Supplementary Material for: 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 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.

背景/目的：经典霍奇金淋巴瘤（Classical Hodgkin lymphoma, cHL）是最常见的淋巴瘤亚型之一，其肿瘤细胞来源于发生凋亡逃逸的功能缺陷生发中心（germinal center, GC）B细胞。微小RNA（microRNAs, miRNAs）在B细胞成熟过程中发挥关键调控作用，其异常表达与cHL的发病机制密切相关。本研究旨在通过高通量筛选方法，鉴定与cHL生长相关的致癌性miRNAs。
方法：本研究构建包含63种miRNA抑制载体的慢病毒混合文库，以重复实验的方式在3株cHL细胞系中筛选对细胞生长至关重要的miRNAs。同时，以包含222种序列的条形码化空载体慢病毒文库作为阴性对照感染cHL细胞系。通过下一代测序技术实时追踪各载体的丰度随时间的变化。采用单个绿色荧光蛋白（green fluorescent protein, 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调控作用。利用本课题组已发表的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和蛋白质印迹实验验证miRNAs对靶基因的靶向调控作用。
结果：在6次重复感染实验中，4种miRNA抑制载体（即miR-449a-5p、miR-625-5p、let-7f-2-3p及miR-21-5p）的丰度在至少4次实验中出现显著下降。与之相反，空载体对照组在6次感染实验中，无1种载体的丰度在3次及以上实验中出现显著下降。在cHL细胞系中表达丰度最高的miR-21-5p，其表达水平显著高于正常GC B细胞。GFP竞争实验证实，抑制miR-21-5p可显著降低HL细胞的生长能力。膜联蛋白V（Annexin-V）染色结果显示，感染miR-21-5p抑制剂的细胞在病毒感染后第7天和第9天的凋亡率显著升高，与细胞生长受抑制的结果一致。4个与miR-21-5p调控的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，且相较于正常GC B细胞，其在cHL细胞系中的表达水平显著下调。对于其中两种高丰度靶基因BTG2和PELI1，我们通过荧光素酶报告基因实验验证了miR-21-5p对其的靶向调控作用；同时，通过蛋白质印迹实验在蛋白水平验证了PELI1的靶向调控关系。
结论：本研究通过miRNA功能缺失型高通量筛选，鉴定出4种具有致癌作用的miRNAs，并针对在cHL中高表达的miR-21-5p验证了筛选结果。相较于GC B细胞，miR-21-5p在cHL中呈高表达，其可能通过靶向BTG2和PELI1，保护cHL细胞免于凋亡。