Haplotypes of the HLA-G 3' Untranslated Region Respond to Endogenous Factors of HLA-G+ and HLA-G- Cell Lines Differentially.

The immune checkpoint HLA-G prevents maternal rejection of the fetus and contributes in cancer invasion and acceptance of allografts. The 5' and 3' regulatory regions of the HLA-G gene are polymorphic and balancing selection probably maintains this variability. It is proposed that nucleotide variations may affect the level of HLA-G expression. To investigate this issue we aimed to analyze how haplotypes of the 3' untranslated region (3'UTR) with highest worldwide frequencies, namely UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, UTR-18 and UTR-7, impact the expression of a luciferase reporter gene in vitro. Experiments performed with the HLA-G positive cell lines JEG-3 (choricarcinoma) and FON (melanoma), and with the HLA-G negative cell lines M8 (melanoma) and U251MG (glioblastoma) showed that the HLA-G 3'UTR polymorphism influences the response to endogenous cellular factors and may vary according to the cell type. UTR-5 and UTR-7 impact the activity of luciferase the most whereas UTR-2, UTR-3, UTR-4, and UTR-18 have intermediate impact, and UTR-1 has the lowest impact. These results corroborate the previous associations between amounts of plasma sHLA-G levels and 3'UTR haplotypes in healthy individuals and reinforce that 3'UTR typing may be a predictor of the genetic predisposition of an individual to express different levels of HLA-G

Impact of the 14 bp InDel on the luciferase reporter gene expression when both construction types (1Fter-4R and 1Fter-2R primers) were considered in each cell line independently or in the four cell lines in combination.

<p>Impact of the 14 bp InDel on the luciferase reporter gene expression when both construction types (1Fter-4R and 1Fter-2R primers) were considered in each cell line independently or in the four cell lines in combination.</p

Significant differences in the modulation of the expression of the luciferase reporter gene considering either the cell type or the status of HLA-G expression, with 1Fter-2R and 1Fter-4R constructions and haplotypes combined.

<p>Points indicate the mean and vertical bars indicate the 95% confidence interval. The number of experiments considering each cell line or the status of HLA-G expression is indicated between parentheses. MW: Mann-whitney test; KW: Kruskal-Wallis test. (A) Modulation of luciferase gene expression in each cell line separately. The decrease in the luciferase gene activity in U251MG (HLA-G-) and FON cells (HLA-G+) was not statistically different. (B) Modulation of luciferase gene expression in the HLA-G- (M8 + U251MG) cells <i>vs</i> HLA-G + (FON + JEG-3) cells combined.</p

Site-directed mutagenesis replacing a T with a C at position +3035 in UTR-5 and UTR-7 (1Fter-2R constructions) does not significantly influence the levels of luciferase responses.

<p>The two-tailed Mann-Whitney test was used to compare the impact of wild type UTR-5 and UTR-7 to the impact of mutated UTR-5 and UTR-7 respectively in JEG-3 and U251MG cells. Data is presented as mean <b>+/-</b> SEM.</p

Modulation of the expression of the reporter gene luciferase by 3’UTR haplotypes in four cell lines expressing HLA-G or not.

<p>Modulation of the expression of the reporter gene luciferase by 3’UTR haplotypes in four cell lines expressing HLA-G or not.</p

Modulation of luciferase reporter gene expression in FON+, JEG-3, M8 and U251MG cells considering the constructions 1Fter-2R <i>vs</i> 1Fter-4R, is not statistically different, regardless of the haplotypes.

<p>Points indicate the means and vertical bars indicate the 95% confidence interval. The number of experiments considering each 1Fter construction is indicated between parentheses. MW: Mann-whitney test; KW: Kruskal-Wallis test. (A) Modulation of luciferase gene expression in the four cell lines combined. (B) Modulation of luciferase gene expression in each cell line independently. P values for Dunn’s multiple comparison test are indicated above and under the horizontal bar for the 1Fter-2R and 1Fter-4R constructions respectively (** p value < 0.01; *** p value < 0.001).</p

Significant differences in the modulation of the expression of the luciferase reporter gene in FON+, JEG-3, M8 and U251MG cells considering each 3’UTR haplotype independently, with the 1Fter-2R and 1Fter-4R constructions combined.

<p>Points indicate the means and vertical bars indicate the 95% confidence interval. The number of experiments considering each 3’UTR haplotype is indicated between parentheses. KW: Kruskal-Wallis test; * p value <0.05; ** p value < 0.01; *** p value < 0.001. (A) Modulation of luciferase expression in the four cell lines combined. (B) Modulation of luciferase expression in the HLA-G+ (FON + JEG-3) cell lines combined. (C) Modulation of luciferase expression in the HLA-G- (M8 + U251MG) cell lines combined. In (A) and (B) luciferase expression is significantly more intensely downregulated by UTR-5 and UTR-7 than by other haplotypes.</p

<i>HLA-G</i> 3’UTR polymorphisms.

<p>(A) Variations in <i>HLA-G</i> mRNA (red) along the 3’UTR. The less frequent variants are positioned below the most frequent one in the nucleotide sequence. Polymorphic sites are framed in bold <u>when the frequency of the</u> minor allele worldwide is higher than 1%. Purple single arrows point to the three predicted polyadenylation signals. Vertical lines with horizontal arrows indicate the 5’ end of the <i>HLA-G</i> nucleotide sequences for 1Fter, 2R and 4R primers used <u>in</u> pMIR-REPORT^™ constructions. (B) Sequence comparison of the most frequent 3’UTR haplotypes (21 worldwide populations; 2n = 3870) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169032#pone.0169032.ref025" target="_blank">25</a>] analyzed in the present study. Frequencies are indicated on the right part. UTR-1 and UTR-2 differ at 5 positions (pink rectangles). UTR-6 and UTR-18 (analyzed in the present work) only differ at one position and depending on the UTR typing strategy that was used (i.e., targeting or not +3227 SNP), UTR-18 could be confused with UTR-6 in several studies.</p