Long Non-Coding RNAs Are Commonly Deregulated In Hodgkin Lymphoma

Hodgkin’s lymphoma (HL) is a B-cell neoplasm characterized by a minority of neoplastic cells within an extensive inflammatory background. Long non-coding RNAs (lncRNAs) have diverse roles in transcriptional, posttranscriptional and epigenetic mechanisms in normal cell physiology. Deregulation of lncRNA expression has been implicated in a number of cancers. Up to date it is unclear to what extent lncRNAs are involved in the pathogenesis of Hodgkin lymphoma (HL).

We designed a custom made microarray based on a published lncRNA catalog of 10,509 lncRNA loci (Cabili et al., 2011). The array also contains probes for all protein coding genes allowing simultaneous detection of the lncRNA and mRNA expression pattern. We analyzed 6 HL cell lines and three samples of purified tonsillar naive, germinal center (GC) and memory B cells. To get more insight into the potential function of individual differentially expressed lncRNAs we determined lncRNA enrichment in cytoplasmic and nuclear fractions of two HL cell lines. Furthermore, we studied to what extent lncRNAs are enriched in the Argonaute 2 (Ago2) containing complexes as an indication for interaction with miRNAs.

In a comparison between the three different normal B-cell subsets, we observed 565 differentially expressed lncRNA probes. Overall, naive and memory B cell subsets showed a highly similar expression pattern while GC B cells showed a distinct lncRNA expression pattern. A significant differential expression between HL and normal GC B-cells was observed for 717 lncRNA probes. Interestingly, 109 of those overlap with the 565 lncRNAs differentially expressed between the normal B cell subsets. Differential expression for 4 in HL up and 2 downregulated lncRNAs was confirmed using qRT-PCR. To get a first indication about their potential function, we evaluated the subcellular location of the lncRNAs by comparing the abundance in nuclear and cytoplasmic fractions relative to the total fraction. Overall, 26% of the expressed lncRNAs were consistently enriched in the nuclear fraction and 10% were predominantly enriched in the cytoplasmic fraction. Of the 717 differentially expressed lncRNAs 82 were consistently enriched in the nucleus and 16 in the cytoplasm. Our Ago2 pull-down experiments revealed that 9% of the mRNAs and 3% of the expressed lncRNAs are consistently enriched in Ago2 complexes. In total, we observed evidence for miRNA binding for 17 of the in HL differentially expressed lncRNAs.

In conclusion, we identified a specific lncRNA expression pattern in GC B cells and observed for more than 700 lncRNAs a differentially expression pattern between HL and normal GC B-cells. For 98 lncRNAs we found a specific subcellular enrichment and for 17 we observed enrichment in the Ago2 fraction, indicative of miRNA-lncRNA interactions. Follow-up studies will determine to what extent these lncRNAs contribute to the pathogenesis of Hodgkin lymphoma.