Circ_0007841 promotes the progression of multiple myeloma via miR-338-3p/BRD4 axis

The pathogenesis of multiple myeloma (MM) is not completely known. Herein, we explored the function and the working mechanism of circular RNA circ_0007841 in MM progression. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to detect the expression of circ_0007841, microRNA-338-3p (miR-338-3p) and bromodomain containing 4 (BRD4). The proliferation and metastasis of MM cells were examined by cell counting kit-8 (CCK8) assay and transwell assays. Flow cytometry was conducted to assess the cell cycle and the apoptosis of MM cells. The targets of circ_0007841 and miR-338-3p were predicted by circinteractome and targetscan softwares, and these predictions were confirmed by dual-luciferase reporter assay and RNA-pull down assay. The protein levels of BRD4, phosphorylated-phosphatidylinositol 3-kinase (p-PI3K), PI3K, p-AKT serine/threonine kinase (p-AKT) and AKT were measured by Western blot assay. Exosomes were extracted using Exosome isolation kit.


Conclusion
Circ_0007841 acted as an oncogene to promote the proliferation, cell cycle and motility and restrain the apoptosis of MM cells through sequestering miR-338-3p to up-regulate the expression of BRD4.

Background
Multiple myeloma (MM) is a kind of hematologic cancer that derived from plasma cells.
Normal plasma cells were transformed into malignant myeloma cells in MM patients [1].
The treatment methods for MM patients include chemotherapy, radiotherapy and target therapy [2][3][4]. Uncovering the molecular mechanism behind the progression of MM and intercellular interaction is important to find more effective treatment methods for MM patients.
Non-coding RNAs (ncRNAs) are a class of RNAs that unable to code proteins, and they are abundant in human genome to regulate the cellular processes including proliferation, metastasis and apoptosis [5]. Circular RNAs (circRNAs) are a kind of ncRNAs that characterized by covalently closed loop structure [6]. CircRNAs are more stable than linear RNAs and they are resistant to exonuclease due to their loop structure [7]. CircRNAs engaged in the pathogenesis of cancers through serving as microRNAs (miRNAs) sponges to modulate the abundance of downstream genes linked to proliferation, metastasis and apoptosis [8,9]. The roles of circRNAs in hematological cancers have been reported before [10,11]. However, the functions of circRNAs in MM remain to be uncovered.
MiRNAs belong to another class of ncRNAs that involved in the progression of cancers through inducing degradation or translational repression of target messenger RNAs (mRNAs) [12]. The dysregulation of miRNAs was involved in the pathogenesis of MM [13,14]. MiR-338-3p suppressed the development of many cancers [15][16][17][18]. As for MM, Cao et al. reported that miR-338-3p suppressed the proliferation and accelerated the apoptosis of 4 MM cells via CDK4 [19]. Nevertheless, the function of miR-338-3p in MM is largely unexplored.
Bromodomain containing 4 (BRD4) is a crucial epigenetic protein, and it has been reported to elevate the levels of oncogenic proteins and accelerate the progression of cancers [20]. Zheng et al. claimed that H19 accelerated the development of MM through up-regulating BRD4 via sponging miR-152-3p [21]. Here, the direct interaction between miR-338-3p and BRD4 was first found in MM, and the function of BRD4 in MM was illustrated.
In this study, circ_0007841 was abnormally up-regulated in MM. The biological functions and the underlying mechanism of circ_0007841 in MM were further explored.

Bioinformatic Prediction And Dual-luciferase Reporter Assay
The targets of circ_0007841 and miR-338-3p were predicted by circinteractome and targetscan software, respectively.
The wild-type sequence in circ_0007841 predicted to bind to miR-338-3p, along with the mutant-type sequence, was amplified and cloned into pGL3 luciferase reporter vector (Promega, Madison, WI, USA), termed as circ_0007841 WT or circ_0007841 MUT. MM cells were co-transfected with miR-NC or miR-338-3p and circ_0007841 WT or circ_0007841 MUT. After 48-h transfection, MM cells were harvested and the luciferase activity was detected with the dual-luciferase reporter assay system (Promega).
The confirmation of the target relationship between miR-338-3p and BRD4 was conducted according to the similar procedures.

RNA-pull Down Assay
Biotin RNA Labeling Mix (Roche, Shanghai, China) was used in this study. The wild-type and mutant-type binding sites of circ_0007841 that were predicted to bind to miR-338-3p were biotinylated to obtain Bio-circ_0007841 WT and Bio-circ_0007841 MUT. MM cells were disrupted and incubated with Bio-NC, Bio-circ_0007841 WT or Bio-circ_0007841 MUT. The abundance of miR-338-3p was measured by qRT-PCR.

Exosome Isolation
Exosome isolation kit (Qiagen, Frankfurt, Germany) was used to extract exosomes from the culture medium of MM cells according to previous studies [22,23].

Statistical analysis
All statistical data were shown as mean ± standard deviation (SD). Data were analyzed using GraphPad Prism 7.0. The differences between two groups or among more than two 8 groups were assessed through using Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's test. The comparison between groups was considered significant when P value less than 0.05. Linear correlation was analyzed using Spearman's correlation coefficient.

Results
Circ_0007841 promotes the progression of MM Circ_0007841 was abnormally up-regulated in bone marrow (BM)-derived plasma cells from MM patients compared with that in healthy individuals (Fig. 1A). Meanwhile, the level of circ_0007841 was higher in MM cells than that in normal plasma cells (nPCs, Fig. 1B).
Circ_0007841 specific small interfering RNAs were used to knockdown circ_0007841 to uncover its biological functions in MM cells. As mentioned in Fig. 1C, the level of circ_0007841 was down-regulated with the transfection of si-circ_0007841#1, si-circ_0007841#2 or si-circ_0007841#3. Among these three siRNAs, si-circ_0007841#1 was chose for the following assays due to its highest knockdown efficiency (Fig. 1C). According to the results of CCK8 assay, si-circ_0007841#1 transfection inhibited the proliferation of MM cells ( Fig. 1D and 1E). The cell cycle of MM cells was arrested in G1/S transition in si-circ_0007841#1 group than that in si-NC group (Fig. 1F). What's more, circ_0007841 interference notably suppressed the migration and invasion of MM cells ( Fig. 1G and 1H).
The apoptosis of MM cells was promoted in si-circ_0007841#1 group compared with that in si-NC group (Fig. 1I). Overall, circ_0007841 accelerated the proliferation, cell cycle, metastasis and inhibited the apoptosis of MM cells.

MiR-338-3p Could Directly Interact With circ_0007841 In MM Cells
To address the mechanism by which circ_0007841 functioned in MM cells, circinteractome website was used to seek the targets of circ_0007841. As shown in Fig. 2A, miR-338-3p possessed the complementary sites with circ_0007841. However, the interaction between miR-338-3p and circ_0007841 has never been reported. The luciferase activity was dramatically reduced in circ_0007841 WT group when co-transfecting with miR-338-3p ( Fig. 2B and 2C). RNA-pull down assay revealed that miR-338-3p could be pulled-down when using Bio-circ_0007841 WT, proving the target relationship between miR-338-3p and circ_0007841 ( Fig. 2D and 2E). An obvious decrease in the level of miR-338-3p was observed in BM-derived plasma cells from MM patients in contrast to that in normal volunteers (Fig. 2F). Additionally, there was a prominent reduction in the expression of miR-338-3p in MM cells than that in nPCs cells (Fig. 2G). The expression of miR-338-3p was negatively correlated with the level of circ_0007841 in BM-derived plasma cells from MM patients (Fig. 2H). The overexpression efficiency of circ_0007841 was high in MM cells, and circ_0007841 accumulation caused a notable decrease in the level of miR-338-3p in MM cells ( Fig. 2I and 2J). In summary, circ_0007841 could inversely regulate the expression of miR-338-3p through direct interaction.
Circ_0007841 plays an oncogenic role through targeting miR-338-3p in MM cells H929 and OPM2 cells were transfected with si-NC, si-circ_0007841#1, si-circ_0007841#1 + in-miR-NC or si-circ_0007841#1 + in-miR-338-3p to disclose the functions of circ_0007841 and miR-338-3p. As mentioned in Fig. 3A, si-circ_0007841#1 transfection increased the level of miR-338-3p, and the introduction of in-miR-338-3p reversed the influence of circ_0007841 silencing in the expression of miR-338-3p. We also detected the proliferation ability, cell cycle, motility and apoptosis of MM cells with same transfection.
Si-circ_0007841#1-mediated inhibitory effect on the proliferation of MM cells was counteracted by the interference of miR-338-3p ( Fig. 3B and 3C). Additionally, cell cycle of MM cells was arrested at G1/S transition in si-circ_0007841#1 group, and this suppressive impact in the cell cycle of MM cells was attenuated by the addition of in-miR-338-3p ( Fig. 3D and 3E). The migration and invasion of MM cells were suppressed by the knockdown of circ_0007841, and the metastasis ability was recovered in si-circ_0007841#1 and in-miR-338-3p co-transfected group (Fig. 3F and 3G). Si-circ_0007841#1-induced apoptosis of MM cells was suppressed by the addition of in-miR-338-3p (Fig. 3H). Overall, circ_0007841 could promote the malignant potential of MM cells through sponging miR-338-3p.
BRD4 is validated as a target of miR-338-3p in MM cells BRD4 was predicted as a direct target of miR-338-3p by targetscan database (Fig. 4A). As exhibited in Fig. 4B and 4C, the luciferase activity was markedly decreased in miR-338-3p and BRD4 3'untranslated region (3'UTR) WT co-transfected group, while miR-338-3p transfection had no effect on the luciferase activity in BRD4 3'UTR MUT group compared with that in miR-NC and BRD4 3'UTR MUT co-transfected group. BRD4 was conspicuously up-regulated in BM-derived plasma cells of MM patients compared with that in healthy individuals (Fig. 4D). Meanwhile, BRD4 was also found to be up-regulated in MM cells than that in nPCs cells (Fig. 4E). The expression correlation between BRD4 and circ_0007841 or miR-338-3p was analyzed using Spearman's correlation coefficient. As shown in Fig. 4F and 4G, there was an inverse correlation between the levels of BRD4 and miR-338-3p, while the expression of BRD4 was positively correlated with the level of circ_0007841.

BRD4 overexpression attenuates the effects of miR-338-3p accumulation on MM cells
MiR-338-3p and BRD4 were co-transfected into MM cells to explore whether miR-338-3p exerted an anti-tumor role in MM cells through targeting BRD4. As shown in Fig. 5A, the addition of BRD4 overexpression plasmid recovered the expression of BRD4 in MM cells that was down-regulated by the accumulation of miR-338-3p. MiR-338-3p overexpression inhibited the proliferation, cell cycle and metastasis of MM cells, and these inhibitory effects were attenuated by the addition of BRD4 overexpression plasmid (Fig. 5B-5G). The apoptosis of MM cells was induced by the transfection of miR-338-3p, and the introduction of BRD4 overexpression plasmid recovered the viability of MM cells (Fig. 5H). In conclusion, miR-338-3p accumulation-mediated influence on MM cells was overturned by the introduction of BRD4.
The activation of PI3K/AKT signal pathway is modulated by circ_0007841/miR-338-3p/BRD4 axis The activation of PI3K/AKT signal pathway is linked to the promotion of cell proliferation and metastasis and the inhibition of cell apoptosis. Herein, we examined the phosphorylation levels of PI3K and AKT to illustrate the influence of circ_0007841/miR-338-3p/BRD4 axis in the activation of PI3K/AKT signaling. Circ_0007841 silencing downregulated the level of BRD4, and the level of BRD4 was recovered in si-circ_0007841#1 and in-miR-338-3p co-transfected group ( Fig. 6A and 6B). The activation of PI3K/AKT signaling was suppressed with the silencing of circ_0007841, and the addition of in-miR-338-3p recovered the phosphorylation levels of PI3K and AKT ( Fig. 6A and 6C). Meanwhile, H929 and OPM2 cells were transfected with miR-NC, miR-338-3p, miR-338-3p + pcDNA or miR-338-3p + BRD4. As mentioned in Fig. 6D and 6E, miR-338-3p overexpression downregulated the level of BRD4, and the introduction of BRD4 overexpression plasmid regained the level of BRD4 in MM cells. The addition of BRD4 alleviated the inhibitory influence of miR-338-3p overexpression on the activation of PI3K/AKT signaling in MM cells ( Fig. 6D and 6F). Taken together, circ_0007841 accelerated the progression of MM through miR-338-3p/BRD4/PI3K/AKT axis.

Mesenchymal stromal cells (MSCs)-generated exosomes accelerate the malignant potential of MM cells via circ_0007841
MSCs exert crucial roles in the progression of MM. Herein, we explored whether exosomes derived from MSCs could regulate the proliferation, cell cycle, metastasis and apoptosis of MM cells via circ_0007841. MSCs were isolated from the adjacent tissues of MM and normal tissues. The expression of circ_0007841 was higher in MSCs and MSCs-derived exosomes from adjacent tissues than that in normal tissues ( Fig. 7A and 7B). The markers of exosomes (CD63 and CD81) were notably up-regulated in exosomes of MSCs instead of cell lysate (Fig. 7C). As mentioned in Fig. 7D, we established a working model as previously described to explore if MSCs-derived exosomes could regulate the proliferation, cell cycle, motility and apoptosis of MM cells [24]. In this model, only exosomes could be transmitted through the filter to the upper chambers. As presented in Fig. 7E-7J, si-circ_0007841#1 transfection inhibited the malignant behaviors of MM cells in Mock + si-circ_0007841#1 group compared with that in Mock + si-NC group. Besides, MSCs-derived exosomes (MSCs + si-NC group) promoted the proliferation, cell cycle, metastasis and inhibited the apoptosis of MM cells than that in Mock + si-NC group, and these effects were attenuated by the silencing the circ_0007841, suggested that MSCs-derived exosomes could promote the progression of MM via circ_0007841. What's more, the exosomes generated from MSCs accelerated the activation of PI3K/AKT signaling, while this effect was counteracted with the transfection of si-circ_0007841#1 (Fig. 7K). Collectively, MSCsderived exosomes could facilitate the progression of MM via circ_0007841.

Discussion
MM is an incurable cancer at present. Because many MM patients were diagnosed at late 13 stage, the treatment outcomes were unsatisfactory [25]. Therefore, finding crucial markers in MM is urgent to improve the therapeutic effects.
CircRNAs featured by closely loop structure and they are widely distributed in human tissues. Due to the stability and the universality of the distribution, circRNAs are identified as ideal biomarkers for cancers and other diseases [26]. For example, the high expression of circ_0004277 was associated with the better prognosis of AML patients [27]. Xia et al. claimed that circ-CBFB was highly expressed in chronic lymphocytic leukemia, and circ-CBFB accelerated the proliferation and suppressed the apoptosis of chronic lymphocytic leukemia cells [10]. Circ_0007841 was found to be overexpressed in BM-derived plasma cells of MM patients and MM cells. Further studies suggested that circ_0007841 promoted the proliferation, cell cycle, metastasis and inhibited the apoptosis of MM cells. These findings demonstrated that circ_0007841 might be an important biomarker for MM patients, which was in agreement with the former article [28]. However, the regulatory mechanism by which circ_0007841 promoted the progression of MM was unclear.
MiRNAs are single-stranded ncRNAs, and they are implicated in cell proliferation, metastasis and apoptosis through base pairing with mRNAs [29]. Additionally, circRNAs could act as miRNAs sponges to function [30]. suppressed the metastasis of colorectal cancer cells through targeting smoothened [33].
MiR-338-3p was also found to be down-regulated in MM, and it suppressed the proliferation and facilitated the apoptosis of MM cells via CDK4 [19]. MiR-338-3p was identified as a novel target of circ_0007841 in MM cells in our study. Subsequently, rescue experiments were performed to explore whether circ_0007841 functioned through 14 sponging miR-338-3p. We found that si-circ_0007841#1-mediated effects in MM cells were alleviated by the transfection of in-miR-338-3p, suggested that circ_0007841 acted as an oncogene in MM through targeting and down-regulating miR-338-3p.
To uncover the potential mechanism that was responsible for the functions of miR-338-3p in MM cells, the downstream genes of miR-338-3p were searched using targetscan software. BRD4 was confirmed as a candidate gene of miR-338-3p. BRD4 was regulated by circ_0007841/miR-338-3p axis in MM cells. High expression of BRD4 promoted the progression of high-grade serous ovarian cancer [34]. Besides, BRD4 was found to be a target of H19/miR-152-3p axis to promote the progression of MM [21]. Rescue experiments revealed that BRD4 overexpression attenuated the inhibitory effects of miR-338-3p transfection on the proliferation, cell cycle and metastasis and the promoting effect on the apoptosis of MM cells, proved that BRD4 acted as a target of miR-338-3p to promote the progression of MM.
PI3K/AKT signal pathway was related to cell proliferation, viability, apoptosis and autophagy. Accumulating articles have reported the association between the proproliferative influence of circRNAs in cancer cells and the activation of PI3K/AKT pathway.
For example, circ-IGF1R promoted the proliferation and blocked the apoptosis of hepatocellular carcinoma cells through activating PI3K/AKT pathway [35]. Liu et al. found that circ_8073 accelerated the proliferation of caprine endometrial epithelial cells through activating PI3K/AKT/mTOR pathway via miR-449a/CEP55 axis [36]. Further studies demonstrated that the activation PI3K/AKT signaling could be regulated by circ_0007841/miR-338-3p/BRD4 axis in MM cells. Additionally, we found that MSCsgenerated exosomes could accelerate the progression of MM via circ_0007841.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of The Fifth Affiliated Hospital of Zhengzhou University and written informed consents were collected from all patients and hospitals.

Availability of data and materials
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no conflicts of interest.

Funding
The project was funded by the Henan Science and Technology Project (No. 202102310372)

Authors' contributions
Yan Wang and Quande Lin performed experiments, analyzed data, and wrote the manuscript. Chunge Song and Ruojin Ma designed research, performed experiments, and analyzed data. Xiaojie Li conceived and designed research.