DNA methylation was shown previously to be a crucial mechanism responsible for transcriptional deregulation in the pathogenesis of classical Hodgkin lymphoma (cHL). To identify epigenetically inactivated miRNAs in cHL, we have analyzed the set of miRNAs downregulated in cHL cell lines using bisulfite pyrosequencing. We focused on miRNAs with promoter regions located within or <1000 bp from a CpG island. Most promising candidate miRNAs were further studied in primary Hodgkin and Reed-Sternberg (HRS) cells obtained by laser capture microdissection. Last, to evaluate the function of identified miRNAs, we performed a luciferase reporter assay to confirm miRNA: mRNA interactions and therefore established cHL cell lines with stable overexpression of selected miRNAs for proliferation tests. We found a significant reverse correlation between DNA methylation and expression levels of mir-339-3p, mir-148a-3p, mir-148a-5p and mir-193a-5 demonstrating epigenetic regulation of these miRNAs in cHL cell lines. Moreover, we demonstrated direct interaction between miR-148a-3p and IL15 and HOMER1 transcripts as well as between mir-148a-5p and SUB1 and SERPINH1 transcripts. Furthermore, mir-148a overexpression resulted in reduced cell proliferation in the KM-H2 cell line. In summary, we report that mir-148a is a novel tumor suppressor inactivated in cHL and that epigenetic silencing of miRNAs is a common phenomenon in cHL.

中文翻译：

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在经典霍奇金淋巴瘤中表观遗传失活的肿瘤抑制mir-148a。

以前已证明DNA甲基化是经典霍奇金淋巴瘤（cHL）发病机理中导致转录失调的关键机制。为了鉴定cHL中的表观遗传失活的miRNA，我们使用亚硫酸氢盐焦磷酸测序分析了在cHL细胞系中下调的miRNA集。我们专注于启动子区域位于CpG岛之内或<1000 bp的miRNA。最有希望的候选miRNA在通过激光捕获显微切割术获得的原发性霍奇金和Reed-Sternberg（HRS）细胞中进行了进一步研究。最后，为了评估已鉴定的miRNA的功能，我们进行了荧光素酶报告基因分析，以确认miRNA：mRNA的相互作用，并因此建立了具有稳定过表达所选miRNA的cHL细胞系，以进行增殖测试。我们发现mir-339-3p，mir-148a-3p，mir-148a-5p和mir-193a-5的DNA甲基化与表达水平之间存在显着的反向相关性，证明了这些miRNA在cHL细胞系中的表观遗传调控。此外，我们证明了miR-148a-3p与IL15和HOMER1转录本以及mir-148a-5p与SUB1和SERPINH1转录本之间。此外，mir-148a过表达导致KM-H2细胞系中的细胞增殖减少。总之，我们报道mir-148a是一种在cHL中失活的新型肿瘤抑制因子，miRNA的表观遗传沉默是cHL中的常见现象。