genes regulated by mir-19a-3p.We found that,compared with the control group,the expression of PTEN and LC3Ⅱ/I were decreased,while the expression of p-Akt/Akt,p-mTOR/mTOR and p62 were increased in the miR-19a-3p mimic group and the SNCA-HM Exo group(P<0.05).However,miR-19a-3p inhibitor could reverse this effect(P<0.05).【Conclusion】SNCA-HM Exo regulates PTEN through miR-19a-3p,activates PI3K/Akt/mTOR signaling pathway,and then inhibits autophagy in SHSY5Y cells.