Patients
Plasma cells from MM patients (n = 41) and healthy volunteers (n = 41) in The Fifth Affiliated Hospital of Zhengzhou Univercity were collected to detect the expression of circ_0007841, miR-338-3p and BRD4.
Cell Culture
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℃.
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 (Takara, Dalian, China) and All-in-OneTM miRNA First stand cDNA Synthesis Kit (GeneCopoeia, Rockville, MD, USA). Housekeeping genes served as the controls in this study. The quantification of circ_0007841, miR-338-3p and BRD4 was carried out with the 2−ΔΔCt method. The specific primers enrolled in this study were 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’), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Forward, 5’-GCGACACCCACTCCTCCAC-3’; Reverse, 5’-TCCACCACCCTGTTGCTGTAG-3’).
Cell Transfection
Three small interfering RNAs (siRNAs) targeting circ_0007841, including si-circ_0007841#1, si-circ_0007841#2 and si-circ_0007841#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).
Cell Counting Kit-8 (CCK8) Assay
MM cells were plated in 96-well plates 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.
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. Prior to propidine iodide (PI; Solarbio, Beijing, China) staining, RNase was used to remove RNA in the samples. The percentage of MM cells in different phases was detected on a flow cytometer.
For apoptosis analysis, 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 used to identify apoptotic MM cells on a flow cytometer.
Transwell Assays
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, and the migrated MM cells were fixed and counted.
In transwell invasion assay, the upper chambers were pre-coated with 50 µL Matrigel (Sigma) to mimic the extracellular matrix. The other steps were similar as mentioned above.
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.
Western Blot Assay
Proteins were obtained using whole cell lysis buffer (Roche, Basel, Switzerland). 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, 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). Gray analysis was conducted to quantify the expression of proteins. 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
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 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.