MicroRNA High Throughput Loss-of-Function Screening Reveals an Oncogenic Role for miR-21-5p in Hodgkin Lymphoma

Ye Yuan, Fubiao Niu, Ilja M. Nolte, Jasper Koerts, Debora de Jong, Bea Rutgers, Jan Osinga, Maria Azkanaz, Martijn Terpstra, Leonid Bystrykh, Arjan Diepstra, Lydia Visser, Agnieszka Dzikiewicz-Krawczyk, Klaas Kok, Joost Kluiver, Anke van den Berg

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Abstract

Background/Aims: Classical Hodgkin lymphoma (cHL) is among the most frequent lymphoma subtypes. The tumor cells originate from crippled germinal center (GC)-B cells that escaped from apoptosis. MicroRNAs (miRNAs) play important roles in B-cell maturation and aberrant expression of miRNAs contributes to the pathogenesis of cHL. Our aim was to identify oncogenic miRNAs relevant for growth of cHL using a high-throughput screening approach. Methods: A lentiviral pool of 63 miRNA inhibition constructs was used to identify miRNAs essential to cell growth in three cHL cell lines in duplicate. As a negative control we also infected cHL cell lines with a lentiviral barcoded empty vector pool consisting of 222 constructs. The abundance of individual constructs was followed over time by a next generation sequencing approach. The effect on growth was confirmed using individual GFP competition assays and on apoptosis using Annexin-V staining. Our previously published Argonaute 2 (Ago2) immunoprecipitation (IP) data were used to identify target genes relevant for cell growth /apoptosis. Luciferase assays and western blotting were performed to confirm targeting by miRNAs. Results: Four miRNA inhibition constructs, i.e. miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p, showed a significant decrease in abundance in at least 4 of 6 infections. In contrast, none of the empty vector constructs showed a significant decrease in abundance in 3 or more of the 6 infections. The most abundantly expressed miRNA, i.e. miR-21-5p, showed significantly higher expression levels in cHL compared to GC-B cells. GFP competition assays confirmed the negative effect of miR-21-5p inhibition on HL cell growth. Annexin-V staining of cells infected with miR-21-5p inhibitor indicated a significant increase in apoptosis at day 7 and 9 after viral infection, consistent with the decrease in growth. Four miR-21-5p cell growth-and apoptosis-associated targets were AGO2-IP enriched in cHL cell lines and showed a significant decrease in expression in cHL cell lines in comparison to normal GC-B cells. For the two most abundantly expressed, i.e. BTG2 and PELI1, we confirmed targeting by miR-21-5p using luciferase assays and for PELI1 we also confirmed this at the protein level by western blotting. Conclusion: Using a miRNA loss-of-function high-throughput screen we identified four miRNAs with oncogenic effects in cHL and validated the results for the in cHL abundantly expressed miR-21-5p. MiR-21-5p is upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1. (C) 2018 The Author(s) Published by S. Karger AG, Basel

Original languageEnglish
Pages (from-to)144-159
Number of pages16
JournalCellular physiology and biochemistry
Volume49
Issue number1
DOIs
Publication statusPublished - 2018

Keywords

  • Classical Hodgkin lymphoma (cHL)
  • High-throughput screen
  • miR-21-5p
  • Apoptosis
  • BTG2
  • PELI1
  • REED-STERNBERG CELLS
  • POOR-PROGNOSIS
  • BREAST-CANCER
  • EXPRESSION
  • INHIBITION
  • APOPTOSIS
  • OUTGROWTH
  • PROFILE
  • TARGET
  • SMAD7

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