Expression of miR-148a, miR-152 and HLA-G mRNA in choriocarcinoma cell lines.

<p>(A) Quantitative real-time PCR analysis for miR-148a (black columns) and miR-152 (white columns) in BEWO, JAR and JEG-3 cell lines, presented relative to U6. (B) Quantitative real-time PCR analysis of HLA-G mRNA in BEWO, JAR and JEG-3 cell lines, presented relative to hUBC. One out of three representative experiments is shown.</p

Expression of miR-148a, miR-152 and HLA-G mRNA in various human tissues.

<p>(A–C) Quantitative real-time PCR analysis of miR-148a (A), miR-152 (B) and the mRNA of HLA-G (C) in different healthy tissues, presented relative to miR-16 (A and B) or hUBC (C). (D–E) Ratio of HLA-G mRNA levels divided by the miR-148a levels (D) or by miR-152 levels (E). One out of three representative experiments is shown.</p

MiR-148a and miR-152 down-regulate HLA-G expression and reduce LILRB1 binding.

<p>721.221/HLA-G (G variant, A, or C variant, B) were transduced with the control miRNA, miR-148a or miR-152. The various cells were then stained with anti HLA-G mAb and analyzed by FACS. One out of three representative experiments is shown. (C) FACS histograms re HLA-G staining of 721.221/HLA-G cells not expressing the 3′UTR of HLA-G. One out of three representative experiments is shown. (D–E) LILRB1-Ig staining of 721.221/HLA-G (G variant, D, or C variant, E) and of 21.221/HLA-G cells not expressing the 3′UTR of HLA-G (F) transduced with control miRNA, with miR-148a or with miR-152. Black histogram: cells transduced with a control miRNA. Dark grey histogram: cells transduced with miR-148a. Light grey histogram: cells transduced with miR-152. One out of three representative experiments is shown. (G) Quantitative real-time PCR analysis of HLA-G mRNA levels in 721.221/HLA-G cells (G variant), presented relative to hUBC. One out of three representative experiments is shown.</p

The miRNA-mediate reduction of HLA-G expression enhances NK cell cytotoxicity.

<p>(A–B) 721.221 and 721.221/HLA-G cells, expressing either the G variant (A) or the C variant (B), transduced with control miRNA, miR-148a or miR-152, were used as target cells in killing assays against NK clones expressing LILRB1. The effector: target (E∶T) ratio was 1∶1. Values are mean ± s.d. for triplicate samples. * <i>P</i><0.02 (two-tailed Student's <i>t</i>-test). One out of three representative experiments is shown.</p

MiR-148a and miR-152 specifically target the 3′UTR of HLA-G.

<p>(A) Luciferase activity in RKO cells transduced with control miRNA (black columns), miR-148a (white columns) or miR-152 (grey columns). Results are presented relative to control reporter activity. G/C variant, reporter containing the WT sequence of the 3′UTR of HLA-G, which contains guanine or cytosine at position +3142, as indicated in the figure. Mut, reporter mutated at the seed sequence of HLA-G 3′UTR. Values are mean ± s.d. of triplicate samples. * <i>P</i><0.05 (two-tailed Student's <i>t</i>-test). One out of three representative experiments is shown. (B) FACS histogram showing GFP levels (indicative for miRNA expression) of the miRNA-infected 721.221 cells. (C–D) Quantitative real-time PCR analysis of miR-148a (C) or miR-152 (D) in 721.221/HLA-G cells expressing the G variant transduced with the relevant miRNA or a control miRNA, as indicated in the figure. Results presented relative to U6. One out of three representative experiments is shown.</p

MiR-148a and miR-152 potentially target the 3′UTR of HLA-G.

<p>(A) Immunohistochemisty performed on first trimester placental tissue sections. Right panel: negative control. Left panel: HLA-G expression is detected in the trophoblast cell columns and extravillous trophoblasts. (B) Alignment of the 3′UTR of HLA-G (G variant) with the predicted miRNAs: miR-152 left panel, miR-148a right panel. (C) Alignment of the 3′UTR of HLA-G (C variant) with the predicted miRNAs: miR-152 left panel, miR-148a right panel.</p