MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies.

Background: Hodgkin's lymphoma (HL) is characterized by a minority of neoplastic cells, the so‐called Reed–Sternberg (RS) cells, and an admixture of reactive cells including lymphocytes, plasma cells, eosinophils and histiocytes. Cytokines produced in HL, either by RS or infiltrating cells, might explain the presence and maintenance of an impaired immune response. Chemokines (cytokines with chemoattractant properties) produced by RS cells play a major role in leukocyte trafficking. These molecules with specific receptor affinities contribute, for example by attracting TH2‐like T cells, to the maintenance of a favorable environment for survival of RS cells. Results and discussion: Cross‐talk between RS cells and reactive elements involves several cytokine/chemokines that process proliferative [interleukin (IL)‐13 and IL‐17], immunosuppressive (IL‐10 and transforming growth factor‐β) and background formation (IL‐5, TARC, MDC, IP‐10, RANTES, Mig and others) messages. These autocrine and paracrine interactions lead to an environment where RS cells are able to proliferate, escape from apoptosis and survive host anti‐tumor defense. Conclusions: The proper modulation of these complex pathways may allow the development of new strategies in HL therapy.