PRDM1负向调控MDR1在non-GCB型弥漫大B细胞淋巴瘤化疗耐药中的作用及其分子机制研究

Diffuse large B cell lymphoma (DLBCL) is the most common aggressive form of non-Hodgkin’s lymphoma in adults. The non-germinal center B cell-like subtype of diffuse large B cell lymphoma (non-GCB DLBCL) presents aggressive clinical courses and poor prognosis compared with other type of DLBCL. In our preliminary experiments, a doxorubicin (DOX) resistant non-GCB DLBCL cell line was generated through long-term incubation of cells in the medium with gradually increasing concentrations of DOX. Using a Functional Gene Grouping PCR array, MDR1 was.identified as the most obvious up-regulated gene in drug-resistance non-GCB DLBCL cell line, meanwhile, the expression of PRDM1 was significantly lower than that of the parental cells. Immunohistochemical analysis revealed that relative MDR1 expression was higher than that of PRDM1 in human DLBCL tissue samples. A negative correlation between MDR1 and PRDM1 was found. This study is based on our previous research，in order to confirm PRDM1 can bind to the promoter of MDR1 and inhibit the expression of MDR1 through cell lines, animal models and clinical samples，further explain the role and molecular mechanism of PRDM1 regulate MDR1 in non-GCB DLBCL with chemotherapy resistance. Our findings may provide new ideas and potential therapeutic strategy for reducing drug resistance and relapse in non-GCB DLBCL patients.

弥漫大B细胞淋巴瘤（DLBCL）是成人最常见的非霍奇金淋巴瘤，相比于其他类型，non-GCB型患者化疗耐药及复发的发生率高，极大的影响临床疗效及预后。为探究其化疗耐药和复发的机制，前期研究中我们从诱导成功的non-GCB型DLBCL耐药细胞及其亲本细胞中筛选出的差异表达的基因，发现耐药细胞中MDR1上调最为明显，但PRDM1的表达却较亲本细胞显著下降，同时组织芯片的结果也证实DLBCL中PRDM1和MDR1的表达呈负相关，由此我们推测PRDM1可能抑制MDR1的表达。本课题立足于前期研究基础，在细胞系、动物模型及临床标本中进一步证实PRDM1是MDR1的负调控因子，PRDM1可结合MDR1的启动子序列并抑制MDR1的表达，深入阐释PRDM1负调控MDR1参与non-GCB型DLBCL的化疗耐药的作用及分子机制，为临床上治疗复发耐药的non-GCB型DLCBL提供新的靶标和思路。

弥漫大B细胞淋巴瘤（DLBCL）是成人最常见的非霍奇金淋巴瘤，相比于其他类型，non-GCB型患者化疗耐药及复发的发生率高，极大的影响临床疗效及预后。本项目探讨了non-GCB型弥漫大B细胞淋巴瘤（DLBCL）对阿霉素的耐药机制。本研究采用渐增浓度的阿霉素长时间培养OCI-Ly3细胞，筛选性培养成功获得了可在含100ng/mL的阿霉素的培养基中正常生长的non-GCB型弥漫大B细胞淋巴瘤细胞株，命名为OCI-Ly3/DOX-A100。PCR芯片发现MDR1基因是OCI-Ly3/DOX-A100耐药细胞抵抗阿霉素的关键基因之一；双荧光素酶报告基因检测显示PRDM1能抑制MDR1的转录水平；ChIP实验证实PRDM1能结合MDR1基因启动子区域-1132 至-996的DNA结构域；在耐药程度不同的耐药细胞株OCI-Ly3/DOX中，随着细胞耐药指数的增加，NF-κB的活性逐渐下降，PRDM1的表达逐渐下调，MDR1的表达呈上调趋势。采用PRDM1慢病毒颗粒感染OCI-Ly3/DOX-A100细胞，结果发现过表达PRDM1后，细胞的MDR1表达水平快速下降，耐药性下降，细胞对阿霉素的敏感性升高；裸鼠移植瘤模型治疗实验证实，与单用阿霉素或MDR1抑制剂zosuquidar相比较，采用zosuquidar联合阿霉素治疗裸鼠移植瘤，能显著抑制移植瘤的生长；免疫组化结果发现，在临床DLBCL组织标本中，MDR1的相对表达水平高于PRDM1的相对表达水平，二者的表达呈反向关系。结论：成功诱导non-GCB型DLBCL耐药细胞株OCI-Ly3/DOX，耐药指数约为亲本细胞OCI-Ly3的40倍；PRDM1是MDR1的负调控因子，PRDM1可结合MDR1的启动子序列并抑制MDR1的表达；MDR1是参与OCI-Ly3/DOX细胞耐药的重要分子；OCI-Ly3/DOX细胞的耐药性可被MDR1抑制剂Zosuquidar（ZSQ）逆转，OCI-Ly3/DOX细胞中恢复表达PRDM1，可逆转其耐药性；弱化的NF-κB信号可下调PRDM1的表达，解除对MDR1的转录抑制，触发MDR1的转录表达；临床标本中，PRDM1的表达与MDR1的表达呈负相关关系。