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.
Highlights
The non-classical HLA class Ib molecule human leukocyte antigen G (HLA-G) exerts some properties, which are distinct from that of classical HLA class Ia molecules, such as limited allelic variability, existence of membranebound and soluble isoforms due to alternative splicing and a restricted physiologic expression to mainly immune-privileged tissues and cells [1]
The predicted binding sites of both novel miRs identified a second hotspot (Figure 1C, red box) for the miR-mediated post-transcriptional control of HLA-G expression next to the binding site of miR-148 family members (Figure 1C, blue box). These novel HLA-G-regulating miRs were confirmed by the miR-specific enrichment from a cell lysate of the HLA-G mRNA+/protein- renal cell carcinoma (RCC) cell line MZ2905RC using the miTRAP technique [31]
Employing a MS2 looptagged and in vitro transcribed HLA-G 3’-untranslated region (UTR) as bait, an enrichment of miR-628-5p and miR-548q was observed by qPCR (Figure 2A)
Summary
The non-classical HLA class Ib molecule HLA-G exerts some properties, which are distinct from that of classical HLA class Ia molecules, such as limited allelic variability, existence of membranebound and soluble isoforms due to alternative splicing and a restricted physiologic expression to mainly immune-privileged tissues and cells [1]. HLA-G can modulate immune cell responses by its interaction with the inhibitory lymphocyte receptors, the immunoglobulin-like transcript (ILT), ILT4 and the killer immunoglobulin-like receptor KIR2DL4 present on different immune cell populations [2, 3]. This leads to an inhibition of the immune effector cell-mediated cytotoxicity. Under pathophysiologic conditions constitutive HLA-G surface expression was frequently upregulated in hematopoietic and solid tumors including renal cell carcinoma (RCC) [1, 7,8,9] which could often be correlated to an unfavorable prognosis and poor clinical outcome of tumor patients. The aberrant expression of HLA-G is regulated by different molecular mechanisms including epigenetic, transcriptional, post-transcriptional as well as post-translational control [12] rather than structural alterations
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
