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

Figures & data

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

	Total	Maternal smoking during pregnancy	No maternal smoking during pregnancy
Total, n (%)	25 (100)	8 (32)	17 (68)
Gender, n (%)
Female	13 (52)	3 (37.5)	10 (59)
Male	12 (48)	5 (62.5)	7 (41)
Gestational Age in weeks, mean (SD)	38.7 (0.88)	38.4 (1.20)	38.9 (0.66)
Birthweight in g, mean (SD)	3080 (540)	3091 (682)	3071 (484)
Maternal Age in years, mean (SD)	27.3 (6.66)	24.3 (7.94)	28.8 (5.68)

Table 2 Multivariable linear regression model of individual miRNA expression

	miR-16		miR-21		miR-146a
	Reg. coeff.	p	Reg. coeff.	p	Reg. coeff.	p
Maternal Cigarette Smoking During Pregnancy
No	Reference		Reference		Reference
Yes	−0.058	<0.0001	−0.094	<0.0001	−0.039	0.003
Gender
Female	Reference		Reference		Reference
Male	−0.008	0.4	−0.003	0.9	0.001	0.9
Gestational Age, per week	0.004	0.5	0.007	0.5	0.005	0.5
Birthweight, per gram	−8.85 × 10^−06	0.4	−1.26 × 10^−06	0.9	−7.23 × 10^−06	0.5
Maternal Age, per year	−0.0003	0.7	−0.002	0.1	−0.001	0.2

Note: Each miRNA is modeled individually and the models are controlled for all co-variates in the table.

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

miRNA	Predicted target mRNA	Minimum free energy of duplex (kcal/mol)	Experimentally confirmed target mRNA	Source of target confirmation
miR-16	BCL2L2 EDA	−23.1 −25.3	Caprin-1, HGMA-1	Kaddar et al. 2009
miR-21	PLAG1 SATB1	−22.7 −23.8	PTEN PLAG1	Meng et al. 2007 Tran et al. 2007
miR-146a	TRAF6	−26.1	CCL8/MCP-2 TRAF6	Rom et al. 2010 Taganov et al. 2006; Hou et al. 2009

Note: In order to be considered a “predicted target,” the target must appear in the “top 100” targets for the respective miRNA using all of the following target prediction tools: miRanda (September 2008 Release), PicTar (based on Lall, et al. 2006) and TargetScan 5.1. Minimum free energies of the miRNA-mRNA duplexes were determined using RNA-hybrid.