Circ_0007841 promotes the progression of multiple myeloma through targeting miR-338-3p/BRD4 signaling cascade

Abstract

Background: The pathogenesis of multiple myeloma (MM) is not completely known. Uncovering the potential mechanism of MM initiation and progression is essential for identifying novel diagnostic and therapeutic targets. 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). Cell proliferation ability was analyzed through cell counting kit-8 (CCK8) assay, colony formation assay and flow cytometry. Transwell assays were conducted to measure the migration and invasion abilities of MM cells. Cell apoptosis was also assessed by flow cytometry. The interaction between miR-338-3p and circ_0007841 or BRD4 was confirmed by dual-luciferase reporter assay and RNA-pull down assay.

Results: Circ_0007841 was highly expressed in bone marrow (BM)-derived plasma cells of MM patients and MM cell lines than that in healthy volunteers and normal plasma cell line nPCs. Circ_0007841 promoted the proliferation, cell cycle and metastasis and impeded the apoptosis of MM cells. miR-338-3p was a direct target of circ_0007841 in MM cells and circ_0007841 accelerated the progression of MM through targeting miR-338-3p. BRD4 could directly bind to miR-338-3p in MM cells and miR-338-3p exerted an anti-tumor role through targeting BRD4. Circ_0007841 promoted the activation of PI3K/AKT signaling via miR-338-3p/BRD4 axis. Exosomes generated from mesenchymal stromal cells (MSCs) elevated the malignant behaviors of MM cells via circ_0007841.

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.

Keywords: BRD4; Exosome; Multiple myeloma; circ_0007841; miR-338-3p.

Fig. 1

Circ_0007841 elevates the malignant behaviors of MM cells. a The enrichment of circ_0007841 was examined in BM-derived plasma cells of MM patients and normal volunteers by qRT-PCR. b The expression of circ_0007841 was measured in MM cell lines and normal plasma cell line nPCs by qRT-PCR. c, d The level of circ_0007841 was detected in H929 and OPM2 cells transfected with si-NC, si-circ_0007841#1, si-circ_0007841#2 or si-circ_0007841#3 by qRT-PCR. e–k MM cells were transfected with si-NC or si-circ_0007841#1. e, f CCK8 assay was employed to assess the proliferation ability of MM cells. g Colony formation assay was performed for the determination of cell proliferation ability in transfected MM cells. h Flow cytometry was carried out to detect the influence of circ_0007841 silencing on the cycle of MM cells. i, j The metastasis ability of MM cells was evaluated by transwell assays. k The apoptosis of MM cells was analyzed by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 2

miR-338-3p could directly interact with circ_0007841 in MM cells. a miR-338-3p was predicted as a candidate target of circ_0007841 by circinteractome software. b, c Dual-luciferase reporter assay was conducted to verify whether miR-338-3p could bind to circ_0007841 in MM cells. d, e RNA-pull down assay was performed to confirm the target relationship between miR-338-3p and circ_0007841 in MM cells. f, g The expression of miR-338-3p was detected in BM-derived plasma cells of MM patients and healthy volunteers, MM cells and nPCs cells by qRT-PCR. h The correlation between the expression of miR-338-3p and circ_0007841 was analyzed using Spearman’s coefficient. i, j The abundance of circ_0007841 and miR-338-3p was examined in H929 and OPM2 cells transfected with Vector or circ_0007841 by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

Circ_0007841 plays an oncogenic role through targeting miR-338-3p in MM cells. a–i MM 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. a The level of miR-338-3p was examined in MM cells by qRT-PCR assay. b, c The proliferation of MM cells was measured through conducting CCK8 assay. d The proliferation capacity in transfected MM cells was assessed by colony formation assay. e, f The percentage of MM cells in G0/G1, S or G2/M phase was analyzed using flow cytometry. g, h The migration and invasion abilities of MM cells were evaluated by transwell assays. i The apoptosis rate of MM cells in different groups was analyzed by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

BRD4 is validated as a target of miR-338-3p in MM cells. a The complementary sites between miR-338-3p and the 3′UTR of BRD4 were predicted by targetscan software. b, c The luciferase activity was measured in H929 and OPM2 cells transfected with miR-NC or miR-338-3p and BRD4 3′UTR WT or BRD4 3′UTR MUT. d The protein level of BRD4 in BM-derived plasma cells of MM patients and healthy volunteers was detected by Western blot assay. e The level of BRD4 in H929, OPM2 and nPCs cells was evaluated by Western blot assay. f, g The linear relationship between BRD4 and miR-338-3p or circ_0007841 was analyzed using Spearman’s coefficient. h The expression of BRD4 was detected in MM cells transfected with miR-NC or miR-338-3p by Western blot assay. i The protein level of BRD4 was detected in MM cells transfected with Vector, circ_0007841, circ_0007841 + miR-NC or circ_0007841 + miR-338-3p by Western blot assay. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

BRD4 overexpression attenuates the effects of miR-338-3p accumulation on MM cells. a–i MM cells were transfected with miR-NC, miR-338-3p, miR-338-3p + pcDNA or miR-338-3p + BRD4. a qRT-PCR was employed to measure the expression of BRD4 in MM cells. b, c CCK8 assay was applied to assess the proliferation ability of MM cells. d Colony formation assay was performed to analyze the influences of miR-338-3p and BRD4 on the proliferation of MM cells. e, f Flow cytometry was conducted to detect the cell cycle of MM cells. g, h Transwell assays were performed to detect the metastasis of MM cells. i The apoptosis rate of MM cells was examined by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Circ_0007841 activates PI3K/AKT signal pathway through targeting miR-338-3p/BRD4 axis. a–c Western blot assay was performed to detect the levels of BRD4 and PI3K/AKT signaling-related proteins in MM cells transfected with si-NC, si-circ_0007841#1, si-circ_0007841#1 + in-miR-NC or si-circ_0007841#1 + in-miR-338-3p, and gray analysis was used to assess the abundance of these proteins. d–f The expression of BRD4 and PI3K/AKT signaling-associated proteins in MM cells transfected with miR-NC, miR-338-3p, miR-338-3p + pcDNA or miR-338-3p + BRD4 was examined by Western blot assay. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

MSCs-generated exosomes accelerate the malignant potential of MM cells via circ_0007841. a, b The expression of circ_0007841 was detected in the MSCs and MSCs-generated exosomes from the adjacent tissues of MM and normal tissues by qRT-PCR. c Western blot assay was performed to detect the protein levels of exosome-related markers, including CD63 and CD81, in cell lysate and exosomes. d The model showed that MM cells were co-cultured with MSCs, and only exosomes could move from the lower chambers to the upper chambers. e–l MM cells transfected with si-NC or si-circ_0007841#1 were co-cultured with MSCs or not. e, f CCK8 assay was performed to assess the proliferation of MM cells. g The proliferation of MM cells was evaluated by colony formation assay. h The cell cycle of MM cells was detected through conducting flow cytometry. i, j The abilities of migration and invasion of MM cells were assessed by transwell assays. k The apoptosis of MM cells was examined through performing flow cytometry. l The levels of p-PI3K, PI3K, p-AKT and AKT were detected in MM cells by Western blot assay. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001