Identification of novel microRNAs regulating HLA-G expression and investigating their clinical relevance in renal cell carcinoma

Abstract

The non-classical human leukocyte antigen G (HLA-G) is expressed at a high frequency in renal cell carcinoma (RCC) and is associated with a higher tumor grade and a poor clinical outcome. This might be caused by the HLA-G-mediated inhibition of the cytotoxicity of T and NK cells. Therefore a selective targeting of HLA-G might represent a powerful strategy to enhance the immunogenicity of RCC lesions. Recent studies identified a number of HLA-G-regulating microRNAs (miRs) and demonstrated an inverse expression of some of these miRs with HLA-G in RCC in vitro and in vivo. However, it was postulated that further miRs might exist contributing to the tightly controlled selective HLA-G expression.By application of a miR enrichment assay (miTRAP) in combination with in silico profiling two novel HLA-G-regulatory miRs, miR-548q and miR-628-5p, were identified. Direct interactions of both miRs with the 3′ untranslated region of HLA-G were confirmed with luciferase reporter gene assays. In addition, qPCR analyses and immunohistochemical staining revealed an inverse, expression of miR-628-5p, but not of miR-548q to the HLA-G protein in primary RCC lesions and cell lines. Stable overexpression of miR-548q and miR-628-5p caused a downregulation of HLA-G mRNA and protein. This leads in case of miR-548q to an enhanced NK cell-mediated HLA-G-dependent cytotoxicity, which could be reverted by ILT2 blockade suggesting a control of the immune effector cell activity at least by this miR. The identification of two novel HLA-G-regulatory miRs extends the number of HLA-G-relevant miRs tuning the HLA-G expression and might serve as future therapeutic targets.

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

“…It has been previously shown that miR-133a regulates HLA-G expression through a non-polymorphic binding site whereas miR-148a, miR-148b, and miR-152 influence HLA-G expression depending on the SNP present at position +3142. More recently miR-628-5p and miR-548q have been demonstrated to target the segment encompassing the variable sites +3003 and +3010 [24]. Notably, there is no consensus about how +3142 SNP [22, 33] impacts on the binding profile of microRNAs, and we have not found a significant effect of the +3035 C-T polymorphism, which suggest that the influence of a genetic background should be considered in a specific microRNA environment.…”

“…On the other hand, published data has pointed out that the 3’ untranslated region (UTR) shared by the HLA-G1 to HLA-G6 alternative transcripts (~ 370 bases, Fig 1A) has a crucial role in modulating the expression of HLA-G. A first important finding is that this segment is targeted by six already identified microRNAs, namely miR-148a, miR-148b, miR-152 [22], miR-133a [23], miR-628-5p and miR-548q [24] which can downregulate HLA-G expression. Another finding is that the 3’UTR is polymorphic, with a genetic diversity likely influenced by balancing selection as observed in the case of the HLA-G promoter region.…”

Molecular checkpoints controlling natural killer cell activation and their modulation for cancer immunotherapy

“…Several miRNAs regulate HLA-G expression: miR-548q, miR628-5p and miR152. 128, 129 miR-548q and miR628-5p interact with the 3′ untranslated region of HLA-G directly and overexpression of these miRNAs enhanced NK-cell-mediated HLA-G-dependent cytotoxicity. 128 HLA-G expression is upregulated and miR-152 is downregulated by HBV infection in human samples.…”

“…128, 129 miR-548q and miR628-5p interact with the 3′ untranslated region of HLA-G directly and overexpression of these miRNAs enhanced NK-cell-mediated HLA-G-dependent cytotoxicity. 128 HLA-G expression is upregulated and miR-152 is downregulated by HBV infection in human samples. 129 HBV-infected hepatocarcinoma cells express more HLA-G and are resistant to NK cytotoxicity.…”

Emerging role of microRNA 628-5p as a novel biomarker for cancer and other diseases

“…It was considered that miRNA 628-5p is a fine tuner because of its lower affinity to the HLA-G 3#UTR. 21 miRNA 628-5p downregulation has been evidenced in glioblastoma (GBM) tumor tissue compared to normal controls; 23 however, further study of miRNA expression signatures found miRNA 628-5p to be ''protective'' or beneficial to patient outcome. 24 In addition, cell cycle genes, Ttk, Top2a, and Col4a1, were found to be regulated partially by miRNA 628-5p, highlighting a possible regulatory role in this cancer.…”