lncRNA-81002通过调控ceRNA网络和自噬活性介导多发性骨髓瘤蛋白酶体抑制剂耐药的机制研究

Whereas the therapeutic benefit of proteasome inhibitors(PIs) remains unchallenged, acquired drug resistance remains a major obstacle toward achieving sustained clinical responses. Here we exposed drug-sensitive myeloma cells to successively increased concentrations of bortezomib or carfilzomib to generate drug-resistant cells. We then identified lncRNAs differentially expressed in PIs-resistant cells by RNA-seq. A novel lncRNA, lncRNA-81002 was involved in PIs resistance based on its high level in PIs-resistant myeloma cells compared with drug-naive cells. LncRNA-81002 increased proteasome activityby acting as competing endogenous RNA(ceRNA). Moreover, lncRNA-81002 was critical fortranscription of LC3 that plays an important role in autophagy. Our study suggested that downregulation of lncRNA-81002 may offer a therapeutic advantage for myeloma treatment by inhibiting proteasomes and reducing autophagy. Our further study will propose a mechanism to explain how lncRNA-81002 promotes PIs resistance by acting as ceRNA and enhancing autophagy. A proof-of-principle will be provided to demonstrate that lncRNA-81002 offers potential as novel therapeutic agents to overcome PIs resistance.

多发性骨髓瘤(MM)患者的蛋白酶体抑制剂(PIs)耐药机制尚不清楚，文献报道蛋白酶体活性增加和自噬活性增加均与此密切相关。我们在前期研究中通过RNA-seq在PIs耐药的MM细胞中筛选出异常表达的新转录本lncRNA-81002，并发现它既可作为竞争性内源RNA(ceRNA)增加蛋白酶体活性，又可通过促进LC3转录导致自噬活性增加，从而提示lncRNA-81002可能通过调控泛素-蛋白酶体及自噬-溶酶体两条途径影响MM对PIs的敏感性。本课题拟利用临床标本和已建立的PIs耐药MM细胞株，在细胞、分子和动物模型水平，利用生物信息学分析、分子生物学和细胞生物学技术，进一步确证lncRNA-81002在MM蛋白酶体抑制剂耐药中的作用，并阐明此耐药机制中的关键调控因子和具体调控方式，从而提出lncRNA-81002可作为增加MM对PIs敏感性的潜在作用靶点，为解决临床中PIs耐药问题提供新的思路。

项目研究背景：.多发性骨髓瘤(multiple myeloma, MM)蛋白酶体抑制剂(Proteasome Inhibitors, PIs)耐药机制尚不清楚。长链非编码RNA(Long non-coding RNA, LncRNA)可以通过调节组蛋白修饰、DNA甲基化、染色质重塑、转录因子活性和RNA前体剪接等对基因表达在表观遗传水平、转录水平和转录后水平进行调控。本研究目的在于探究lncRNA在MM蛋白酶体抑制剂耐药中的作用及其分子机制。.主要研究内容：.1..明确lncRNA-81002升高是MM对PIs敏感性降低的原因之一。.2..探讨lncRNA-81002在MM蛋白酶体抑制剂耐药中的作用。.3..揭示lncRNA-81002在MM蛋白酶体抑制剂耐药中的作用靶点，阐明lncRNA-81002在在MM蛋白酶体抑制剂耐药中具体调控方式和作用机制。.重要结果及关键数据：.1..在蛋白酶体抑制敏感和耐药骨髓瘤细胞中利用RNA-seq筛选差异表达的lncRNA，扩大样本量，通过qPCR确证差异表达lncRNA。.2..通过功能获得性研究和功能缺失性研究确证lncRNA-81002在多发性骨髓瘤蛋白酶体耐药中的作用。.3..明确了lncRNA-81002在MM蛋白酶体抑制剂耐药中的作用靶点，阐明了lncRNA-81002在泛素-蛋白酶体通路和自噬-溶酶体通路这两条通路中的具体调控方式和作用机制。.4..建立裸鼠骨髓瘤模型验证lncRNA-81002在多发性骨髓瘤蛋白酶体耐药中的作用，评估lncRNA-81002作为提高MM蛋白酶体抑制剂敏感性的潜在靶点。.研究意义：.本项目探讨了lncRNA-81002在MM蛋白酶体抑制剂耐药中的作用及其分子机制，从而提出lncRNA-81002作为增加MM对PIs敏感性的潜在作用靶点，为解决临床中PIs耐药问题提供新的思路。