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

<b><i>Background/Aims:</i></b> 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. <b><i>Methods:</i></b> 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. <b><i>Results:</i></b> 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. <b><i>Conclusion:</i></b> 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.

**背景与目的：** 经典霍奇金淋巴瘤（cHL）是最常见的淋巴瘤亚型之一。其肿瘤细胞源自逃脱凋亡的功能受损的生发中心（GC）B细胞。微小RNA（miRNAs）在B细胞成熟过程中发挥重要作用，而miRNAs的异常表达与cHL的发病机制密切相关。本研究旨在通过高通量筛选（high-throughput screening）方法，鉴定与cHL生长相关的致癌性miRNAs。
**方法：** 本研究使用包含63个miRNA抑制构建体（miRNA inhibition constructs）的慢病毒库（lentiviral pool），对3株cHL细胞系开展重复感染实验，以筛选对细胞生长必需的miRNAs。同时，我们使用包含222个构建体的带有条形码的空载体慢病毒库（lentiviral barcoded empty vector pool）感染cHL细胞系作为阴性对照（negative control）。通过下一代测序（next generation sequencing）技术动态追踪单个构建体的丰度变化。采用单个绿色荧光蛋白（GFP）竞争实验验证其对细胞生长的影响，并通过膜联蛋白-V（Annexin-V）染色检测其对细胞凋亡的作用。我们使用已发表的Argonaute 2（Ago2）免疫沉淀（IP）数据，筛选与细胞生长/凋亡相关的靶基因。通过荧光素酶报告基因实验（Luciferase assays）和蛋白质印迹法（western blotting）验证miRNAs的靶向调控作用。
**结果：** 4个miRNA抑制构建体，即miR-449a-5p、miR-625-5p、let-7f-2-3p和miR-21-5p，在至少4/6次感染实验中呈现出丰度显著下降。与之相反，在6次感染实验中，没有任何空载体构建体在3次及以上实验中出现丰度显著下降。表达丰度最高的miRNA为miR-21-5p，其在cHL细胞中的表达水平显著高于生发中心B细胞。GFP竞争实验证实，抑制miR-21-5p可对HL细胞生长产生负面影响。用miR-21-5p抑制剂感染的细胞经膜联蛋白-V染色后显示，病毒感染后第7天和第9天的细胞凋亡率显著升高，这与细胞生长受到抑制的结果一致。4个与miR-21-5p细胞生长和凋亡相关的靶基因在cHL细胞系中经AGO2免疫沉淀富集，且与正常生发中心B细胞相比，其在cHL细胞系中的表达水平显著降低。对于其中两个表达丰度最高的靶基因BTG2和PELI1，我们通过荧光素酶报告基因实验证实了miR-21-5p对其的靶向调控作用，同时通过蛋白质印迹法在蛋白水平验证了PELI1的靶向调控关系。
**结论：** 本研究通过miRNA功能缺失（loss-of-function）高通量筛选，鉴定出4个在cHL中具有致癌作用的miRNAs，并对在cHL中高表达的miR-21-5p的结果进行了验证。与生发中心B细胞相比，miR-21-5p在cHL中表达上调，其可能通过靶向BTG2和PELI1，保护cHL细胞免于凋亡。