microRNA profiling in mantle cell lymphoma cell lines before and after 5-azadC treatment. Homo sapiens

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin’s lymphoma (NHL). In cancers, tumour suppressive microRNAs may be silenced by DNA hypermethylation. By microRNA profiling, miR-155-3p was significantly upregulated upon demethylation treatment of MCL cell lines with 5-aza-2’-deoxycytidine (5-azadC). Methylation-specific PCR, verified by pyrosequencing, showed complete methylation of miR-155-3p in one MCL cell line (REC-1). 5-azadC treatment of REC-1 led to demethylation and re-expression of miR-155-3p. Over-expression of miR-155-3p led to increased sub-G1 apoptotic cells and reduced cellular viability, demonstrating its tumour suppressive properties. By luciferase assay, lymphotoxin-beta (LT-β) was validated as a miR-155-3p target. In 31 primary MCL, miR-155-3p was found hypermethylated in 6(19%) cases. To test if methylation of miR-155-3p was MCL-specific, miR-155-3p methylation was tested in an additional 191 B-cell, T-cell and NK-cell NHLs, yielding miR-155-3p methylation in 66(34.6%) including 36(27%) non-MCL B-cell, 24(53%) T-cell and 6(46%) of NK-cell lymphoma. Moreover, in 72 primary NHL samples with RNA, miR-155-3p methylation correlated with miR-155-3p downregulation (p=0.030), and LT-β upregulation (p=0.004). Collectively, miR-155-3p is tumour suppressive microRNA hypermethylated in MCL and other NHL subtypes. As miR-155-3p targets LT-β, which is an upstream activator of the non-canonical NF-kB signalling, miR-155-3p methylation is potentially important in lymphomagenesis Overall design: Total RNA isolated from MINO and JEKO-1 before and after 5-azadC treatment were converted into cDNA by MegaplexTM RT Primers and TaqMan® MicroRNA Reverse Transcription Kit. cDNA was pre-amplified using MegaplexTM PreAmp Primer and loaded onto 384-well format Taqman® human microRNA array A V2.0 & B V3.0. Real-time PCR was performed on 7900HT Real-Time PCR system and raw data were analyzed normalizing to mean of three endogenous controls (U6snRNA, RNU44 and RNU48). Relative microRNA levels were determined by ΔΔCt using endogenous controls and untreated controls using SDS 2.4 and RQ manager 1.2. All experimental procedures and analyses were performed according to manufacturer’s instruction, using reagents, system and softwares acquired from Applied Biosystems (Foster City, USA).

套细胞淋巴瘤（Mantle cell lymphoma, MCL）是一类侵袭性B细胞非霍奇金淋巴瘤（non-Hodgkin’s lymphoma, NHL）。在肿瘤发生进程中，抑癌微小RNA（microRNA, miR）可通过DNA高甲基化发生转录沉默。通过微小RNA谱分析，研究人员在经5-氮杂-2’-脱氧胞苷（5-aza-2’-deoxycytidine, 5-azadC）处理的MCL细胞系中，发现miR-155-3p的表达水平显著上调。经焦磷酸测序验证的甲基化特异性PCR检测显示，MCL细胞系REC-1中miR-155-3p存在完全甲基化。对REC-1细胞施以5-azadC处理可诱导其去甲基化，并重新激活miR-155-3p的表达。过表达miR-155-3p可使亚G1期凋亡细胞比例升高，同时降低细胞活力，证实了其抑癌生物学特性。通过荧光素酶报告基因实验，研究人员证实淋巴毒素β（lymphotoxin-beta, LT-β）为miR-155-3p的直接靶基因。在31例原发性MCL样本中，有6例（19%）检测到miR-155-3p高甲基化。为验证miR-155-3p甲基化是否具有MCL特异性，研究人员额外检测了191例B细胞、T细胞及NK细胞来源的NHL样本，结果显示其中66例（34.6%）存在miR-155-3p甲基化，包括36例（27%）非MCL型B细胞淋巴瘤、24例（53%）T细胞淋巴瘤及6例（46%）NK细胞淋巴瘤。此外，在72例带有可用RNA的原发性NHL样本中，miR-155-3p甲基化与miR-155-3p表达下调呈显著相关（p=0.030），同时与LT-β表达上调呈显著相关（p=0.004）。综上，miR-155-3p是一种抑癌微小RNA，在MCL及其他NHL亚型中存在高甲基化现象。由于miR-155-3p的靶基因LT-β是非经典核因子κB（NF-κB）信号通路的上游激活因子，因此miR-155-3p甲基化在淋巴瘤发生发展过程中可能具有重要的生物学意义。实验整体设计：从经5-azadC处理前后的MINO与JEKO-1细胞中提取总RNA，利用Megaplex™逆转录引物与TaqMan®微小RNA逆转录试剂盒将其反转录为cDNA。随后采用Megaplex™预扩增引物对cDNA进行预扩增，将扩增产物点样至384孔板格式的Taqman®人类微小RNA芯片A V2.0与B V3.0中。在7900HT实时荧光定量PCR系统上完成实时PCR检测，原始数据以三种内参基因（U6 snRNA、RNU44及RNU48）的平均值进行标准化处理。利用ΔΔCt法，结合内参基因与未处理对照组，通过SDS 2.4与RQ manager 1.2软件计算微小RNA的相对表达量。所有实验操作与数据分析均严格按照制造商提供的说明书进行，所用试剂、实验系统及软件均购自美国福斯特城的应用生物系统公司（Applied Biosystems）。