miR-221/222通过抑制自噬性死亡和促进MMSC的形成和扩增双重机制介导多发性骨髓瘤的耐药

Induction of autophagic cell death is an important new therapeutic strategy for drug-resistant multiple myeloma (MM). Our previous study has shown differential expression of miR-221/222 in dexamethasone-resistant and -sensitive MM cell lines, as well as in MM patient samples. The target genes of miR-221/222 as revealed by bioinformatic analysis, including ULK2, ATG2B, ATG4A and ATG12, are key molecules involved in autophagy. Moreover, our preliminary study has demonstrated that miR-221/222 inhibit the expression of PTEN, a negative regulator of PI3K/Akt signaling. Recent studies have demonstrated the regulation of CSCs (cancer stem cells) characteristics by PI3K/Akt/mTOR signaling pathway. Therefore, our hypothesis is that miR-221/222 mediates drug resistance in multiple myeloma through inhibiting autophagic cell death, as well as promoting formation and amplication of MMSCs (multiple myeloma stem cells) via PTEN/PI3K/Akt/mTOR signaling pathway. In the proposed study, we will use both overexpression and knockdown strategies to verify the target genes of miR-221/222 predicted by bioinformatics assay. Specifically, we will investigate the effect of deregulated expression of miR-221/222 on autophagic cell death, as well as formation and amplication of MMSCs. Next, the in vitro data will be evaluated in vivo using MM mouse models. Our aim is to elucidate the role of miR-221/222 in MM drug resistance and relapse. Ultimately, this proposed study will provide the rationale for the combination of miRNA-targeted treatment strategy with chemotherapy to cure MM.

诱导自噬性死亡是治疗耐药MM细胞的重要新策略。项目组前期研究发现miR-221/222在地塞米松耐药和敏感的MM细胞系和临床标本中存在着明显差异表达，其预测靶基因中包含多个参与自噬的关键分子。前期研究还发现miR-221/222下调PI3K/Akt抑制分子PTEN的表达。最新研究证实PI3K/Akt/mTOR信号通路参与调节CSC的多种生物学特性。据此推测异常表达的miR-221/222通过抑制自噬性死亡和调节PTEN/PI3K/Akt/mTOR信号通路促进MMSC形成和扩增的双重机制介导MM耐药。本项目拟采用miRNA表达/拮抗实验结合临床资料的分析对生物信息学预测的靶基因加以验证，进而研究异常表达的miR-221/222对自噬抵抗以及MMSC形成和扩增的影响，然后通过小鼠模型进行体内实验，最终确定此miRNA在MM耐药和复发中的作用，并探讨以此miRNA为靶点联合化疗治愈MM的可行性。

多发性骨髓瘤（MM）是血液系统第二常见的恶性肿瘤，以克隆性浆细胞异常增生为特征。尽管随着新药的不断研发和应用，MM患者的生存时间明显延长，但该疾病目前仍被认为是不可治愈的肿瘤，绝大多数患者在疾病的病程中都会出现复发或者对前期治疗的耐药。地塞米松是MM联合化疗方案中最常用的化疗药物，原发性或者继发性的对地塞米松耐药的MM患者，往往伴有不良的预后。虽然，糖皮质激素受体（GR）的缺失，凋亡与抗凋亡蛋白的失调，以及信号分子之间的相互作用与地塞米松耐药相关，但目前引起地塞米松耐药关键机制尚不清楚。本项目研究发现miR-221/222 在MM 病人的骨髓浆细胞中的表达明显高于健康对照，尤其在复发、耐药的MM患者。miR-221/222的高表达可促进 MM细胞对地塞米松的耐药。证实ATG12、p27和PTEN是miR-221/222 的靶基因，miR-221/222不仅可通过抑制PTEN导致PI3K/Akt/mTOR信号通路活化促进MM增殖，还可抑制ATG12/p27-mTOR-自噬通路抑制自噬。除此之外，我们发现地塞米松通过下调MM 细胞miR-221/222的表达活化ATG12/p27-mTOR自噬途径引起MM发生自噬性死亡的新机制。最终，通过体、内外实验证实靶向miR-221/222诱导MM自噬性死亡可逆转MM细胞对地塞米松耐药。该研究成果为揭示miR-221/222 在MM发生发展及耐药中的作用提供了一定的理论基础，为临床上使用miR-221/222 的表达水平预估患者对于糖皮质激素的治疗的反应性或治疗预后提供了一定的研究基础，为临床上基于调控miRNA 的表达以及诱导自噬性死亡应用于耐药MM 患者的治疗提供了一定的研究基础。