title: Clinical relevance of miR-mediated HLA-G regulation and the associated immune cell infiltration in renal cell carcinoma.
abstract: 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 proteinin situandin vitro. Stable miR overexpression caused a downregulation of HLA-G protein enhancing the NK and LAK cell-mediated cytotoxicity inin vitroCD107a 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.
authors.affiliation: Institute of Medical Immunology; Martin Luther University Halle-Wittenberg ; Halle, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Jasinski-Bergner
authors.affiliation: Institute of Pathology; Friedrich Alexander University Erlangen-Nuremberg ; Erlangen, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Stoehr
authors.affiliation: Institute of Medical Immunology; Martin Luther University Halle-Wittenberg ; Halle, Germany.
authors.forename: Juergen
authors.initials: J
authors.lastname: Bukur
authors.affiliation: Institute of Medical Immunology; Martin Luther University Halle-Wittenberg ; Halle, Germany.
authors.forename: Chiara
authors.initials: C
authors.lastname: Massa
authors.affiliation: Institute of Molecular Medicine; Martin Luther University Halle-Wittenberg ; Halle, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Braun
authors.affiliation: Institute of Molecular Medicine; Martin Luther University Halle-Wittenberg ; Halle, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hüttelmaier
authors.affiliation: Institute of Pathology; Friedrich Alexander University Erlangen-Nuremberg ; Erlangen, Germany.
authors.forename: Verena
authors.initials: V
authors.lastname: Spath
authors.affiliation: Institute of Pathology; Friedrich Alexander University Erlangen-Nuremberg ; Erlangen, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Wartenberg
authors.affiliation: Clinic of Urology; Friedrich Alexander University Erlangen-Nuremberg ; Erlangen, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Legal
authors.affiliation: Clinic of Urology; Friedrich Alexander University Erlangen-Nuremberg ; Erlangen, Germany.
authors.forename: Helge
authors.initials: H
authors.lastname: Taubert
authors.affiliation: Clinic of Urology; Friedrich Alexander University Erlangen-Nuremberg ; Erlangen, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Wach
authors.affiliation: Clinic of Urology; Friedrich Alexander University Erlangen-Nuremberg ; Erlangen, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Wullich
authors.affiliation: Institute of Pathology; Friedrich Alexander University Erlangen-Nuremberg ; Erlangen, Germany.
authors.forename: Arndt
authors.initials: A
authors.lastname: Hartmann
authors.affiliation: Institute of Medical Immunology; Martin Luther University Halle-Wittenberg ; Halle, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Seliger
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