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    • On the other hand cell lines VUB and

    2018-10-20

    On the other hand, cell lines VUB01 and VUB14 showing the highest methylation grade, expressed more HLA-G mRNA compared to the low methylated cell lines VUB19_DM1 and VUB31_FSHD suggesting that next to promoter CpG methylation other mechanisms were involved in HLA-G expression control. Cis-regulatory sequences as well as polymorphisms within the atypical HLA-G promoter were described to be implicated in the transcriptional gene control but were not investigated in this study (Moreau et al., 2009; Castelli et al., 2014; Ferreira et al., 2016). The fact that we detected the isoforms -G1, -G2/4, -G3 and -G5 in all hESC samples is in contradiction with our previous study (Verloes et al., 2011), where we had found a different isoform expression pattern within the three lines VUB01, VUB02 and VUB04_CF. The most likely explanation for this discrepancy is the different sensitivity of the methods used in these two studies. Although we used the same primer sequences for both studies, we now used a fluorescence-based fragment analysis instead of gel electrophoresis with ethidium bromide, which enhances the sensitivity to PCR products present in low quantities. Apart from the transcriptional control described above, HLA-G expression is also regulated at the post-transcriptional level by its 3′UTR sequence. This sequence contains several polymorphic sites and regulatory elements that influence HLA-G transcript expression (Castelli et al., 2010, 2014; Donadi et al., 2011). Because we found that the methylation patterns could not explain the differences in expression levels in our hESC lines, we set to investigate these polymorphisms in the 3′UTR sequence and their binding miRNAs. We studied eight 3′UTR polymorphic sites, three of which (the 14bp INDEL, +3187 SNP and +3142 SNP) are known to affect HLA-G expression (Castelli et al., 2010; Sabbagh et al., 2014). Amongst our lines, we found the three possible 14bp genotypes. Remarkably, the two VUB hESC lines with a 14bp +/+ genotype clearly showed lower mRNA levels of HLA-G. This influence of the 14bp genotype on the mRNA levels in hESC is in accordance with the study by Hviid et al., where trophoblast sodium channel and biopsies containing the 14bp sequence were associated with a lower level of HLA-G mRNA for most isoforms (Hviid et al., 2003). Furthermore, preeclampsia placentas, showing reduced HLA-G mRNA levels, have been associated with a 14bp +/+ genotype (O\'Brien et al., 2001). An AUUUG sequence within this 14bp sequence has been speculated to contribute to this lower mRNA expression by having an AU-pentamer-like effect that stimulates deadenylation and subsequent mRNA degradation (Rousseau et al., 2003). Next to the 14bp polymorphism, the +3187A/G SNP has also been reported to influence HLA-G. Our two 14bp +/+ hESC lines were homozygous A/A for this SNP. This increased number of A(denines) in the AU-rich motif has been described as the underlying mechanism for reduced mRNA levels through decreased mRNA stability (Yie et al., 2008). In addition, both VUB19_DM1 and VUB31_FSHD were homozygous G(uanine) for the SNP +3142 C/G. The presence of G(uanine) has previously been linked to lower HLA-G levels and the mechanism behind this modulation appears to be by targeted miRNA binding (Tan et al., 2007; Castelli et al., 2009). Numerous miRNAs have been identified that preferentially target different SNPs in the HLA-G region by in silico modelling, and for a small number of these, in vivo modelling data exist (Tan et al., 2007; Castelli et al., 2009). We only studied a number of these miRNAs, namely those that we found to be expressed in hESC based on published and unpublished data (Suh et al., 2004; Lakshmipathy et al., 2007; Tsai et al., 2010). We did not observe statistically significant differences in miR-148a, miR-148b, miR-152 and miR-19a levels between the lines. It has previously been described that miR-148a, miR-148b and miR-152 all have a high affinity for the +3142G allele and thus might be involved in the down-regulation of total mRNA and isoforms in the 14bp +/+ lines by the mechanism of allele specific mRNA degradation (Castelli et al., 2009). The mechanism of HLA-G mRNA degradation has been previously described in the study of Manaster et al., where 721.221 HLA-G expressing cells transfected with miR-148a and -152 showed reduced HLA-G mRNA levels (Manaster et al., 2012). However, they demonstrated no effect of the C/G polymorphism. Conversely, the study from Zhu et al. reported no effect on HLA-G mRNA expression in JEG-3 cells (G/G) overexpressing miR-152 (Zhu et al., 2010; Zhu et al., 2010) while the study by Tan et al. only investigated the effect of miR-148a at soluble HLA-G protein level in JEG-3 cells (Tan et al., 2007). Based on these data, we cannot draw any conclusion on the effect of miR-148a, -148b and -152 in hESC HLA-G regulation. In addition, the functional effect and mechanism of miR-19a on HLA-G mRNA and protein expression have not yet been investigated.