Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
Int J Mol Sci. 2023 Jul 29;24(15):12166. doi: 10.3390/ijms241512166.
Exosomes are key mediators of intercellular communication. They are secreted by most cells and contain a cargo of protein-coding genes, long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), which modulate recipient cell behavior. Herein, we collected blood samples from Holstein cows at days 30 (mid-lactation) and 250 (dry period) of pregnancy. Prolactin, follicle-stimulating hormone, luteinizing hormone, estrogen, and progesterone levels showed an obvious increase during D250. We then extracted exosomes from bovine blood samples and found that their sizes generally ranged from 100 to 200 nm. Further, Western blotting validated that they contained CD9, CD63, and TSG101, but not calnexin. Blood-derived exosomes significantly promoted the proliferation of mammary epithelial cells, particularly from D250. This change was accompanied by increased expression levels of proliferation marker proteins PCNA, cyclin D, and cyclin E, as detected by EdU assay, cell counting kit-8 assay, and flow cytometric cell cycle analysis. Moreover, we treated mammary epithelial cells with blood-derived exosomes that were isolated from the D30 and D250 periods. And RNA-seq of two groups of cells led to the identification of 839 differentially expressed genes that were significantly enriched in KEGG signaling pathways associated with apoptosis, cell cycle and proliferation. In bovine blood-derived exosomes, we found 12,747 protein-coding genes, 31,181 lncRNAs, 9374 transcripts of uncertain coding potential (TUCP) candidates, and 460 circRNAs, and 32 protein-coding genes, 806 lncRNAs, 515 TUCP candidates, and 45 circRNAs that were differentially expressed between the D30 and D250 groups. We selected six highly expressed and four differentially expressed circRNAs to verify their head-to-tail splicing using PCR and Sanger sequencing. To summarize, our findings improve our understanding of the key roles of blood-derived exosomes and the characterization of exosomal circRNAs in mammary gland development.
外泌体是细胞间通讯的关键介质。它们由大多数细胞分泌，包含货物的蛋白质编码基因、长非编码 RNA（lncRNA）和环状 RNA（circRNA），调节受体细胞的行为。在此，我们收集了荷斯坦奶牛妊娠第 30 天（泌乳中期）和第 250 天（干奶期）的血液样本。在 D250 时，催乳素、卵泡刺激素、黄体生成素、雌激素和孕酮水平明显升高。然后，我们从牛血液样本中提取外泌体，发现它们的大小通常在 100 到 200nm 之间。进一步的 Western blot 验证了它们包含 CD9、CD63 和 TSG101，但不包含 calnexin。来自血液的外泌体显著促进了乳腺上皮细胞的增殖，特别是来自 D250 的细胞。这种变化伴随着增殖标记蛋白 PCNA、细胞周期蛋白 D 和细胞周期蛋白 E 的表达水平增加，通过 EdU 检测、细胞计数试剂盒-8 检测和流式细胞术细胞周期分析检测到。此外，我们用从 D30 和 D250 期分离的血液来源的外泌体处理乳腺上皮细胞。对两组细胞的 RNA-seq 导致鉴定出 839 个差异表达基因，这些基因在与凋亡、细胞周期和增殖相关的 KEGG 信号通路中显著富集。在牛血液来源的外泌体中，我们发现了 12747 个蛋白质编码基因、31181 个 lncRNA、9374 个不确定编码潜能（TUCP）候选转录本和 460 个 circRNA，以及 32 个蛋白质编码基因、806 个 lncRNA、515 个 TUCP 候选转录本和 45 个 circRNA，它们在 D30 和 D250 组之间存在差异表达。我们选择了六个高表达和四个差异表达的 circRNA，通过 PCR 和 Sanger 测序验证它们的头尾拼接。总之，我们的研究结果提高了我们对血液来源的外泌体的关键作用以及外泌体 circRNA 在乳腺发育中的特征的认识。
