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

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...

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Vydáno v:Cellular physiology and biochemistry Ročník 49; číslo 1; s. 144 - 159
Hlavní autoři: Yuan, Ye, Niu, Fubiao, Nolte, Ilja M., Koerts, Jasper, de Jong, Debora, Rutgers, Bea, Osinga, Jan, Azkanaz, Maria, Terpstra, Martijn, Bystrykh, Leonid, Diepstra, Arjan, Visser, Lydia, Dzikiewicz-Krawczyk, Agnieszka, Kok, Klaas, Kluiver, Joost , van den Berg, Anke
Médium: Journal Article
Jazyk:angličtina
Vydáno: Basel, Switzerland S. Karger AG 01.09.2018
Cell Physiol Biochem Press GmbH & Co KG
Témata:
3' Untranslated Regions
Antagomirs - metabolism
Apoptosis
Breast cancer
BTG2
Cell cycle
Cell growth
Cell Line, Tumor
Cell Proliferation - genetics
Classical Hodgkin lymphoma (cHL)
Deoxyribonucleic acid
DNA
Gene expression
HEK293 Cells
High-Throughput Nucleotide Sequencing
High-throughput screen
Hodgkin Disease - metabolism
Hodgkin Disease - pathology
Humans
Immediate-Early Proteins - genetics
Immediate-Early Proteins - metabolism
Infections
Lymphoma
MicroRNAs
MicroRNAs - antagonists & inhibitors
MicroRNAs - genetics
MicroRNAs - metabolism
miR-21-5p
Nuclear Proteins - genetics
Nuclear Proteins - metabolism
Oncogenes - genetics
Original Paper
PELI1
Tumor Suppressor Proteins - genetics
Tumor Suppressor Proteins - metabolism
Ubiquitin-Protein Ligases - genetics
Ubiquitin-Protein Ligases - metabolism
United States > US
High-throughput screen
PELI1
Classical Hodgkin lymphoma (cHL)
BTG2
miR-21-5p
Apoptosis
ISSN:1015-8987, 1421-9778, 1421-9778
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Shrnutí: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.
Bibliografie:ObjectType-Article-1
SourceType-Scholarly Journals-1
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ISSN:1015-8987
1421-9778
1421-9778
DOI:10.1159/000492850