Reply to Diet-responsive MicroRNAs Are Likely Exogenous

This is a response to a letter by Zempleni et al. (1).

We here address concerns raised in a letter to the Journal of Biological Chemistry by Zempleni et al. (1). First, exosomal encapsulation of milk miRNAs confers limited protection against degradation by the digestive system (Fig. 5 of Ref. 2). Exosomal transport into intestinal cells has only been demonstrated in vitro (3) and is not supported by our in vivo data. Second, detection of dietary miRNAs has been linked to sample contamination and oversensitivity of sequencing methods (4). Furthermore, analysis of the cited paper reveals that total plant miRNAs represent only 0.07 and 0.007% on average of small RNA sequences in human and porcine milk, respectively, suggesting they are artifactual or present in trace amounts. Third, miRNAs are only subject to degradation at high sponge site expression (5) and upon extensive sequence complementarity between miRNA and target, rarely reached in mammalian cells (6). It is unlikely that in murine tissues, sufficient complementarity is achieved to degrade miR-375 post-uptake and prevent its subsequent detection. Fourth, we studied milk miRNA uptake at early (D3) and late (D14) lactation. While we cannot exclude that uptake occurs on other days, we believe this is unlikely. Fifth, much of this research involves in vitro or non-physiological in vivo analysis (7). In our study, we never detect any significant evidence of uptake (Fig. 3 of Ref. 2). Although we cannot exclude that a low miR-375 copy number remains undetectable, analysis of the highly endocytotic liver reveals no difference in target gene expression (Fig. 4 of Ref. 2), demonstrating that low copy number is unlikely to lead to canonical miRNA gene regulation.