TNF-α刺激的IEC-6细胞源性外泌体miRNAs表达谱差异

Different expression profiles of TNF-α stimulated IEC-6 cells-derived exosomal miRNAs

摘要:

目的： 分析肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）刺激的IEC-6小肠上皮细胞源性外泌体miRNAs的表达差异。方法： 以TNF-α（终浓度50 ng/mL）刺激IEC-6细胞4 h（TNF-α-exosome组）后收集上清液，外泌体提取试剂盒结合超速离心法分离提取上清液中的外泌体；使用透射电镜、纳米微粒跟踪分析（nanoparticle tracking analysis，NTA）技术和Western blot鉴定外泌体；提取外泌体miRNAs进行高通量测序，比较control-exosome组（IEC-6细胞以不含TNF-α的无外泌体血清培养基正常孵育）和TNF-α-exosome组外泌体miRNAs表达谱差异；采用TargetScan、miRDB和miRWalk 3个软件对有显著差异表达的miRNAs进行潜在靶基因预测；Cytoscape软件绘制显著差异表达miRNAs靶基因调控网络图；DAVID网站注释工具对潜在靶基因进行功能注释，以此进一步分析潜在靶基因的生物学功能。结果： 在透射电镜下可以观察到大量外泌体结构，其分布不均匀，直径50~200 nm，呈圆形或椭圆形的脂质双层杯状囊泡；NTA检测显示其粒径主峰为141.9 nm，浓度为3.41×10⁹particle/mL；Western blot结果显示外泌体表面标志蛋白CD63表达阳性；高通量测序筛选出6个显著差异表达（差异倍数>2，P<0.05）的外泌体miRNAs。与control-exosome组比较，TNF-α-exosome组rno-miR-125a-5p（差异倍数=3.689 0）、rno-miR-351-5p（差异倍数=3.803 1）、rno-miR-125b-5p（差异倍数=2.933 0）、rno-miR-542-3p（差异倍数=2.995 6）、rno-miR-30c-5p（差异倍数=3.065 0）和miR-20a-5p（差异倍数=2.890 8）均明显下调（P=0.001，P=0.002，P=0.025，P=0.019，P=0.022，P=0.032）；运用靶基因预测网站对6个差异表达miRNAs靶基因预测，共得到潜在靶基因158个，并绘制miRNAs靶基因网络调控图；DAVID功能注释结果显示这些下调miRNAs主要参与了1个京都基因和基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路和12个基因本体论（gene ontology，GO）_生物学过程（biological process，BP）、15个GO_细胞组分（cell component，CC）、3个GO_分子功能（molecular function，MF）。结论： TNF-α诱导的IEC-6细胞来源外泌体中包含丰富的miRNAs，其表达谱有明显差异，高通量测序和相关数据库的联合运用对小肠上皮细胞外泌体miRNA的功能作用有了更深的了解，为小肠黏膜损伤相关疾病的发病机制研究提供了新视角。

Abstract:

Objective: To analyze the differential expression of IEC-6 small intestinal epithelial-derived exosomal miRNAs stimulated by TNF-α. Methods: IEC-6 cells were stimulated with TNF-α (final concentration of 50 ng/mL) for 4h (the TNF-α-exosome group), and the supernatant was collected. The exosomes in the supernatant were extracted by the total exosome isolation kit combined with ultracentrifugation. Transmission electron microscopy, nanoparticle tracking analysis (NTA) and Western blot were performed to identify exosomes. High throughput sequencing was used to compare the differential expression of exosome miRNAs between the control-exosome group (IEC-6 normally incubated with TNF-α-free medium) and the TNF-α-exosome group. Target Scan, miRDB and miRWalk were used to predict target genes of differential miRNAs, cytoscape was applied for the construction of miRNAs-target genes network and DAVID annotation tools were applied on target genes to analyze the biological functions of genes. Results: The transmission microscopy showed that there were a large number of exosomes, which were round or elliptical lipid bilayer vesicles, heterogeneously distributed, with a diameter about 50 nm to 200 nm. NTA showed that the main peak of the particle size was 141.9 nm, the concentration was 3.41×10⁹particle/mL. Western blot results showed that the exosome marker protein CD63 was positive. High-throughput sequencing screened six differentially expressed exosomal miRNAs (|log₂ (fold change) |>2, P<0.05). Compared with the control-exosome group, rno-miR-125a-5p (|log₂ (fold change) |=3.689 0) and rno-miR-351-5p (|log₂ (fold change) |=3.803 1) in the TNF-α-exosome group were significantly down-regulated, and rno-miR-125b-5p (|log₂ (fold change) |=2.933 0), rno-miR-542-3p (|log₂ (fold change) |=2.995 6), rno-miR-30c-5p (|log₂ (fold change) |=3.065 0) and rno-miR-20a-5p (|log₂ (fold change) |=2.890 8) in the TNF-α-exosome group were obvious down-regulated. Six differentially expressed miRNAs target genes were predicted by using the miRNA target gene prediction website and 158 genes were obtained and the network of six miRNAs were constructed. DAVID annotation showed that the target genes of down-regulated miRNAs were mainly involved in 1 Kyoto encyclopedia of genes and genomes (KEGG) pathway and 12 gene ontology_biological process (GO_BP), 15 gene ontology_cell component (GO_CC), 3 gene ontology_molecular function (GO_MF). Conclusion: Exosomes derived from TNF-α-induced small intestinal epithelial cells contain rich miRNAs, and their expression profiles are significantly different. With the help of high throughput sequencing and related database, more information about the exosomal miRNAs derived from small intestinal epithelial cells and its function are revealed, and a new perspective for the pathogenesis of small intestinal mucosal injury-related diseases are provided.