Bioinformatics and Computational Biology Program, Iowa State University, Ames, IA, USA.
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA.
BMC Genomics. 2021 Oct 17;22(1):749. doi: 10.1186/s12864-021-07964-w.
Bovine milk contains extracellular vesicles (EVs) that play a role in cellular communication, acting in either an autocrine, paracrine, or an exocrine manner. The unique properties of the EVs protect the cargo against degradation. We profiled the ncRNAs (non-coding RNA) present in the EVs from seven dairy products - raw whole milk, heat-treated skim milk, homogenized heat-treated skim milk, pasteurized homogenized skim milk, pasteurized heavy whipping cream, sweet cream buttermilk and cultured buttermilk with four replicates each, obtained at different processing steps from a commercial dairy plant. EVs and their cargo were extracted by using a validated commercial kit that has been shown to be efficient and specific for EVs. Further, to find the annotation of ncRNAs, we probed bovine and other organism repositories(such as miRBase, miRTarBase, Ensemble) to find homolog ncRNA annotation in case the annotations of ncRNA are not available in Bos Taurus database.
Specifically, 30 microRNAs (miRNAs), were isolated throughout all the seven milk samples, which later when annotated with their corresponding 1546 putative gene targets have functions associated with immune response and growth and development. This indicates the potential for these ncRNAs to beneficially support mammary health and growth for the cow as well as neonatal gut maturation. The most abundant miRNAs were bta-miR-125a and human homolog miR-718 based on the abundance values of read count obtained from the milk samples.bta-miR-125a is involved in host bacterial and viral immune response, and human homolog miR-718 is involved in the regulation of p53, VEGF, and IGF signaling pathways, respectively. Sixty-two miRNAs were up-regulated and 121 miRNAs were down-regulated throughout all the milk samples when compared to raw whole milk. In addition, our study explored the putative roles of other ncRNAs which included 88 piRNAs (piwi-interacting RNA), 64 antisense RNAs, and 105 lincRNAs (long-intergenic ncRNAs) contained in the bovine exosomes.
Together, the results indicate that bovine milk contains significant numbers of ncRNAs with putative regulatory targets associated with immune- and developmental-functions important for neonatal bovine health, and that processing significantly affects the ncRNA expression values; but statistical testing of overall abundance(read counts) of all miRNA samples suggests abundance values aren't much affected. This can be attributed to the breakage of exosomal vesicles during the processing stages. It is worth noting, however, that these gene regulatory targets are putative, and further evidence could be generated through experimental validation.
牛奶中含有细胞外囊泡（EVs），它们在细胞通讯中发挥作用，以自分泌、旁分泌或外分泌的方式发挥作用。EVs 的独特特性保护货物免受降解。我们对来自 7 种乳制品的 EVs 中存在的 ncRNAs（非编码 RNA）进行了分析 - 生全脂牛奶、热处理脱脂牛奶、均质热处理脱脂牛奶、巴氏消毒均质脱脂牛奶、巴氏消毒重搅打奶油、甜奶油白脱牛奶和培养白脱牛奶，每个处理步骤各有 4 个重复，从商业乳制品厂获得。使用经过验证的商业试剂盒提取 EVs 和它们的货物，该试剂盒已被证明对 EVs 有效且具有特异性。此外，为了找到 ncRNA 的注释，我们探测了牛和其他生物体库（如 miRBase、miRTarBase、Ensemble），以找到 ncRNA 注释的同源物，以防在 Bos Taurus 数据库中找不到 ncRNA 的注释。
具体来说，在所有 7 种牛奶样本中都分离出了 30 个 microRNAs（miRNAs），后来在对其对应的 1546 个假定基因靶标进行注释时，这些 miRNAs 具有与免疫反应和生长发育相关的功能。这表明这些 ncRNAs 有可能对奶牛的乳腺健康和生长以及新生肠道成熟有益。根据从牛奶样本中获得的读数计数的丰度值，最丰富的 miRNAs 是 bta-miR-125a 和人类同源物 miR-718。bta-miR-125a 参与宿主细菌和病毒免疫反应，而人类同源物 miR-718 分别参与 p53、VEGF 和 IGF 信号通路的调节。与生全脂牛奶相比，所有牛奶样本中 62 个 miRNA 上调，121 个 miRNA 下调。此外，我们的研究还探讨了其他 ncRNAs 的假定作用，包括 88 个 piRNAs（piwi-interacting RNA）、64 个反义 RNA 和 105 个 lincRNAs（长间隔 ncRNAs），这些 RNA 存在于牛的外泌体中。
总之，结果表明，牛奶中含有大量具有免疫和发育功能的假定调节靶标的 ncRNAs，这些功能对新生牛的健康很重要，并且加工过程显著影响 ncRNA 的表达值；但是，对所有 miRNA 样本的总体丰度（读数计数）进行统计检验表明，丰度值受影响不大。这可以归因于在加工阶段外囊泡的破裂。值得注意的是，这些基因调控靶标是假定的，通过实验验证可以产生更多证据。
