Table 1.
Maternal and infant characteristics.
Fig 1.
Heatmap of top miRNA candidates up-regulated in SGA/IUGR placentas compared to control placentas.
Red indicates up-regulation and green indicates down-regulation of miRNA expression. This heatmap represents good differentiation of SGA/IUGR from AGA (individual samples listed across the x-axis) based upon the top 50 miRNAs identified as up-regulated in the SGA/IUGR group by microarray (depicted along the y-axis). Selected top candidates, including miR-10b and -363 are called out to the right of the heatmap.
Fig 2.
qRT-PCR validation of miRs of interest in human placental samples.
The left column represents comparison of AGA to SGA samples, and the right column represents comparison of AGA to IUGR samples. The first row depicts data for miR-10b (Panels 2A, 2B), the second row for miR-363 (Panels 2C, 2D), and the third row for miR-149 (Panels 2E, 2F). All data are represented as means ± SD. Asterisks indicate significance, with p-values<0.05 by Student’s t-test.
Fig 3.
miR expression after 24 hours of 50% nutrient restriction in HTR8 trophoblast cells.
(3A) miR-10b expression increases after 24 hours of NR, (3B) miR-363 expression decreases after 24 hours of NR, and (3C) mir-149 expression increases after 24 hours of NR in HTR8 trophoblast cells, but does not achieve statistical significant (p<0.07). All data are expressed as means ± SD. Asterisks denote significance, with p-value<0.05 by Student’s t-test for comparison of 24hr level expression to baseline expression.
Fig 4.
Inhibition of miR-10b in HTR8 cells affects expression of targets E-cadherin and KLF4.
(4A) miR-10b expression levels in HTR8 cells at baseline control conditions, and after transfection with inhibitor. (4B) E-cadherin gene expression by qRT-PCR decreases after inhibition of miR-10b in HTR8 cells, compared to control conditions. (4C) KLF4 gene expression by qRT-PCR increases after inhibition of miR-10b in HTR8 cells. All data expressed as means ± SD. Asterisks indicate significance with p-value<0.05, by Student’s t-test.
Fig 5.
Inhibition of miR-363 in HTR8 cells results in increased amino acid transporter expression.
(5A) miR-363 expression levels in HTR8 cells at baseline control conditions and after transfection with inhibitor. (5B) SNAT1 gene expression by qRT-PCR increases after inhibition of miR-363 in HTR8 cells, compared to at control conditions, as does SNAT2 (5C). Protein levels of SNAT1 (5D) and SNAT2 (5E) in HTR8 cells as measured by western immunoblotting also increase after inhibition of miR-363. All data expressed as means ± SD. Asterisks indicate significance with p-value<0.05, by Student’s t-test.
Fig 6.
Inhibition of miR-149 in HTR8 cells increases gene and protein expression of target gene LAT2.
(6A) miR-149 expression levels in HTR8 cells at baseline control conditions and after transfection with inhibitor. (6B) LAT2 gene expression by qRT-PCR increases after inhibition of miR-149 in HTR8 cells, compared to control conditions, as does protein expression of LAT2 (6C). All data expressed as means ± SD. Asterisks indicate significance with p-value<0.05, by Student’s t-test.
Fig 7.
MiR-10b functionally binds its complementary mRNA sequence in HTR8 cells, as measured by the dual-luciferase assay.
Renilla expression is decreased with miR-10b vector, as compared to negative control and scrambled sequence expression. Firefly expression is not different between groups, indicating similar transfection efficiency. All data expressed as means ± SD. Asterisks indicate significance with p-value<0.05, by Student’s t-test.
Table 2.
Differentially expressed miRs in AGA versus SGA placentas in validation microarray.
Fig 8.
Predicted significant pathways on putative target genes of differentially expressed miRNAs in SGA versus AGA placenta.
Predicted pathways on putative target genes (3-UTR region) of top differentially expressed miRNAs in SGA versus AGA placentas. Both the initial screening and subsequent expanded validation microarray sets consistently had these same pathways (in italics) highly represented among the differentially expressed miRs. We also highlight target genes of interest (e-cadherin, KLF4, LAT2, SNAT1, and SNAT2) validated in our studies, as well as target genes identified by pathway analyses.