环状 RNA 结合蛋白 HNRNPU 编码的一种新型蛋白通过调控骨髓微环境和可变剪接促进多发性骨髓瘤进展。
A novel protein encoded by circHNRNPU promotes multiple myeloma progression by regulating the bone marrow microenvironment and alternative splicing.
机构信息
Nanjing Hospital of Chinese Medicine affiliated to Nanjing University of Chinese Medicine, Nanjing, China.
School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing, China.
出版信息
J Exp Clin Cancer Res. 2022 Mar 8;41(1):85. doi: 10.1186/s13046-022-02276-7.
BACKGROUD
Multiple myeloma (MM) is an incurable plasma cell malignancy in the bone marrow (BM), while immunoglobulin D type of MM (IgD MM) is a very rare but most severe subtype in all MM cases. Therefore, systemic study on IgD MM is purposeful to disclose the recurrent and refractory features in both IgD and other types of MM, and beneficial to the development of potent therapeutic strategy on MM.
METHODS
Agilent SBC-ceRNA microarray chips were employed to examine 3 normal plasma cell samples (NPCs), 5 lgD MM samples and 5 lgG MM samples, respectively. Sanger sequencing, RNase R digestion and qPCR assays were used to detect the existence and expression of circHNRNPU. BaseScope™ RNA ISH assay was performed to test circHNRNPU levels in paraffin-embedded MM tissues. The protein encoded by circHNRNPU was identified by LC-MS/MS, which was named as circHNRNPU_603aa. The function of circHNRNPU_603aa on cellular proliferation and cell cycle was assessed by MTT test, colony formation assay, flow cytometry and MM xenograft mouse model in vivo. RIP-seq, RIP-PCR and WB analysis for ubiquitination were performed to explore the potential mechanism of circHNRNPU_603aa in MM. Exosomes were isolated from the culture supernatant of MM cells by ultracentrifugation and characterized by Transmission Electron Microscope and WB confirmation of exosomes markers Alix and CD9.
RESULTS
CircHNRNPU was one of the top most abundant and differentially expressed circRNA in IgD MM relative to lgG and NPCs samples. Increased circHNRNPU was associated with poor outcomes in four independent MM patient cohorts. Intriguingly, MM cells secreted circHNRNPU, which encoded a protein named as circHNRNPU_603aa. Overexpressed circHNRNPU_603aa promoted MM cell proliferation in vitro and in vivo, in contrast knockdown of circHNRNPU_603aa by siRNA abrogated these effects. Due to circHNRNPU_603aa including RNA-binding RGG-box region, it regulated SKP2 exon skipping, thereby competitively inhibited c-Myc ubiquitin so as to stabilize c-Myc in MM. MM cells secreted circHNRNPU through exosomes to interfere with various cells in the BM microenvironment.
CONCLUSION
Our findings demonstrate that circHNRNPU_603aa is a promising diagnostic and therapeutic marker in both MM cells and BM niche.
背景
多发性骨髓瘤（MM）是骨髓（BM）中不可治愈的浆细胞恶性肿瘤，而免疫球蛋白 D 型 MM（IgD MM）是所有 MM 病例中最严重的亚型之一。因此，对 IgD MM 进行系统研究有助于揭示 IgD 和其他类型 MM 中的复发性和难治性特征，有利于开发针对 MM 的有效治疗策略。
方法
采用 Agilent SBC-ceRNA 微阵列芯片分别检测 3 例正常浆细胞样本（NPCs）、5 例 IgD MM 样本和 5 例 IgG MM 样本。采用 Sanger 测序、RNase R 消化和 qPCR 检测 circHNRNPU 的存在和表达。采用 BaseScope™ RNA ISH 检测石蜡包埋 MM 组织中 circHNRNPU 的水平。通过 LC-MS/MS 鉴定 circHNRNPU 编码的蛋白质，命名为 circHNRNPU_603aa。通过 MTT 试验、集落形成试验、流式细胞术和 MM 异种移植小鼠模型在体内评估 circHNRNPU_603aa 对细胞增殖和细胞周期的功能。进行 RIP-seq、RIP-PCR 和 WB 分析以探索 circHNRNPU_603aa 在 MM 中的潜在机制。通过超速离心从 MM 细胞的培养上清液中分离外泌体，并通过透射电子显微镜和外泌体标记物 Alix 和 CD9 的 WB 确认进行表征。
结果
circHNRNPU 是 IgD MM 与 IgG 和 NPCs 样本相比最丰富和差异表达的 circRNA 之一。circHNRNPU 的增加与四个独立的 MM 患者队列的不良预后相关。有趣的是，MM 细胞分泌了 circHNRNPU，它编码了一种名为 circHNRNPU_603aa 的蛋白质。过表达 circHNRNPU_603aa 促进了体外和体内 MM 细胞的增殖，而 siRNA 敲低 circHNRNPU_603aa 则消除了这些效应。由于 circHNRNPU_603aa 包含 RNA 结合 RGG 盒区域，它调节 SKP2 外显子跳跃，从而竞争性抑制 c-Myc 泛素化，从而稳定 MM 中的 c-Myc。MM 细胞通过外泌体分泌 circHNRNPU，以干扰 BM 微环境中的各种细胞。
结论
我们的研究结果表明，circHNRNPU_603aa 是 MM 细胞和 BM 龛中的有前途的诊断和治疗标志物。
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