SEPTIN2 and STATHMIN Regulate CD99-Mediated Cellular Differentiation in Hodgkin's Lymphoma

Hodgkin’s lymphoma (HL) is a lymphoid neoplasm characterized by Hodgkin’s and Reed-Sternberg (H/RS) cells, which is regulated by CD99. We previously reported that CD99 downregulation led to the transformation of murine B lymphoma cells (A20) into cells with an H/RS phenotype, while CD99 upregulation induced differentiation of classical Hodgkin’s lymphoma (cHL) cells (L428) into terminal B-cells. However, the molecular mechanism remains unclear. In this study, using fluorescence two-dimensional differential in-gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), we have analyzed the alteration of protein expression following CD99 upregulation in L428 cells as well as downregulation of mouse CD99 antigen-like 2 (mCD99L2) in A20 cells. Bioinformatics analysis showed that SEPTIN2 and STATHMIN, which are cytoskeleton proteins, were significantly differentially expressed, and chosen for further validation and functional analysis. Differential expression of SEPTIN2 was found in both models and was inversely correlated with CD99 expression. STATHMIN was identified in the A20 cell line model and its expression was positively correlated with that of CD99. Importantly, silencing of SEPTIN2 with siRNA substantially altered the cellular cytoskeleton in L428 cells. The downregulation of STATHMIN by siRNA promoted the differentiation of H/RS cells toward terminal B-cells. These results suggest that SEPTIN2-mediated cytoskeletal rearrangement and STATHMIN-mediated differentiation may contribute to changes in cell morphology and differentiation of H/RS cells with CD99 upregulation in HL.霍奇金淋巴瘤（Hodgkin’s lymphoma, HL）是一类以霍奇金细胞和里-施（Reed-Sternberg, H/RS）细胞为特征的淋巴组织肿瘤，其发生发展由CD99分子调控。我们此前的研究表明，CD99表达下调可使小鼠B淋巴瘤细胞（A20）转化为具有H/RS细胞表型的细胞；而CD99表达上调则可诱导经典型霍奇金淋巴瘤（classical Hodgkin’s lymphoma, cHL）细胞（L428）向终末B细胞分化。但上述过程的分子机制目前仍不明确。 本研究通过荧光双向差异凝胶电泳结合基质辅助激光解吸电离飞行时间质谱（matrix-assisted laser desorption/ionization time of flight mass spectrometry, MALDI-TOF MS），分别分析了L428细胞中CD99上调以及A20细胞中小鼠CD99抗原样2（mouse CD99 antigen-like 2, mCD99L2）下调后的蛋白质表达变化。生物信息学分析显示，作为细胞骨架蛋白的隔蛋白2（SEPTIN2）与丝切蛋白（STATHMIN）存在显著差异表达，遂选取二者开展后续验证与功能分析。 在两个细胞模型中均检测到SEPTIN2的差异表达，且其表达水平与CD99呈负相关；而STATHMIN仅在A20细胞模型中被鉴定到，其表达水平与CD99呈正相关。值得注意的是，利用小干扰RNA（siRNA）沉默SEPTIN2可显著改变L428细胞的细胞骨架结构；通过siRNA下调STATHMIN的表达则可促进H/RS细胞向终末B细胞分化。 上述结果提示，SEPTIN2介导的细胞骨架重排以及STATHMIN参与的分化调控过程，可能参与了HL中CD99上调所诱导的H/RS细胞形态改变与分化进程。