Samples

This study analyzed the CPEB2 and ARPC5 expression in the bone marrow samples collected from 20 MM patients who were treated at the First Affiliated Hospital, Department of Hematology, Hengyang Medical School, University of South China. Also, 20 healthy normal donors (iron deficiency anemia patients, due to menorrhagia or hemorrhoid blood loss, and excluded malignant tumors) were recruited in the First Affiliated Hospital, Department of Hematology, Hengyang Medical School, University of South China and their bone marrow samples were obtained. The percentage of CD138+ cells in the bone marrow of MM patients was obtained from the pathology report, and the purity of CD138+ plasma cells is over 90%. CD138 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were used to extract CD138+ primary plasma cells from the bone marrow samples of MM patients and healthy normal donors. Each subject signed written informed consent. This study was approved by the Ethics committee of the First Affiliated Hospital, Department of Hematology, Hengyang Medical School, University of South China.

Cell culture

MM cell OPM2 was purchased from Biovector (Beijing, China), and RPMI-8226, NCl-H929, U266, MM1S and normal plasma cells (nPCs) were bought from ATCC (Manassas, VA, USA). Cells were cultured at 37 °C with 5% CO2 in RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 10% FBS (Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA).

Quantitative real-time PCR (qRT-PCR)

RNA was isolated from cells using TRIzol reagent (Invitrogen), and cDNA was obtained by HiScript III RT SuperMix (Vazyme, Nanjing, China). Then, cDNA was amplified in PCR system by SYBR Green (Vazyme) with specific primers (Table 1). Gene expression was calculated 2−ΔΔCt method with GAPDH as internal control.

Table 1 Primer sequences used for qRT-PCRWestern blot

RIPA buffer (Beyotime, Shanghai, China) was used to extract total proteins. After quantified, protein was separated by 10% SDS-PAGE gel and transfer onto PVDF membranes. The membrane was blocked with nonfat milk, and incubated with anti-CPEB2 (ab222070, 1:100, Abcam, Cambridge, MA, USA), anti-ARPC5 (ab51243, 1:5000, Abcam) or anti-GAPDH (ab9485, 1:2500, Abcam). After hatched with secondary antibody (ab205718, 1:50,000, Abcam), membrane was treated with BeyoECL Moon (Beyotime) to observe protein signals. The gray value was analyzed by ImageJ software.

Cell transfection

Lipofectamine 3000 (Invitrogen) was used to transfect with CPEB2 or ARPC5 lentivirus short hairpin RNA (sh-CPEB2 or sh-ARPC5), pcDNA overexpression vector (pc-CPEB2 or pc-ARPC5), and their negative controls (sh-NC and pc-NC) (synthesized by RiboBio, Guangzhou, China) into cells in-line with the manufacturer’s instructions.

Cell counting kit 8 (CCK8) assay

MM cells were seeded into 96-well plates and cultured overnight. At the indicated time points, CCK8 solution (Beyotime) was used for incubation with cells for 4 h. Optical density (OD) values were analyzed using microplate reader at 450 nm.

Soft-agar colony formation assay

MM cells suspended with 0.3% agar in complete medium were seeded in a 24-well plate containing complete medium with 0.5% agar. Cells were cultured for 2 weeks, and then the stained colonies were counted with AlphaView software.

Flow cytometry

After transfection for 48 h, MM cells were harvested and suspended with binding buffer. After that cells were stained with Annexin V-FITC and propidium iodide using corresponding assay kit (Solarbio, Beijing, China). Apoptosis analysis was performed by flow cytometer (BD Bioscience, San Jose, CA, USA).

Tube formation assay

The medium collected from transfected MM cells were harvested to prepare conditioned medium. HUVECs suspended with conditioned medium were seeded into 96-well plates pre-coated with Matrigel (BD Bioscience). Tube formation was observed after 8 h under a microscope and documented with ImageJ software.

Fluorescence in situ hybridization (FISH) assay

OPM2 cells were fixed with 4% paraformaldehyde and then hybridized with FISH-CPEB2 probe and FISH-ARPC5 probe (Genepharma, Shanghai, China) overnight. After that cell nuclei was stained with DAPI, and fluorescence image was analyzed by confocal microscopy.

Actinomycin D treatment

After transfection, MM cells were harvested with 20 μg/mL actinomycin D (Cell Signaling Technology, Danvers, MA, USA) as previously described [16]. At 0, 2, 4, 6 and 8 h post-reaction, cells were collected to measure ARPC5 mRNA expression by qRT-PCR.

Cycloheximide (CHX) chase assay

Transfected MM cells were treated with 100 mM CHX (Millipore, Billerica, MA, USA) as previously described [11] for 0, 1, 3 and 6 h, respectively. At each incubation time, cells were used for protein extraction to detect ARPC5 protein expression using western blot analysis.

RNA immunoprecipitation (RIP) assay

MM cells were transfected with pcDNA3.1(+)-CPEB2-Flag and incubated for 72 h. Then, cells were treated with RIP lysis buffer (Millipore), and cell lysates were pre-cleaned with protein G Sepharose beads. After incubated with anti-Flag antibody or anti-IgG antibody, the immunoprecipitated RNA was extracted for qRT-PCR to examine GAPDH and ARPC5 expression.

Animal experiments

The BALB/c nude mice (Vital River, Beijing, China) were randomly divided into two groups: sh-NC group and sh-CPEB2 group (six mice in each group). OPM2 cells were transfected with sh-CPEB2/sh-NC, and then injected subcutaneously into mice. Tumor volume was measured every 7 d following injection. Mice were euthanized after 28 days and tumor tissues were collected. In addition to being weighed and examined, the tumor tissue was also made into paraffin sections for Ki-67 immunohistochemical (IHC) staining using anti-Ki-67 (ab15580, 1:200, Abcam) and SP Kit (Solarbio). Our study was approved by the Animal Ethics committee of the First Affiliated Hospital, Department of Hematology, Hengyang Medical School, University of South China.

Statistical analysis

Data were presented as the mean ± SD and analyzed by Student’s t test (for two groups) or one-way ANOVA followed by Tukey post hoc test (for multiple groups). GraphPad 7.0 software was applied for data analysis. P < 0.05 was considered statistically significant.