miR-217下调通过增强PEA15的表达促进STAT1介导的多发性骨髓瘤细胞CAM-DR进程

Cell adhesion-mediated drug resistance is an important reason for the incurability of multiple myeloma. Anti-apoptosis is the vital reason in the process of CAM-DR. PEA15, an anti-apoptotic protein, is closely correlated with cell apoptosis. It is reported that STAT1 has a vital role in the regulation of drug resistance by regulating the expression of apoptosis-related protein. Our research group has recently identified that miR217, PEA15 and STAT1 were all associated with CAM-DR. Furthermore, we found that PEA15 was a target gene of miR-217 and PEA15 could interact with STAT1. Therefore, we speculate that miR-217 may be involved in regulation of STAT1 and its apoptotic target genes via targeting PEA15, and thereby participating in CAM-DR. Based on these, we first aim to analyze the role of PEA15 in CAM-DR. Then, we intend to explore the effect of miR-217 on the expression of PEA15 and CAM-DR. Subsequently, we will investigate the regulatory mechanism of PEA15-STAT1 interaction and its effect on CAM-DR phenotype. Finally, we will comprehensively analyze the significance of PEA15-STAT1 interaction in the process of STAT1-mediated CAM-DR phenotype. Our research will shed new light on the mechanism of CAM-DR and provide theoretical basis for clinical treatment of multiple myeloma.

细胞粘附介导的耐药（CAM-DR）是多发性骨髓瘤不可治愈的重要原因。抗凋亡是CAM-DR发生的重要原因。凋亡抑制蛋白PEA15与细胞的凋亡耐受有密切关系。信号转导与转录激活因子1（STAT1）可以通过调节凋亡信号分子的表达在调控肿瘤细胞耐药过程中发挥重要作用。前期研究发现，miR-217、PEA15、STAT1均与CAM-DR相关，miR-217可靶向抑制PEA15的表达，并且PEA15可以与STAT1相互作用。因此推测：miR-217可能通过调节PEA15继而影响STAT1及其下游凋亡信号分子的表达参与CAM-DR进程。课题组拟分析PEA15、miR-217对PEA15的调节以及PEA15与STAT1的相互作用的调节机制在CAM-DR进程中的作用；然后全面分析miR-217对PEA15的调节在STAT1介导的CAM-DR进程中的意义，从而进一步揭示CAM-DR的机制。

细胞粘附介导的耐药(CAM-DR)是导致多发性骨髓瘤不可治愈的重要原因。其中PEA15的表达水平及在细胞中的内化循环与CAM-DR形成密切相关。根据实验室前期工作基础及相关领域的研究进展，课题组利用纤连蛋白FN或骨髓基质细胞HS-5与人多发性骨髓瘤细胞RPMI8226、NCI-H929构建粘附模型，结果显示在骨髓瘤细胞粘附培养过程中， PEA15的表达明显高于悬浮细胞；利用化疗药物硼替佐米刺激细胞，粘附培养的骨髓瘤细胞活力明显高于悬浮培养的细胞，粘附组凋亡相关蛋白表达显著低于悬浮培养的骨髓瘤细胞。接下来，在骨髓瘤细胞中干扰PEA15的表达，CCK-8及克隆形成实验结果显示细胞增殖缓慢，细胞活力显著下降。流式细胞仪结果显示，干扰PEA15表达的骨髓瘤细胞，大部分细胞的周期阻滞在G1期，进入S期细胞数量明显减少，周期相关蛋白CDK2、cyclinD亦表达下降。在骨髓瘤细胞中干扰PEA15的表达，并使用硼替佐米刺激细胞，PEA15低表达的细胞其凋亡数量显著高于对照组，并且PEA15低表达的细胞其凋亡相关蛋白cleaved-caspase3、cleaved-PARP的表达亦显著增加。为了进一步探讨PEA15参与调控骨髓瘤细胞生长的分子机制，免疫共沉淀实验表明PEA15与STAT1在多发性骨髓瘤细胞中存在相互作用。在骨髓瘤细胞中干扰PEA15表达，STAT1表达水平增加，磷酸化STAT1表达下降，STAT1下游靶基因Bcl-2蛋白水平下降，Bax蛋白水平增加。研究结果提示：骨髓瘤细胞中，PEA15与STAT1有直接相互作用，并通过抑制STAT1磷酸化，下调STAT1下游靶基因Bcl-2的表达，参与调控骨髓瘤细胞对硼替佐米的敏感性。.课题的研究结果将为我们进一步揭示CAM-DR发生发展的具体分子机制，并确认PEA15与STAT1的相互作用在MM细胞CAM-DR形成过程中的重要作用。.按照合同约定，课题研究成果以论文形式提交。在该基金共资助下，目前已发表论文2篇，参与学术交流2次，协助培养研究生3名。