Predicted binding of miR-148a, miR-148b, and miR-152 to HLA-G. The seed region of the target site is shown in bold letters, and +3142C/G is indicated by an arrow. The minimum free energy (MFE) of the RNA duplex was analyzed by RNAhybrid.

Pairwise linkage disequilibrium (r²) map of HLA-G in the Chicago families. The LD plot was made by LDPlotter.

HLA-G +3142C/G affects the targeting of miR-148a, miR-148b, and miR-152 to HLA-G and interacts with mother’s asthma status to determine risk of asthma in the child. a, Luciferase assays showing the allele-specific targeting of the three miRNAs to HLA-G in 16HBE14o- cells. pluc-HLAG-G (G), pluc-HLAG-C (C), or pluc-HLA-G-Del (Del) luciferase plasmid was cotransfected with negative control miRNA, miR-148a, miR-148b, or miR-152. At least six replicate assays were performed for each transfection. For each sample, luciferase activity was normalized by renilla activity and then normalized again by the median value of the pluc-HLAG-C plasmid within each miRNA transfection group. The P values for the difference in luciferase activity of the three plasmids are as follows. For NC miRNA transfection: G versus C, P>.05; G versus Del, P>.05. For miR-148a transfection: G versus C, P=.0002; G versus Del, P<.0001. For miR-148b transfection: G versus C, P<.0001; G versus Del, P<.0001. For miR-152 transfection: G versus C, P=.0002; G versus Del, P=.0016. b, Endogenous HLA-G expression is inhibited by miR-148a in JEG3 cells. JEG3 cells, which are +3142GG, were transfected with either negative control miRNA, miR-148a, or HLA-G siRNA as a positive control. A total of seven replicate assays were performed for each RNA. miR-148a and HLA-G siRNA significantly reduced the level of sHLA-G in JEG3 supernatant compared with the NC miRNA. sHLA-G level was normalized to the median of the HLA-G siRNA group. c, miR-148a and miR-148b levels in primary cytotrophoblast (CTB) cells from six individuals and primary bronchial epithelial (BE) cells from three individuals, determined by miRNA real-time PCR. miR-148a (P=.0016) and miR-148b (P=.0006) levels were significantly lower in CTB cells than in BE cells. d, +3142C/G interacts with maternal asthma status and is associated with asthma in the COAST children. Dashed lines are the children of mothers with asthma (N=58), and solid lines are the children of mothers without asthma (N=119). The interaction P=.0011.