The placenta plays an essential role in fetal development. Thus, understanding the role of factors determining miRNA expression variation in this tissue can shed light on the fundamental mechanisms of human developmental regulation and variability. Our study design helps to address this question by minimizing sampling effects on the results. The placentas were obtained from a single location, all processed according to the same protocol, and all collected at the same time point (birth). Sampling was further averaged in each individual by taking five independently dissected tissue fragments. For each sample, we recorded 26 demographic variables of mother and newborn infants, allowing us to assess their influence of placental miRNA expression variation.
Our results demonstrate that of three investigated non-environmental demographic variables, two substantially influence the expression of common posttranscriptional regulators, miRNAs, in the human placenta: population identity and sex of the newborn. Population has the most substantial influence explaining up to 11% of the total miRNA variance, and the relative miRNA expression divergence among four populations investigated in the study is consistent with their genetic divergence (Two-sided Mantel permutation test, Spearman’s correlation coefficients rho = 0.771, p = 0.08; Fig S3, Additional file 7) . Since genetic divergence is largely thought to reflect the accumulation of phenotypically neutral mutations , it is therefore conceivable that miRNA variation among populations is similarly influenced by the phenotypically neutral changes. This notion aligns with previous work suggesting that mRNA expression divergence includes a substantial proportion of functionally and phenotypically neutral changes [29, 34].
Regulatory effects of population-associated miRNA expression differences estimated using mRNA expression data derived mainly from the same tissue revealed significant excess of expressional repression among predicted targets for one of the six miRNA clusters only. This result appears to contrast the reported widespread population-specific downregulation of miRNA targets described in cell lines . While part of this discrepancy might be due to the limited statistical power of our study, the rest could be caused by unequal extent of the evolutionarily constraint in tissues and cell lines. As other regulators controlling multiple targets, miRNAs are under substantial evolutionary constraint [35, 36]. Assuming that most of randomly arising population-specific miRNA expression differences are non-adaptive, the ones with large regulatory effects are likely to be detrimental and will not be observed in a natural tissue, such as placenta. The artificial growth conditions of the cell lines could, however, allow the manifestation of large-scale population-associated regulatory effects of miRNA variation.
Several technical factors might have further restricted our ability to detect miRNA-driven regulation of their predicted mRNA targets. Such factors include a mismatch between computational and experimentally verified miRNA target predictions, sequestering of target mRNA out of the translational pool without degradation, and the complex and often a tissue-specific interplay between miRNAs and other regulators [37, 38]. Biologically, our study includes a limited number of populations and biological replicates and certainly does not cover all population-associated aspects of miRNA regulatory effects. Evolutionarily, as mentioned above, the proportion of population-associated miRNA differences leading to functionally meaningful effects might be minor, analogous to genetic and mRNA divergence [4, 29]. It has to be noted, however, that despite these limitations, the fact that our study reveals many population-associated miRNA expression differences indicates the importance of further studies investigating the functional significance of this phenomenon.
Previous investigation of mRNA expression in human placenta reported 41 genes with sex-associated expression, 12 of them (30%) localized on sex chromosomes . The substantial prevalence for sex chromosome localization was not, however, the case for SON-associated differences in miRNA expression: of 32 miRNAs, four (13%) localize on sex chromosomes. Sex-associated differences in miRNA expression were similarly reported in human tissues other than the placenta. Specifically, miRNA analysis across postnatal brain development revealed 40 miRNAs with significant sex-biased expression differences in the prefrontal cortex regions, 93% of them female-biased . Further, investigation of four adult human tissues – brain, colorectal mucosa, peripheral blood, and cord blood – revealing 73 female-biased and 163 male-biased expressed miRNAs . Notably, two of 32 SON-associated miRNAs overlapped with miRNAs showing corresponding sex-biased expression in the adult brain, and two overlapped with miRNAs showing such a bias in the peripheral blood. In addition to human studies, sex-biased miRNA expression was reported in mouse brain , mouse liver , rat liver , developing rat cortex , and other mammalian somatic tissues . Previous studies singled out hormonal regulation as the main driving mechanism of miRNA sex-biased expression [43, 47]. In our study, functional analysis of target genes downregulated by SON-associated miRNAs in placenta similarly reveled terms related to hormonal processes, but also in other biological pathways.
In addition to the identification of population and SON effects, our data allowed us to examine a well-characterized phenomenon of imprinted miRNA expression in the human placenta . Previously reported imprinted expression of the miRNA cluster located on chromosome 19 (C19MC) [31, 32] was also evident in our data. Previous work further linked the amplitude of the imprinting effect with the mother’s BMI . Our analysis of demographic variables indicated that the relationship between C19MC cluster imprinting and mother’s BMI depends on the mother’s weight but not the height.