Background

Multiple myeloma (MM) is a molecularly and cytogenetically heterogeneous hematological malignancy heavily dependent on bone marrow (BM) microenvironment, which is characterized by the clonal proliferation of malignant plasma cells [1]. Despite the advanced development of targeted drugs, such as immune modulators and proteasome inhibitors, have greatly improved outcomes of MM patients over the decades, MM remains incurable [2, 3]. As a most severe type among all the subtypes of MM, immunoglobulin D multiple myeloma (IgD MM) is very rare comprising only 1 to 2% of all MM cases featured by diagnosis in relatively young patients, and often accompanied by multiple adverse prognosis, such as extraosseous lesions, renal failure, extramedullary involvement, amyloidosis [4, 5]. The most distinguish characteristic of lgD MM is poor outcome of refractory status, only 13 ~ 21-month overall survival (OS) compared with 3 ~ 6-year overall median survival in common MM [6,7,8]. Therefore, systemic investigation on IgD MM is purposeful to reveal the recurrent and refractory features in both IgD and other types of MM, and beneficial to develop potent therapeutic strategy on MM.

The advanced research achievement of the microenvironmental interactions between MM cells and the BM niche, and their roles in the progression of disease and acquisition of drug resistance, has promoted the development of novel therapeutic drugs for MM treatment [9,10,11,12]. The intercellular interaction in BM niche through exosomes and circular RNAs (circRNAs) attracts extensive attention [13]. CircRNAs, back-spliced products of exonic or intronic sequence of precursor mRNA (pre-mRNA), are a fascinating class of conserved single-stranded RNA molecules [14,15,16]. It has been reported that circRNAs act as essential players in cancer initiation, progression and drug resistance [17, 18]. In particular, circRNAs may influence tumor microenvironment through intercellular communication due to its abundance in exosomes and human body fluids [19]. Therefore, circRNAs are now being considered as promising biomarkers for cancer [20]. Many studies have suggested that circRNAs are translatable, which are translated into previously unknown protein isoforms [5A). We subsequently performed Gene Ontology (GO) function significance enrichment analysis for better understanding of the potential biological effects of these circHNRNPU_603aa-regulated AS events, which were mainly related to cell composition, binding and other functions (Fig. 5B). The pathway enrichment analysis of these circHNRNPU_603aa regulated AS events revealed that their molecular function was centered on ubiquitin mediated proteolysis (Fig. 5C). To further identify the regulation mechanism of circHNRNPU_603aa for AS, we performed de novo discovery of the circHNRNPU_603aa binding motif using the overlap exon sequences of circHNRNPU_603aa regulated AS events and circHNRNPU_603aa binding transcripts from RIP-seq data. Among these circHNRNPU_603aa regulated AS events, the expression of SKP2 exon 5 skip** splice variants was significantly increased in circHNRNPU_603aa-OE cells and ranked the top of AS events (Fig. 5D). SKP2 encodes a member of the F-box protein family, which is characterized by F-box nearly 40 amino acid motif. The F-box proteins constitute one of the four subunits of ubiquitin protein ligase complex called SCF complex, which plays an important role in ubiquitination [35,36,37] and might be related to the molecular function of circHNRNPU_603aa.

Fig. 5
figure 5

Identification of circHNRNPU_603aa-regulated AS events. A Number of AS events in each category. B GO Molecular Function enrichment of circHNRNPU_603aa-regulated AS genes. C The pathway enrichment analysis of above genes revealed that their molecular function was centered on ubiquitin mediated proteolysis. D The expression of SKP2 exon 5 skip** splice variants was significantly increased by increased circHNRNPU_603aa and ranked top of the circHNRNPU_603aa-regulated AS events. E-G Survival proportions of patients corresponding to different probes of SKP2 in APEX cohort. 203626_s_at was related to better survival compared with other probes. H-J The expression of SKP2-NM_001243120.2 in circHNRNPU_603aa-OE and si-circHNRNPU_603aa cells relative to control cells. A two-tailed Student’s t-test was utilized to evaluate statistical significance. K-L Overexpression of circHNRNPU-603aa directly upregulated SKP2-NM_001243120.2 and downregulated SKP2-NM_005983.4 detected by RIP-qPCR. The data are presented as mean ± SD.*p < 0.05, **p < 0.01, ***p < 0.001

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In particular, we found that the probe 203626_s_at, designed on the basis of SKP2- NM_005983.4 splicing variant, was associated with better overall survival compared with other probes in APEX cohort (Fig. 5E-G). We detected the expressions of the two isoforms SKP2-NM_005983.4 and SKP2-NM_001243120.2 in circHNRNPU_603aa-OE cells by qPCR. Interestingly, the expression of SKP2-NM_001243120.2 was increased in circHNRNPU_603aa-OE cells relative to WT cells in both ARP1 and CAG cells (Fig. 5H-I). In comparison, the expression of SKP2-NM_001243120.2 was significantly decreased in si-circHNRNPU_603aa cells compared with NC cells treated with non-targeted siRNA (Fig. 5H-J). Furthermore, we adopted RIP-PCR to confirm circHNRNPU-603aa-regulated exon skip** in MM cells using HA antibody as bait. It was found that circHNRNPU-603aa directly bound to the endogenous SKP2-NM_001243120.2, and elevated circHNRNPU-603aa increased SKP2-NM_001243120.2 expression compared with WT cells (Fig. 5K-L). Collectively, we inferred that circHNRNPU-603aa regulated SKP2 exon skip**, thereby spliced NM_005983.4 into NM_001243120.2, suggesting that circHNRNPU-603aa-regulated SKP2 exon skip** might play an important role in promoting MM progression.

Aberrant splicing of SKP2 contributes to the reduction of c-Myc ubiquitin

To further understand the significance of circHNRNPU-603aa-regulated SKP2 exon skip** and clarify the function of the two splicing isoforms of SKP2 in MM, we first designed a siRNA targeting SKP2-NM_001243120.2 (Fig. 6A-B). As shown in Fig. 6C, the cell proliferation was decreased in si-SKP2-NM_001243120.2 cells relative to NC cells. Since SKP2 is associated with SCF complex participating in c-Myc proteosomal degradation [36], our WB analysis identified that c-Myc expression was decreased in si-SKP2-NM_001243120.2 cells relative to NC cells (Fig. 6D).

Fig. 6
figure 6

Aberrant splicing of SKP2 contributes to the reduction of c-Myc ubiquitin. A Graphic illustration of siRNA targeting SKP2-NM_001243120.2. B qPCR analysis of SKP2-NM_001243120.2 expression in ARP1 and CAG cells. C Inhibition of SKP2-NM_001243120.2 prominently decreased cell proliferation in ARP1 and CAG cells detected by MTT. D WB showed that c-Myc expression was decreased in si-SKP2-NM_001243120.2 cells compared with NC cells. E Graphic illustration of SKP2-NM_005983.4-OE and SKP2-NM_001243120.2-OE plasmids linked with HA and FLAG tag, respectively. F SKP2-NM_001243120.2 FLAG-tagged isoform upregulated c-Myc expression while the SKP2-NM_005983.4 HA-tagged isoform downregulated c-Myc expression in ARP1 and CAG cells. G WB confirmed that c-Myc expression was increased in circHNRNPU_603aa-OE cells and downregulated in si-circHNRNPU_603aa cells. H Co-IP experiment indicated an interaction between SKP2 and c-Myc in MM cells. I SKP2-NM_001243120.2 FLAG-tagged isoform or SKP2-NM_005983.4 HA-tagged isoform overexpressed cells were treated with MG132 for 12 h. An ubiquitination assay was performed using anti-c-Myc magnetic beads, and the ubiquitylated proteins were detected by Ub antibody. The data are presented as mean ± SD.*p < 0.05, **p < 0.01, ***p < 0.001

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As the protein encoded by SKP2-NM_001243120.2 lacks F-Box domain compared with the protein encoded by SKP2-NM_005983.4, which is a component of a SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex mediating the ubiquitination and subsequent proteasomal degradation of c-Myc [36], we further constructed SKP2-NM_005983.4-OE and SKP2-NM_001243120.2-OE plasmids linked with HA and FLAG tags respectively to examine the molecular function of the two splicing isoforms of SKP2 (Fig. 6E). Intriguingly, SKP2-NM_001243120.2 FLAG-tagged isoform upregulated c-Myc expression, while SKP2-NM_005983.4 HA-tagged isoform downregulated c-Myc expression (Fig. 6F). In addition, c-Myc expression was increased in circHNRNPU_603aa-OE cells and decreased in si-circHNRNPU_603aa cells relative to control cells (Fig. 6G). Co-IP assay validated the interaction between SKP2 and c-Myc in MM cells (Fig. 6H). After MM cells were incubated with 20 μM MG132 (a proteasome inhibitor) for 12 h, the ubiquitination was significantly suppressed in SKP2-NM_001243120.2-OE cells compared with SKP2-NM_005983.4-OE cells (Fig. 6I). Therefore, it was speculated that circHNRNPU-603aa mediated the alternative splicing of SKP2 to upregulate circHNRNPU-603aa splicing isoform SKP2-NM_001243120.2, and subsequently competitively inhibited c-Myc ubiquitination and stabilized c-Myc expression (Fig. 6H-I).

MM cells secrete circHNRNPU into the BM microenvironment through exosomes

It is well known that the BM microenvironment is especially important for the oncogenic growth of MM cells, and many studies have explored the effect of circRNAs on the BM microenvironment through intercellular communication [38]. CircRNAs are abundant and stable in exosomes serving as potential biomarkers for cancer detection and transferring biological activity to recipient cells [39]. We extracted the exosomes from the culture supernatant of ARP1 and CAG cells, which were identified by TEM method (Fig. 7A) and WB confirmation of exosomes markers Alix and CD9 (Fig. 7B). As expected, circHNRNPU was detected in exosomes (Fig. 7C-D). We cocultured WT ARP1, WT CAG, HEK-293 and HS-5 cells with CAG circHNRNPU-OE cells using transwell (Fig. 7E), then we found that all the cocultured cells expressed circHNRNPU (Fig. 7F). Under the treatment with GW4869, a well-recognized exosomes inhibitor that could reduce exosomes release [40], circHNRNPU did not migrate into cells in the BM, indicating that circHNRNPU was secreted by MM cells through exosomes (Fig. 7F). Furthermore, application of HA antibody and MS analysis confirmed the specific peptide fragments from circHNRNPU_603aa in the cocultured cells (Fig. 7G-H). After ARP1 and CAG WT cells were cocultured with CAG circHNRNPU-OE cells for 12 h, 24 h, 48 h, IF staining for HA and DAPI showed that the cocultured cells expressed circHNRNPU_603aa in a time-dependent manner (Fig. 7I-J). As depicted in Fig. 7K, the proliferation rate of cocultured ARP1 and CAG cells was significantly increased (p < 0.01) relative to non-cocultured WT cells. Cell cycle analysis also indicated an increased proportion of G2/M phase in cocultured ARP1 and CAG cells relative to non-cocultured cells (Fig. 7L). Summarily, we demonstrated that MM cells could secrete circHNRNPU through exosomes to interfere with various cells in the BM microenvironment (Fig. 8).

Fig. 7
figure 7

MM cells secrete circHNRNPU into the BM microenvironment through exosomes. A Transmission electron microscopy (TEM) was used to characterize exosomes from the culture supernatant of ARP1 and CAG cells. B WB analysis showed the presence of exosome markers Alix and CD9. C-D RNA levels of circHNRNPU and linear HNRNPU ± RNase R were determined by RT-PCR and qRT-PCR. E Graphic illustration of cocultured WT ARP1, CAG, HEK-293, HS-5 cells with the CAG circHNRNPU-OE cells using transwell. F qPCR analysis of circHNRNPU expression in ARP1, CAG, HEK-293, HS-5 cells cocultured with CAG circHNRNPU-OE cells. G WB analysis of circHNRNPU_603aa expression in ARP1, CAG, HEK-293, HS-5 cells cocultured with CAG circHNRNPU-OE cells using HA tag antibody. H The specific peptides from circHNRNPU_603aa were identified by mass spectrometry analysis. I-J Representative confocal images for HA and DAPI showed that circHNRNPU_603aa expression was in time-dependent manner in (I) ARP1 and (J) CAG cells. K MTT assay indicated higher cell proliferation rate of cocultured circHNRNPU-OE MM cells than WT cells. L Cell cycle assay exhibited higher G2/M proportion of cocultured circHNRNPU-OE MM cells than WT cells. The data are presented as mean ± SD.*p < 0.05, **p < 0.01, ***p < 0.001

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Fig. 8
figure 8

Schematic depiction illustrates that MM cells secrete circHNRNPU into BM microenvironment to regulate SKP2 exon skip** and thereby inhibit c-Myc ubiquitin

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Discussion

MM remains an incurable hematologic malignancy due to adverse features, clonal heterogeneity and BM dependency, in which IgD MM is a very rare but most severe subtype [6]. In all MM cases, the low prevalence and insensitivity to diagnostic methods of IgD MM make it intractable [7]. Therefore, effective therapeutic strategies to target both MM cells and BM niche are of great importance to disclose the recurrent and refractory features in IgD and other types of MM. Diagnosis of IgD MM is difficult, since IgD presents minimal or even undetectable M-protein spikes via serum protein electrophoresis (SPEP) [41, 42]. Thus, some cases manifest as hypogammaglobulinemia or present normal SPEP results, which may lead to misdiagnosis of patients in this subgroup [43, 44]. Many studies have focused on IgD MM and circRNAs, which are novel RNA molecules with significant biological functions, therapeutic and diagnostic significance, especially on cellular interaction in the BM niche [45, 46].

In this study, we employed Agilent SBC-ceRNA microarray chips to identify circHNRNPU as the most abundantly and differentially expressed circRNA in IgD MM samples relative to IgG MM samples and NPCs. BaseScopeTM RNA ISH follows a similar workflow and principle to the well-established novel RNAscopeTM RNA ISH technique used for detection of RNA in situ, with hybridization and amplification of target RNA [47, 54]. Changes in clonal dynamics over time during MM progression and drug therapy lead to drug resistance and relapse [55]. Our work has explained that MM cells secrete circHNRNPU into BM microenvironment through exosomes to influence the surrounding cells, and the exact composition and distribution of circRNAs contribute to the clonal evolution. Here, we infer that circRNAs shuttle may play a vital role in clonal competition and therefore lead to treatment failure and relapse in IgD and other types of MM.

The common function model of circRNAs is serving as a miRNA sponge and interacts with associated proteins [67]. Patients with c-Myc translocation have worse progression-free survival (PFS) and overall survival (OS) [68]. In addition, c-Myc alteration is proposed to be a trigger of monoclonal gammopathy of undetermined significance (MGUS) to MM transition [5], and it is regarded as a late genomic event responsible for tumor progression.

Conclusions

Our findings provide a novel and mechanistic insight into circHNRNPU_603aa which is secreted into the BM microenvironment and promotes MM progression through regulating SKP2 exon skip** and subsequently competitively inhibiting c-Myc ubiquitin. CircHNRNPU_603aa may serve as a promising diagnostic marker and potential therapeutic target in MM.