Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Department of Laboratory Medicine, Ruijin Hospital Affiliated to School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Chemotherapy. 2022;67(1):12-23. doi: 10.1159/000520070. Epub 2021 Nov 29.
The chemoresistance mechanism of diffuse large B-cell lymphoma (DLBCL) is still poorly understood, and patient prognosis remains unsatisfactory. This study aimed to investigate drug resistance mechanisms in non-germinal center B-cell-like (non-GCB) DLBCL.
Doxorubicin (DOX)-resistant OCI-Ly3 cells were generated through long-term incubation of cells in a medium with gradually increasing DOX concentrations. The expression levels of genes related to drug metabolism were determined using a functional gene grouping polymerase chain reaction (PCR) array. Drug-resistant proteins were identified using bioinformatics, and molecular association networks were subsequently generated. The association and mechanism of key genes were determined using a dual-luciferase reporter assay System and chromatin immunoprecipitation (ChIP). The expression of drug-resistant genes and target genes was then measured using Western blotting and immunohistochemistry. The correlation between gene expressions was analyzed using Spearman's rank correlation coefficient.
Using the PCR array, MDR1 was identified as the key gene that regulates DOX resistance in OCI-Ly3/DOX-A100, a non-GCB DLBCL cell line. The dual-luciferase reporter assay system demonstrated that MDR1 transcription could be inhibited by PRDM1. ChIP results showed that PRDM1 had the ability to bind to the promoter region (-1,132 to -996) of MDR1. In OCI-Ly3/DOX cells, NF-κB activity and PRDM1 expression decreased with an increase in drug-resistant index, whereas MDR1 expression increased with enhanced drug resistance. Immunohistochemical analysis revealed that relative MDR1 expression was higher than that of PRDM1 in human DLBCL tissue samples. A negative correlation was observed between MDR1 and PRDM1.
In non-GCB DLBCL cells, NF-κB downregulates PRDM1 and thereby promotes MDR1 transcription by terminating PRDM1-induced transcriptional inhibition of MDR1. Such a mechanism may explain the reason for disease recurrence in non-GCB DLBCL after R-CHOP or combined CHOP with bortezomib treatment. Our findings may provide a potential therapeutic strategy for reducing drug resistance in patients with DLBCL.
弥漫大 B 细胞淋巴瘤（DLBCL）的化疗耐药机制仍不清楚，患者预后仍不理想。本研究旨在探讨非生发中心 B 细胞样（non-GCB）DLBCL 的耐药机制。
通过在含有逐渐增加阿霉素（DOX）浓度的培养基中孵育细胞，生成耐 DOX 的 OCI-Ly3 细胞。采用功能基因分组聚合酶链反应（PCR）array 检测与药物代谢相关基因的表达水平。利用生物信息学鉴定耐药蛋白，并随后生成分子关联网络。利用双荧光素酶报告基因检测系统和染色质免疫沉淀（ChIP）确定关键基因的关联和作用机制。采用 Western blot 和免疫组化检测耐药基因和靶基因的表达。采用 Spearman 秩相关系数分析基因表达的相关性。
利用 PCR array，鉴定出 MDR1 是调控 OCI-Ly3/DOX-A100（非 GCB DLBCL 细胞系）中 DOX 耐药的关键基因。双荧光素酶报告基因检测系统显示，PRDM1 可抑制 MDR1 的转录。ChIP 结果表明，PRDM1 具有结合 MDR1 启动子区域（-1132 至-996）的能力。在 OCI-Ly3/DOX 细胞中，随着耐药指数的增加，NF-κB 活性和 PRDM1 表达降低，而 MDR1 表达增加，耐药性增强。免疫组化分析显示，在人 DLBCL 组织样本中，相对 MDR1 表达高于 PRDM1。MDR1 与 PRDM1 呈负相关。
在非 GCB DLBCL 细胞中，NF-κB 下调 PRDM1，从而通过终止 PRDM1 诱导的 MDR1 转录抑制来促进 MDR1 转录。这种机制可能解释了非 GCB DLBCL 患者在接受 R-CHOP 或联合 CHOP 与硼替佐米治疗后疾病复发的原因。我们的研究结果可能为降低 DLBCL 患者的药物耐药性提供潜在的治疗策略。
