低氧及骨髓基质细胞促多发性骨髓瘤耐药的作用及机制研究

It is well known that the bone marrow microenvironment plays an important role in the disease progress and drug resistance of multiple myeloma (MM), but the specific mechanisms are unclear. We have found that the therapeutic effects of bortezomib were significantly weakened by hypoxia and bone marrow stromal cells (BMSCs) with c-Myc elevation in MM cells. Furthermore, by RNA sequencing, we have obtained 3 candidated long noncoding RNAs (lncRNAs) which are controlled by hypoxia and BMSCs, and closely related to c-Myc in MM cells. Therefore, we will conduct further studies in several aspects. Firstly, study the effects of candidated lncRNAs in drug resistance in MM, and further screen out the key lncRNAs. Secondly, search for the targets of key lncRNAs. Thirdly, explore the special mechanisms of key lncRNAs in the drug resistance-related pathways, such as c-Myc. Finally, prove the role of the key lncRNAs in primary MM samples, and find out the compound which can convert drug resistance of MM. In summary, our study will shed light on the role of bone marrow microenvironment and its mechanisms in drug resistance of MM, and provide the novel treatment strategy to improve response rate and reduce relapse.

骨髓微环境在多发性骨髓瘤（MM）的疾病进展和MM细胞耐药过程中起着重要作用，但其相关的分子机制并不清楚。我们的预实验结果提示: 低氧环境和骨髓基质细胞（BMSCs）可上调MM细胞内c-Myc表达并减弱其对硼替佐米治疗的敏感性。对MM细胞行RNA测序分析，发现3个受低氧和BMSCs调控的，且与c-Myc密切相关的长链非编码RNA（lncRNAs）。在此基础上，本项目拟：1）研究候选lncRNAs对MM细胞耐药的影响，筛选出介导耐药的关键lncRNAs；2）寻找关键lncRNAs作用的靶标；3）解析关键lncRNAs对c-Myc等耐药通路的调控作用和具体机制。4）在原代MM标本中验证关键耐药机制，力求发现逆转MM细胞耐药的小分子化合物。本研究将明确微环境促MM耐药的分子机制，为改善MM的治疗和减少疾病复发提供新策略。

多发性骨髓瘤（MM）的疾病特点为是骨髓浆细胞在低氧骨髓微环境中恶性增殖，其具有高度异质的遗传背景和临床表现，预后差。 长链非编码RNA（lncRNA）是一类不编码蛋白，长度大于200nt的RNA分子。lncRNA可参与染色质修饰，转录激活，核内运输等多种生理过程。既往研究报道，低氧相关的lncRNA可参与缺氧/HIF-1相关的癌症进展。低氧微环境对MM的发生、发展、耐药及复发至关重要，所以我们使用RNA高通量测序分析在低氧培养条件下，骨髓瘤细胞系U266内差异表达的lncRNA分子。我们发现HIF-1α可促进骨髓瘤细胞系内lncRNA DARS-AS1的转录。体内及体外实验均证明，DARS-AS1过表达可促进骨髓瘤细胞增殖并抑制细胞凋亡，敲除DARS-AS1可抑制骨髓瘤细胞增殖并促进其凋亡。通过RNA-Pulldown联合蛋白质质谱分析的方法，我们检测到RBM39为DARS-AS1的靶蛋白。过表达RBM39可以部分恢复敲除DARS-AS1后的细胞表型。我们推断DARS-AS1可能通过影响RBM39的表达水平而发挥其生物学功能。进一步研究表明，DARS-AS1可以减弱RBM39与 E3泛素连接酶RNF147的相互作用，从而减少RBM39泛素化降解。下调骨髓瘤细胞系内DARS-AS1或者RBM39水平均可以抑制mTOR信号通路。mTOR通路的激活可促进HIF-1α的翻译，所以DARS-AS1与HIF-1α之间存在正反馈，共同促进MM的疾病进展。 总之，我们发现DARS-AS1可促进骨髓瘤细胞增殖并抑制细胞凋亡。本研究有助于了解骨髓瘤的致病机制，DARS-AS1/RBM39可能是骨髓瘤治疗的新靶标。