Human placental microRNAs dysregulated by cadmium exposure predict neurobehavioral outcomes at birth

Abstract

Background: Prenatal cadmium (Cd) exposure has been implicated in both placental toxicity and adverse neurobehavioral outcomes. Placental microRNAs (miRNAs) may function to developmentally program adverse pregnancy and newborn health outcomes in response to gestational Cd exposure.

Methods: In a subset of the Rhode Island Child Health Study (RICHS, n = 115) and the New Hampshire Birth Cohort Study (NHBCS, = 281), we used small RNA sequencing and trace metal analysis to identify Cd-associated expression of placental miRNAs using negative binomial generalized linear models. We predicted mRNAs targeted by Cd-associated miRNAs and relate them to neurobehavioral outcomes at birth through the integration of transcriptomic data and summary scores from the NICU Network Neurobehavioral Scale (NNNS).

Results: Placental Cd concentrations are significantly associated with the expression level of five placental miRNAs in NHBCS, with similar effect sizes in RICHS. These miRNA target genes overrepresented in nervous system development, and their expression is correlated with NNNS metrics suggestive of atypical neurobehavioral outcomes at birth.

Conclusions: Gestational Cd exposure is associated with the expression of placental miRNAs. Predicted targets of these miRNAs are involved in nervous system development and may also regulate placental physiology, allowing their dysregulation to modify developmental programming of early life health outcomes.

Impact: This research aims to address the poor understanding of the molecular mechanisms governing adverse pregnancy and newborn health outcomes in response to Gestational cadmium (Cd) exposure. Our results outline a robust relationship between Cd-associated placental microRNA expression and NICU Network Neurobehavioral Scales (NNNS) at birth indicative of atypical neurobehavior. This study utilized healthy mother-infant cohorts to describe the role of Cd-associated dysregulation of placental microRNAs as a potential mechanism by which adverse neurobehavioral outcomes are developmentally programmed.

Figure 1:. Placental Cd has associations with specific miRNA expression consistent across two independent cohorts

(a) Volcano plot representing the results of the NHBCS and RICHS meta-analysis. The y-axis shows the −log₁₀(p-values) in the association of each miRNA with log₂-transformed placental cadmium concentrations. The x-axis displays the effect estimates in units of log₂ fold change in each miRNA’s transcript abundance per doubling of placental cadmium. 3 miRNAs are associated with placental cadmium accumulation at an FDR < 0.15; however, none are significant following Bonferroni correction (p-value < 1.50e-04). (b) Estimates of log₂ fold change of miRNA transcript abundances of Cd-associated DEmiRs identified compared across the RICHS and NHBCS cohort analyses and meta-analysis. MiR-10b-3p and miR-10b-5p do not hold meta-analysis estimates considering the opposing direction of effect estimates identified in the cohort level analyses. Error bars in each plot represent the estimated 95% confidence interval, size of the point represents the −log10(p-value) of the estimate, and the color indicates the study from which the data were generated.

Figure 2.. Predicted targets of Cd-associated miRNAs miR-509-3p and miR-193b-5p are overrepresented in biological pathways relevant to nervous system development.

The predicted targets of miR-509-3p and miR-193b-5p commonly reported a potential influence toward nervous system development (q-value < 0.001). MiR-193b was predicted to target 47 genes involved in nervous system development while miR-509-3p was predicted to target 44 unique targets. While all genes listed contribute to the overrepresentation of nervous system development, no predicted gene is a predicted target of multiple miRNA. Of the 91 total mRNA targets involved in nervous system development, 72 were found to be expressed at >1cpm in the RICHS placental total RNA sequencing dataset. Created with www.BioRender.com.

Figure 3.. Quality of Movement and Excitability at birth are correlated with the expression of miR-509-3p and miR-193b-5p.

Scatterplots depicting the relationship between normalized counts of miR-509-3p and miR-193b-5p with respect to individual, z-transformed NNNS metrics (y-axes) (n=114) as determined by simple linear regression. (A) There is a reported β = −0.47 ± 0.380 attenuation in quality of movement scores per-cpm change in miR-509-3p transcript abundance (p ≤ 0.01). (B) There is a reported β = −0.278 ± 0.287 attenuation in quality of movement scores per-cpm change in miR-193b-5p transcript abundance (p = 0.06). (C) There is a reported β = 0.27 ± 0.3 attenuation in excitability measurements per-cpm change in miR-193b-5p transcript abundance (p =0.09). Error for each analysis is reported as the estimated 95% confidence interval, results are considered nominally significant at a p-value threshold of p ≤ 0.1.