Plasma cells from MM patients (n=41) and healthy volunteers (n=41) in The Fifth Affiliated Hospital of Zhengzhou University were collected to detect the expression of circ_0007841, miR-338-3p and BRD4 via qRT-PCR and Western blot assay.
MM cell lines (H929 and OPM2) and normal plasma cell line nPCs were purchased from BeNa Culture Collection (Beijing, China) and maintained in Roswell Park Memorial Institute-1640 (RPMI-1640) medium (Gibco, Carlsbad, CA, USA) added with 10% fetal bovine serum (FBS; Gibco), 100 units/mL penicillin and 100 μg/mL streptomycin. Cell culture plates were placed in a 5% CO2 incubator at 37℃, and cells were collected in the log phase of growth.
Quantitative real-time polymerase chain reaction (qRT-PCR)
After measuring the concentration using NanoDrop 2000 (Invitrogen, Carlsbad, CA, USA), RNA sample (2 ng) was used to synthesize complementary DNA (cDNA) with ReverTra Ace qPCR RT Kit (for circ_0007841, BRD4, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6; Takara, Dalian, China) and All-in-OneTM miRNA First stand cDNA Synthesis Kit (for miR-338-3p; GeneCopoeia, Rockville, MD, USA). U6 served as the internal control for miR-338-3p, while GAPDH acted as the internal reference for circ_0007841 and BRD4. PCR amplification reaction was conducted with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on an ABI 7900 thermocycler (Applied Biosystems). The quantification of circ_0007841, miR-338-3p and BRD4 was carried out with the 2−ΔΔCt method. The specific primers in this study were synthesized from Sangon Biotech (Shanghai, China) and listed as below: circ_0007841 (Forward, 5’-CTAACATCTGTGAAACCATCGT-3’; Reverse, 5’-TCATCACATACACGATAGACTGG-3’), miR-338-3p (Forward, 5’-UCCAGCAUCAGUGAUUUUGUUG-3’; Reverse, 5’-CAACAAAAUCACUGAUGCUGGA-3’), BRD4 (Forward, 5’-GTGGTGCACATCATCCAGTC-3’; Reverse, 5’-CCGACTCTGAGGACGAGAAG-3’), U6 (Forward, 5’-CTCGCTTCGGCAGCACA-3’; Reverse, 5’-AACGCTTCACGAATTTGCGT-3’), GAPDH ( Forward, 5’-GCGACACCCACTCCTCCAC-3’; Reverse, 5’-TCCACCACCCTGTTGCTGTAG-3’).
Three small interfering RNAs (siRNAs) targeting circ_0007841, including si-circ_0007841#1 (5’-UGUUAGUUGCAAUGAAGAGAG-3’), si-circ_0007841#2 (5’-UAAUGAUCAUGCCAAAUACUC-3’) and si-circ_0007841#3 (5’-UCACAUACACGAUAGACUGGC-3’), its negative control (si-NC), circ_0007841 overexpression plasmid (circ_0007841), its control (Vector), BRD4 overexpression plasmid (BRD4), its control (pcDNA), miR-338-3p mimics (miR-338-3p), its control miR-NC, miR-338-3p inhibitor (in-miR-338-3p) and its control in-miR-NC were obtained from Genepharma (Shanghai, China). MM cells were seeded into 24-well plates at a density of 2 × 105 cells/well overnight, and transfection was conducted with Lipofectamine 3000 (Invitrogen).
Cell counting kit-8 (CCK8) assay
MM cells were plated in 96-well plates at the density of 5 × 103 cells/well and cultured overnight. After transfection for indicated time points (0 h, 24 h, 48 h or 72 h), MM cells were incubated with 10 μL CCK-8 (Sigma, St. Louis, MO, USA) for 4 h. The absorbance at 450 nm was detected by a microplate reader (BioTek, Winooski, VT, USA).
Flow cytometry for cell cycle and apoptosis detection
For cell cycle analysis, MM cells were collected using cold phosphate buffer saline (PBS) and then immobilized using 70% cold ethanol solution overnight. Prior to propidium iodide (PI; Solarbio, Beijing, China) staining, RNase was used to remove RNA in the samples. The percentage of MM cells in different phases of cell cycle was detected on the FACSCalibur (Becton Dickinson, San Jose, CA, USA) and analyzed using Cell Quest software (Becton Dickinson).
For apoptosis analysis, after transfection for 72 h, MM cells were collected with PBS, and then these cells were suspended in binding buffer. Annexin V-combined fluorescein isothiocyanate (Annexin V-FITC; Solarbio) and PI (Solarbio) were added to the reaction mixture, and MM cells were simultaneously incubated with Annexin V-FITC and PI at 37℃ for 15 min in a dark room. The apoptotic MM cells were identified by FACSCalibur (Becton Dickinson) and analyzed using Cell Quest software (Becton Dickinson).
In transwell migration assay, cell suspension (MM cells suspended in 100 μL serum-free medium) was added into the upper chambers (Costar, Corning, NY, USA). A total of 600 μL culture medium with 10% FBS was added into the lower chambers. FBS acted as the chemotactic factor in this study. After 24-h incubation, MM cells remained in the upper surface were removed with the cotton swab, and the migrated MM cells were fixed with 4% paraformaldehyde (Sigma) for 20 min and stained with 0.5% crystal violet (Sigma). The number of migrated MM cells in five random visual fields was counted by the microscope (Olympus, Tokyo, Japan).
In transwell invasion assay, the upper chambers were pre-coated with 50 μL Matrigel (Sigma) to mimic the extracellular matrix. The detection of cell invasion was conducted through using these pre-coated transwell chambers following the similar procedure.
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 partial sequence in circ_0007841 that predicted to bind to miR-338-3p, along with the mutant-type sequence with miR-338-3p in circ_0007841 that was synthesized through using Site-directed gene mutagenesis kit (Takara, Dalian, China), 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 10 nM miR-NC or miR-338-3p and 40 ng 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 kit (Promega) using the luminometer (Plate Chameleon V, Hidex, Finland) according to the manufacturer’s instructions. Firefly luciferase activity in each group was normalized to Renilla fluorescence intensity.
The wild-type fragment of BRD4 3’ untranslated region (3’UTR) that predicted to bind to miR-338-3p and the mutant type fragment of BRD4 3’UTR were also amplified and inserted into pGL3 luciferase reporter vector (Promega) to generate BRD4 3’UTR WT and BRD4 3’UTR MUT. Co-transfection of MM cells with BRD4 3’UTR WT or BRD4 3’UTR MUT and miR-NC or miR-338-3p was conducted following the similar procedure.
RNA-pull down assay
RNA-pull down assay was conducted to test the interaction between circ_0007841 and miR-338-3p. Biotin RNA Labeling Mix (Roche, Shanghai, China) was used in this study. The wild-type and mutant-type binding sites in 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.
Western blot assay
Proteins were obtained using whole cell lysis buffer (Roche, Basel, Switzerland) for 30 min on the ice. Protein samples were quantified using Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Then, 30 µg of proteins were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to the polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% w/v nonfat dry milk for 1 h, primary antibodies were used to probe the indicated proteins followed by incubation with the secondary antibody (ab205718; Abcam, Cambridge, MA, USA). The protein bands were measured using the enhanced chemiluminescent (ECL) system (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Gray analysis was conducted to quantify the expression of proteins using ImageJ software. Primary antibodies, including anti-BRD4 (ab128874), anti-phosphorylated-phosphatidylinositol 3-kinase (anti-p-PI3K; ab70912), anti-PI3K (ab32089), anti-p-AKT serine/threonine kinase (p-AKT; ab38449), anti-AKT (ab64148), anti-CD63 (ab59479), anti-CD81 (ab79559) and anti-β-actin (ab8226), were purchased from Abcam.
Exosome isolation kit (Qiagen, Frankfurt, Germany) was used to extract exosomes from the culture medium of MM cells according to previous studies [22, 23].
All statistical data in three independent experiments 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 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.