In human tumors of distinct origin including renal cell carcinoma (RCC), the non-classical human leukocyte antigen G (HLA-G) is frequently expressed, thereby inhibiting the cytotoxic activity of T and natural killer (NK) cells. Recent studies demonstrated a strong post-transcriptional gene regulation of the HLA-G by miR-152, -148A, -148B and -133A. Standard methods were applied to characterize the expression and function of HLA-G, HLA-G-regulatory microRNAs (miRs) and the immune cell infiltration in 453 RCC lesions using a tissue microarray and five RCC cell lines linking these results to clinical parameters. Direct interactions with HLA-G regulatory miRs and the HLA-G 3' untranslated region (UTR) were detected and the affinities of these different miRs to the HLA-G 3'-UTR compared. qPCR analyses and immunohistochemical staining revealed an inverse expression of miR-148A and -133A with the HLA-G protein in situ and in vitro. Stable miR overexpression caused a downregulation of HLA-G protein enhancing the NK and LAK cell-mediated cytotoxicity in in vitro CD107a activation assays revealing a HLA-G-dependent cytotoxic activity of immune effector cells. A significant higher frequency of CD3+/CD8+ T cell lymphocytes, but no differences in the activation markers CD69, CD25 or in the presence of CD56+, FoxP3+ and CD4+ immune cells were detected in HLA-G+ compared to HLA-G- RCC lesions. This could be associated with higher WHO grade, but not with a disease-specific survival. These data suggest a miR-mediated control of HLA-G expression in RCC, which is associated with a distinct pattern of immune cell infiltration.
Keywords: ACTB, β-actin; APM, antigen processing machinery; B7-H1, B7 homolog 1; CDS, coding sequence; Cr, chromium; COPZ2, coatomer protein complex, subunit zeta 2; DAC, 5′-aza-2′-desoxycytidine, GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HLA-G, human leukocyte antigen G; HRP, horseradish peroxidase; IFNγ, interferon gamma; IHC, immunohistochemistry; IL, interleukin; ILT, immunoglobulin-like transcript; LAK, lymphokine-activated killer cell; MDSC, myeloid-derived suppressor cells; MFI, mean-specific fluorescence intensity; NK, natural killer cell; RCC, renal cell carcinoma; SNP, single nucleotide polymorphism; TGF-β, transforming growth factor β; TIL, tumor infiltrating lymphocyte; TMA, tissue microarray; Treg, regulatory T cell; UTR, untranslated region; WB, Western blot analysis; WT, wild type; immune escape; luc, luciferase; mAb, monoclonal antibody; miR, microRNA; miTRAP, miRNA trapping by RNA in vitro affinity purification; microRNA; n.d., not determined; n.o.s., not otherwise specified; ntc., non-template control; non-classical HLA class I molecules; renal cell carcinoma; sHLA-G, soluble HLA-G; tumor-infiltrating lymphocytes; β-gal, β-galactosidase; β2-m, β-2-microglobulin.