Genome-wide analysis of pSTAT3 (Tyr705) in diffuse large B-cell lymphoma. Genome-wide analysis of pSTAT3 (Tyr705) in diffuse large B-cell lymphoma

Diffuse large B cell lymphoma (DLBCL), the most common non-Hodgkin lymphoma, includes two main molecular subtypes: activated B cell-like (ABC) DLBCL and germinal center B cell-like (GCB) DLBCL. ABC DLBCL is more aggressive, with the activated JAK1-STAT3 pathway that promotes cell survival. In order to study the underlying pathogenic mechanism, we performed a ChIP-seq experiment by phospho-STAT3(Tyr705) antibody in ABC DLBCL cell lines, and identified 3,456 STAT3 target genes genome-wide. In parallel, ChIP-seq experiment was performed with STAT3 antibody in α-IgM stimulated naïve B cells for verification of these targets of STAT3 protein. TMD8 was treated by eithor DMSO or AZD1480 (JAK inhibitor) for 4hrs, ChIP-seq experiments were performed by pSTAT3(Tyr705) antibody (#9145S, Cell signaling). Naïve B cells were collected from peripheral blood mononuclear cells by Naive B Cell Isolation kit (miltenyi biotec; 130-091-150), and treated by 10ug/ml α-IgM (southernbiotech; #2020-01) for 24hrs. ChIP-seq experiments were performed by STAT3 antibody (SC-482X, Santa Cruz). Overall design: TMD8 cells were treated by eithor DMSO or AZD1480(JAK inhibitor) for 4hrs. Naïve B cells were collected from peripheral blood mononuclear cells by Naive B Cell Isolation kit (miltenyi biotec; 130-091-150), and treated by 10ug/ml α-IgM (southernbiotech; #2020-01) for 24hrs.

弥漫性大B细胞淋巴瘤（Diffuse large B cell lymphoma, DLBCL）是最常见的非霍奇金淋巴瘤，主要包含两种分子亚型：活化B细胞样（activated B cell-like, ABC）DLBCL与生发中心B细胞样（germinal center B cell-like, GCB）DLBCL。其中活化B细胞样DLBCL侵袭性更强，其激活的JAK1-STAT3信号通路可促进细胞存活。为探究其潜在致病机制，我们针对ABC DLBCL细胞系，使用磷酸化STAT3（Tyr705）抗体开展了染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）实验，在全基因组范围内鉴定出3456个STAT3靶基因。同时，我们在α-IgM刺激的初始B细胞中使用STAT3抗体开展ChIP-seq实验，以验证STAT3蛋白的上述靶标。TMD8细胞分别经二甲基亚砜（DMSO）或AZD1480（JAK抑制剂）处理4小时后，使用pSTAT3（Tyr705）抗体（#9145S, Cell Signaling）开展ChIP-seq实验。初始B细胞通过初始B细胞分离试剂盒（Miltenyi Biotec; 130-091-150）从外周血单核细胞中分离，并用10μg/ml α-IgM（SouthernBiotech; #2020-01）处理24小时，随后使用STAT3抗体（SC-482X, Santa Cruz）开展ChIP-seq实验。本研究整体实验设计如下：将TMD8细胞分别用DMSO或AZD1480（JAK抑制剂）处理4小时；通过初始B细胞分离试剂盒（Miltenyi Biotec; 130-091-150）从外周血单核细胞中分离初始B细胞，并用10μg/ml α-IgM（SouthernBiotech; #2020-01）处理24小时。