Non-coding RNA in raw and commercially processed milk and putative targets related to growth and immune-response

Abstract

Background 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. Results 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. Conclusion 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.

Potential Pathogenic Impact of Cow’s Milk Consumption and Bovine Milk-Derived Exosomal MicroRNAs in Diffuse Large B-Cell Lymphoma

“…Evidence has been presented that epigenetic termination of TNFAIP3 function by miR-125a and miR-125b could strengthen and prolong NF-κB activity, which promotes DLBCL lymphomagenesis [ 442 ]. Shome et al [ 443 ] identified bta-miR-125a as one of the most abundant miRs of commercial milk related to immunological roles that were not affected by pasteurization, homogenization, or heat treatment. The seed sequence of hsa-miR-125a-5p and bta-miR-125a-5p are identical.…”

“…The seed sequence of hsa-miR-125a-5p and bta-miR-125a-5p are identical. The fine-tuning of TNFAIP3 levels mediated by miR-125 expression had a striking impact on K-63 ubiquitination of TRAF2 and RIP1, IκBα degradation, p65 nuclear accumulation, and transcription of NF-κB target genes [ 443 ]. These events defined an antiapoptotic profile compatible with constitutive NF-κB activation and enhanced the fitness of B lymphoma cells.…”

“…Together, miRs of the miR-125 family play a key role in immune cell activation and oncogenesis, and constitutive activation of the NF-κB pathway in DLBCL [ 442 , 443 , 444 ]. In accordance, exosome-derived miR-125b-5p reduced the sensitivity of DLBCL to chemotherapy and rituximab [ 277 , 278 ].…”

Extracellular Vesicles from Animal Milk: Great Potentialities and Critical Issues

“…In the case of milk, another consideration must be made related to the starting material. It is known that industrialisation processes can affect the composition of milk in terms of bioactive molecules in different manners related to the applied procedure [ 30 , 31 ]. A great number of mEV evaluations have been made using raw milk, although its consumption is not allowed in some countries due to hygiene-related problems.…”

Perspectives on Bovine Milk-Derived Extracellular Vesicles for Therapeutic Applications in Gut Health

“…1D ). Additionally, a study examining whether the integrity of EVs is affected during the processing of raw milk has also been reported ( Kleinjan et al, 2021 ; Shome et al, 2021 ). A summary of these papers is shown in Table 2 .…”