不同乳腺癌细胞株胞外囊泡特性和差异miRNA自噬通路靶基因分析

EVs Characteristics and Differential miRNA Autophagy Pathway Target Genes in Different Breast Cancer Cell Lines

目的 研究不同乳腺癌细胞株EVs特性和所含miRNA的数量和功能差异。方法 采用无血清培养基对正常乳腺上皮细胞HMEC和乳腺癌细胞株MDA-MB-231、MCF-7进行培养,通过纳米粒径分析对胞外囊泡颗粒大小和含量分布,利用投射电镜观察胞外囊泡形态,采用Western bolt检测胞外囊泡膜蛋白标记物CD63。通过转录组测序对胞外囊泡中miRNA进行差异分析,利用GO和KEGG分析差异miRNA靶基因。结果 通过多峰粒径分析显示,HMEC与MDA-MB-231、MCF-7胞外囊泡粒径分布、含量有所不同。三株细胞均可检测到具有典型特征胞外囊泡和标记物CD63的表达。HMEC、MCF-7、MDA-MB-231胞外囊泡中共有已知miRNA分别为613、333、698个。以HMEC为对照,进行差异比较分析发现,MCF-7的胞外囊泡中显著上调和下调的miRNA分别为108个和86个,MDA-MB-231的胞外囊泡中显著上调和下调的miRNA分别为135个和246个。GO和KEGG富集分析发现差异miRNA的靶基因涉及多种生物学功能,尤其细胞自噬。结论 不同乳腺癌细胞株胞外囊泡从直径、数量分布、所含miRNA都与正常乳腺上皮细胞存在差异,不同乳腺癌细胞株之间也存在差异。乳腺癌细胞株胞外囊泡的差异miRNA可能对其自身或作用的靶细胞的自噬水平产生不同影响,其机制有待进一步研究揭示。

Objective To study the EVs characteristics and the number and function of miRNA in different breast cancer cell lines.Methods Normal breast epithelial cells HMEC and breast cancer cell lines MDA-MB-231 and MCF-7 were cultured using serum-free medium.The size and content distribution of extracellular vesicles were analyzed by nanoparticle particle size analysis,and the morphology of extracellular vesicles was observed by projection electron microscopy.Extracellular vesicle membrane protein marker CD63 was detected by western bolt.Differential miRNA in extracellular vesicles was analyzed by transcriptome sequencing,and differential miRNA target genes were analyzed by GO and KEGG.Results Multi-modal particle size analysis showed that the particle size distribution and content of extracellular vesicles of HMEC were different from MDA-MB-231 and MCF-7.Extracellular vesicles with typical characteristics and the expression of CD63 were detected in all three cell lines.There were 613,333 and 698 known mirnas in extracellular vesicles of HMEC,MCF-7 and MDA-MB-231,respectively.Compared with HMEC,108 and 86 mirnas were significantly up-regulated and down-regulated in extracellular vesicles of MCF-7,and 135 and 246 mirnas were significantly up-regulated and down-regulated in extracellular vesicles of MDA-MB-231,respectively.GO and KEGG enrichment analysis showed that the target genes of different mirnas were involved in multiple biological functions,especially autophagy.Conclusion The diameter,quantity distribution and miRNA content of extracellular vesicles of different breast cancer cell lines are different from those of normal breast cancer epithelial cells,and there are also differences among different breast cancer cell lines.The different miRNA in extracellular vesicles of breast cancer cell lines may have different effects on the[LM]autophagy level of the vesicles themselves or their target cells,and the mechanism needs to be further studied.