Maternal cigarette smoking during pregnancy is associated with downregulation of miR-16, miR-21, and miR-146a in the placenta

Abstract

Maternal cigarette smoking during pregnancy is associated with poor fetal outcome and aberrant miRNA expression is associated with adverse pregnancy outcomes. In 25 human placentas, we analyzed the expression of four candidate miRNA previously implicated in growth and developmental processes: miR-16, miR-21, miR-146a, and miR-182, and used three immortalized placental cell lines to identify if specific components of cigarette smoke were responsible for alterations to miRNA expression. miR-16, miR-21, and miR-146a were significantly downregulated in cigarette smoke-exposed placentas compared to controls. TCL-1 cells exposed to both nicotine and benzo(a)pyrene exhibited significant, dose-dependent downregulation of miR-146a. These results suggest that miR-146a is particularly responsive to exposures, and that smoking may elicit some of its downstream effects through alteration of miRNA expression.

Acknowledgements

Many thanks to Devin Koestler, Charlotte Wilhelm, Amanda Filiberto and Luc Gagne for their insightful comments and criticisms during the preparation of this manuscript and to Keila Veiga and Alyse LaLiberte for placenta sample and medical history collection.

Financial Support

This work was funded by NIH grants from the NCRRP20RR018728, the NIEHS P42ES013660 and T32ES007272 (MAM) and the Flight Attendants Medical Research Institute Young Clinical Scientist Award.

Figures and Tables

Figure 1 Maternal cigarette smoking during pregnancy is associated with downregulation of miR-16, miR-21 and miR-146a. Quantitative RT-PCR analysis was used to examine the expression of the mature forms of miR-16 (A), miR-21 (B), miR-146a (C) and miR-182 (D), in primary placenta tissue samples from infants whose mothers smoked cigarettes (CS) during pregnancy (n = 8) and infants whose mothers did not smoke during pregnancy (No CS, n = 17). *indicates p < 0.01 and **indicates p < 0.0001 as determined by t-test.

Figure 2 miR-146a is downregulated in TCL-1 cells exposed to nicotine and benzo(a)pyrene. Three placental cell lines (HTR8, 3A and TCL1) were exposed to increasing doses of nicotine (A) and benzo(a)pyrene (B) for 6 days, and the expression of miR-146A was determined through qRT-PCR. Error bars indicated standard error of the mean, *indicates a significant downregulation of miR-146a across doses of nicotine (ANOVA, p < 0.03). **indicates a significant downregulation of miR-146a across doses of benzo(a)pyrene (ANOVA, p < 0.006).

Table 1 Demographics of the study population

Table 2 Multivariable linear regression model of individual miRNA expression

Table 3 mRNA targets predicted and/or experimentally validated for miR-16, miR-21 and miR-146a