Identification of 14-3-3ß gene as a novel miR-152 target using a proteome-based approach.

Recent studies demonstrated that miR-152 overexpression down-regulates the nonclassical human leukocyte antigen (HLA) class I molecule HLA-G in human tumors thereby contributing to their immune surveillance. Using two-dimensional gel electrophoresis followed by MALDI-TOF mass spectrometry, the protein expression profile of HLA-G(+), miR-152(low) cells, and their miR-152-overexpressing (miR(high)) counterparts was compared leading to the identification of 24 differentially expressed proteins. These were categorized according to their function and localization demonstrating for most of them an important role in the initiation and progression of tumors. The novel miR-152 target 14-3-3 protein ß/α/YWHAB (14-3-3ß) is down-regulated upon miR-152 overexpression, although its overexpression was often found in tumors of distinct origin. The miR-152-mediated reduction of the 14-3-3ß expression was accompanied by an up-regulation of BAX protein expression resulting in a pro-apoptotic phenotype. In contrast, the reconstitution of 14-3-3ß expression in miR-152(high) cells increased the expression of the anti-apoptotic BCL2 gene, enhances the proliferative activity in the presence of the cytostatic drug paclitaxel, and causes resistance to apoptosis induced by this drug. By correlating clinical microarray data with the patients' outcome, a link between 14-3-3ß and HLA-G expression was found, which could be associated with poor prognosis and overall survival of patients with tumors. Because miR-152 controls both the expression of 14-3-3ß and HLA-G, it exerts a dual role in tumor cells by both altering the immunogenicity and the tumorigenicity.

Identification of miR-200a-5p targeting the peptide transporter TAP1 and its association with the clinical outcome of melanoma patients.

Tumor escape is often associated with abnormalities in the surface expression of the human leukocyte antigen class I (HLA-I) antigens thereby limiting CD8+ cytotoxic T cell responses. This impaired HLA-I surface expression can be mediated by deficient expression of components of the antigen processing and presentation machinery (APM) due to epigenetic, transcriptional and/or post-transcriptional processes. Since a discordant mRNA and protein expression pattern of APM components including the peptide transporter associated with antigen processing 1 (TAP1) has been frequently described in tumors of distinct origin, a post-transcriptional control of APM components caused by microRNAs (miR) was suggested. Using an in silico approach, miR-200a-5p has been identified as a candidate miR binding to the 3' untranslated region (UTR) of TAP1. Luciferase reporter assays demonstrated a specific binding of miR-200a-5p to the TAP1 3'-UTR. Furthermore, the miR-200a-5p expression is inversely correlated with the TAP1 protein expression in HEK293T cells and in a panel of melanoma cell lines as well as in primary melanoma lesions. High levels of miR-200a-5p expression were associated with a shorter overall survival of melanoma patients. Overexpression of miR-200a-5p reduced TAP1 levels, which was accompanied by a decreased HLA-I surface expression and an enhanced NK cell sensitivity of melanoma cells. These data show for the first time a miR-mediated control of the peptide transporter subunit TAP1 in melanoma thereby leading to a reduced HLA-I surface expression accompanied by an altered immune recognition and reduced patients' survival. Abbreviations: Ab: antibody; ACTB: ß-actin; APM: antigen processing and presentation machinery; ATCC: American tissue culture collection; ß2-m: ß2-microglobulin; BSA: bovine serum albumin; CTL: cytotoxic T lymphocyte; FCS: fetal calf serum; FFL: firefly luciferase; FFPE: formalin-fixed paraffin-embedded; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HC: heavy chain; HLA: human leukocyte antigen; HLA-I: HLA class I; HRP: horseradish peroxidase; IFN: interferon; im-miR: immune modulatory miRNA; LMP: low molecular weight protein; luc: luciferase; MFI: mean fluorescence intensity; MHC: major histocompatibility complex; miR: microRNA; NC: negative control; NK: natural killer; NSCLC: non-small cell lung carcinoma; OS: overall survival; PBMC: peripheral blood mononuclear cells; RBP: RNA-binding proteins; RL: Renilla; RLU: relative light units; TAP: transporter associated with antigen processing; tpn: tapasin; UTR: untranslated region.

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

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.