MicroRNA Analysis by In Situ Hibridization in Hodgkin Lymphoma.

Hodgkin lymphoma (HL) is a neoplasm characterized by the presence of relatively few tumoral cells, Hodgkin and Reed-Sternberg cells in an important non-neoplastic environment. The mature microRNAs (miRNAs) are small RNA molecules (20–25 nt), that act inhibiting the mRNA translation to protein by binding to 3′ UTR region of mRNA. In a previous work we detect a 25 miRNA signature of HL. Some of them inhibit the expression of key genes related to B lymphomagenesis. The aim of the study was to determine if the differential expression of miRNAs in tumoral tissue versus reactive lymph nodes, is due to genetic alterations in the Hodgkin/Reed Sternberg cells or alterations in the non-tumoral microenvironment. Fluorescein (FITC) 5′ labelled locked-nuclei-acid-incorporated (LNA) miRNA ribo probes for miR-21, 134, 138, 155 (miRCURY™ LNA detection, Exiqon) were used in 20 cases of HL formalin-fixed paraffin embedded tissue sections on silane coated slides (Vision BioSystem). Chromogenic in situ hybridization was done in an automated platform Bond Max (Vision Biosystems) with minor modifications. Pre-treatment of the slides was performed with Protease 1 for 10 min at 37°C. A total amount of 300 microliter of 25nM probe was hybridized in 1x sodium chloride-sodium citrate hybridization buffer (SSC) (Innogenetics) up to 50° C for 2 hours. We used a pre-diluted mouse anti-FITC antibody (Vision BioSystems) for 20–60 minutes followed by a goat anti-mouse linked to thousands of horse radish peroxidase (HRP) sites (Refine Detection System, Vision BioSystems). DAB was used as a chromogen reacting for 10 minutes and hematoxilyn was used as a counterstain. By functional and target analysis, we selected three miRNAs, miR-21 (PTEN), miR-134 (J-Chain) and miR-138 (PU.1), from the cHL signature that seemed to have a role in tumorigenesis process and analyzed them by CISH. In all cases a cytoplasmic signal was demonstrated in Hodgkin and Reed-Sternberg cells. Moreover, a nuclear signal was identified in reactive tumor infiltrating lymphocytes for miR-21 and miR-138. This nuclear signal may be due to crossreactivity with primary miRNA in these cells. We analyzed miR-155 as positive control of in situ hibridization and we demonstrated a cytoplasmic signal in Hodgkin and Reed Sternberg cells, as well as in scattered reactive lymphocytes and activated histiocytes as previously reported. In conclusion, miR-21, miR-134 and miR-138 play a role in HL lymphomagenesis since their expression is preferentially localized in Hodgkin/Reed Sternberg cells. The study of the other miRNAs of our signature can help explain the changes that appear in the atypical cells as well in the reactive cellular microenvironment in HL.