Li Fangfang, Long Xinyi, Tang Sishi, Yan Jinhua, Liu Jing, Fu Yunfeng
Department of Hematology, The Third Xiangya Hospital of Central South University, Changsha, 410013, China.
Department of Cell Biology, School of Life Sciences, Central South University, Changsha, 410013, China.
Cell Commun Signal. 2025 Jul 18;23(1):346. doi: 10.1186/s12964-025-02340-7.
Despite many new drugs, multiple myeloma (MM) remains an incurable plasma cell malignancy, and drug resistance is a long-standing topic in this field. Characterized by efficient transcription without being limited by the double helix structure and promoter, extrachromosomal circular DNA (EccDNA) has been proven to be widely involved in cancer development and drug resistance.
We performed circle-seq and mRNA-seq on samples from three MM patients at the time of complete response and relapse to screen EccDNA candidate molecules. Outward PCR and Sanger sequencing were used to identify EccDNA molecules. RT‒qPCR and WB were performed to detect gene expression levels. Fluorescence in situ hybridization (FISH) was carried out to detect the deletion of chromosome 17p (del (17p)). Transmission electron microscopy (TEM) was conducted to observe autophagosomes. Luciferase reporter assays were performed to validate the binding of microRNAs to target genes. Cell viability assays and apoptosis assays were employed to assess drug resistance. Xenograft tumor mouse models were established for in vivo experiments. Immunohistochemistry (IHC) was used to detect protein expression levels.
We successfully identified an EccDNA molecule (EccDNA) in one relapsed MM patient with del(17p) and named it MIR4726. We demonstrated that the overexpression of MIR4726 in MM cells can increase bortezomib resistance. We further confirmed that the precursor miRNA carried by MIR4726 can be efficiently transcribed in MM cells and that MIR4726 drives bortezomib resistance via the MIR4726-5p/NXF1/NKIRAS2 axis. We further revealed that downregulation of NFKB inhibitor interacting Ras like 2 (NKIRAS2) activated the NF-κB pathway and increased autophagy. Moreover, we established a xenograft model of human MM via subcutaneous inoculation. We administered intra-tumoral injection of AgoMIR4726-5p and intraperitoneal injection of bortezomib and found that AgoMIR4726-5p promoted tumor progression and partially drove bortezomib resistance.
In summary, our findings indicate that artificially synthesized MIR4726 is functional in cells and that MIR4726 enhances tumor progression and partially mediates drug resistance by enhancing MIR4726-5p/NXF1/NKIRAS2 axis dependent autophagy.
尽管有许多新药，但多发性骨髓瘤（MM）仍然是一种无法治愈的浆细胞恶性肿瘤，耐药性是该领域长期存在的话题。染色体外环状DNA（EccDNA）具有高效转录的特点，不受双螺旋结构和启动子的限制，已被证明广泛参与癌症发展和耐药性。
我们对3例MM患者完全缓解期和复发期的样本进行了环状序列分析（circle-seq）和信使核糖核酸测序（mRNA-seq），以筛选EccDNA候选分子。采用外向聚合酶链反应（Outward PCR）和桑格测序法鉴定EccDNA分子。进行逆转录定量聚合酶链反应（RT‒qPCR）和蛋白质免疫印迹法（WB）检测基因表达水平。采用荧光原位杂交（FISH）检测17号染色体短臂缺失（del(17p)）。进行透射电子显微镜（TEM）观察自噬体。进行荧光素酶报告基因检测以验证微小RNA与靶基因的结合。采用细胞活力检测和凋亡检测评估耐药性。建立异种移植肿瘤小鼠模型进行体内实验。采用免疫组织化学（IHC）检测蛋白质表达水平。
我们在1例伴有del(17p)的复发MM患者中成功鉴定出一个EccDNA分子（EccDNA），并将其命名为MIR4726。我们证明MM细胞中MIR4726的过表达可增加硼替佐米耐药性。我们进一步证实MIR4726携带的前体微小RNA可在MM细胞中有效转录，且MIR4726通过MIR4726-5p/NXF1/NKIRAS2轴驱动硼替佐米耐药。我们进一步揭示，核因子κB抑制因子相互作用的Ras样蛋白2（NKIRAS2）的下调激活了核因子κB通路并增加了自噬。此外，我们通过皮下接种建立了人MM异种移植模型。我们进行瘤内注射AgoMIR4726-5p和腹腔注射硼替佐米，发现AgoMIR4726-5p促进肿瘤进展并部分导致硼替佐米耐药。
总之，我们的研究结果表明，人工合成的MIR4726在细胞中具有功能，且MIR4726通过增强MIR4726-5p/NXF1/NKIRAS2轴依赖性自噬促进肿瘤进展并部分介导耐药性。
