Comparison of miRNA Profiles of Microdissected Hodgkin/Reed-Sternberg Cells and Hodgkin Cell Lines Versus CD77+ B-Cells Reveals a Distinct Subset of Differentially Expressed miRNAs
Authors: Poppe Bruce , Van Vlierberghe Pieter , Mestdagh Pieter , Feys Tom , De Weer An , De Paepe Pascale , Lambein Kathleen , De Preter Katleen , Van Roy Nadine , Vandesompele Jo , Verhasselt Bruno , Speleman Frank 
Published: April 2020

Abstract
Hodgkin lymphoma (HL) is a rare malignancy arising from pre-apoptotic germinal center B-cells which mainly affects young adults. Classical HL (cHL), the most common form, is unusual in that only a small proportion of the tumour consists of the typical malignant Hodgkin/Reed-Sternberg (HRS) cells, whereas the bulk of the remaining cells consisting of infiltrating reactive cells. The scarcity of tumour cells in lymphoma biopsies and low proliferative index has greatly hampered genetic analyses. The isolation of individual HRS cells through micromanipulation offers a possibility to overcome these problems.
Given the important role that recently emerged for small non-coding RNAs (miRNAs) in cancer development and the development of a platform which allows reliable miRNA profiling starting from as little as 10 ng total RNA, we decided to determine the miRNA signature for 360 miRNAs in a series of 9 classical HL and four common HL cell lines as well as CD77+ germinal center B-cells as normal counter part.First, we profiled a total of 360 miRNAs through automated qRT-PCR using high-throughput quantitative stem-loop RT-PCR in four commonly used cHL cell lines and compared these data to the miRNA signatures obtained from 3 different subsets of CD77+ germinal center B-cells. These analyses revealed 35 up-regulated and 16 down-regulated miRNAs in the cHL cell lines. Next, we wondered whether the miRNA signature obtained from the cHL cell lines would correspond with miRNA expression levels in primary Hodgkin lymphoma patients. Therefore, we determined the miRNA expression signature of microdissected HRS cells from 9 cHL patients and compared these data to the miRNA profiles from CD77+ B-cells. These analyses revealed 30 up-regulated and 73 down-regulated miRNAs in the microdissected HRS cells. Comparing the data from the cHL cell lines and HRS cells, a signature of 17 miRNAs was shown to be consistently up-regulated in both cHL cell lines and HRS cells, whereas a signature of 15 miRNAs was consistently down-regulated. For a number of the most highly under- and over-expressed miRNAs, the differential expression between cHL cell lines and CD77+ B-cells was re-confirmed using monoplex qPCR analysis. Combined knockdown of different over-expressed miRNAs in HL cell lines induced significant inhibition of cell viability, further confirming a potential role of these miRNAs in cHL pathogenesis. In conclusion, this is the first report describing profiling of a large set of 360 miRNAs in pure microdissected populations of HRS cells obtained from frozen biopsies from cHL patients. These miRNA profiles were compared to those from HL cell lines and CD77+ germinal center B-cells yielding a distinct subset of 17 over- and 15 under-expressed miRNAs. This study paves the way for further investigations directed at the role of these miRNAs in the pathogenesis of cHL.