Cell culture
Human myeloma cell lines U266, RPMI 8226 and H929 were obtained from our lab. Primary human BMSCs were separated as previously described9 while myeloma CD138+ plasma cells were purified from bone marrow aspirates using CD138-based immunomagnetic selection (AutoMacs; Miltenyi) following the manufacturer’s instructions. The purity of the CD138+ plasma cells was > 90%. The cells were grown in DMEM basal medium (GIBCOBRL, Grand Island, NY, USA) supplemented with 15% fetal bovine serum (FBS, GIBCO-BRL), 1% penicillin-streptomycin and 1% glutamine in a humidified atmosphere with 5% CO2 at 37℃.
RNA isolation and reverse transcription
Total RNA was isolated from the myeloma cells using Trizol reagent (Invitrogen, USA) following the manufacturer's instructions. We then quantified the total RNA concentration using a nucleic acid protein ultraviolet analyzer (NanoPhotometerTM, IMPLEN, German). We then synthesized complementary DNA (cDNA) from the RNA using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, USA) following standard protocols:42°C for 60 min, 70°C for 5 min. The reaction contained 1500 ng of the RNA samples, 9 μl of RT master mix including 2 μl of 10 mM dNTP mix, 1 μl of 200 u/μl reverse transcriptase, 4 μl of 5×reaction buffer, 1 μl of 20 u/μl RNase inhibitor and 1 μl of 62.5 nM stem loop reverse transcription primers as well as nuclease-free H2O to 20 μl.
Affymetrix chip screening for miRNA
The myeloma cells U266, RPMI 8226 or H929 were co-cultured with primary BMSCs for 24 hours and then we aspirated the suspended myeloma cells gently for chip screening. The experiment was carried out using Affymetrix miRNA 4.0 chip following the manufacturer's instructions. The arrays were scanned by the Affymetrix Scanner 3000 (Affymetrix) while Affymetrix GeneChip Command Console software (version4.0, Affymetrix) was used to analyze the array images to obtain raw data and then performed RMA normalization. Thereafter, data analysis was performed by Genespring software (version 12.5, Agilent Technologies).
Real-time quantitative polymerase chain reaction (qRT-PCR)
We performed qRT-PCR at 95°C for 10 min, 95°C for 15 s, 60°C for 31 s and 72°C for 31 s for 40 cycles. The reaction was conducted in three replicate wells for each sample and the average data were calculated using the 2-△△Ct method. U6 was used as an internal control. The 20 μl PCR reaction system included 10μl SYBR Green Ⅰ Mix, 0.7μl forward primer, 0.7μl reverse primer, 3.0μl cDNA and 5.6μl nuclease-free water.
Cell transfection
miR-30a-5p-mimics and their negative control, miR-30a-5p-inhibitor and its negative control were synthesized by GenePharma Inc. (Shanghai, China). The BCL2L11 overexpression plasmid, shRNA and negative controls were constructed by Jikai Company (Shanghai, China). Plasmid transfection experiment was carried out with lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions.
Luciferase reporter assay
Wild type (WT) or mutant (Mut) BCL2L11 3′UTRs were cloned into the luciferase reporter vector pSI-Check2 (Hanbio Biotechnology, Shanghai, China). 0.16μg BCL2L11-3′UTR luciferase construct and 5pmol miR-30a-5p/negative control were transfected into HEK293T cells using lipofectamine 2000. The cells were harvested after 48h of transfection and then the luciferase activity was measured by promega dual-luciferase system following the manufacturer’s instructions.
Western blot
Anti-BCL2L11 (1:800, Cell Signal Technology, MA, USA), P65, phosphor-P65, phosphor-IκBα, histone (1:800, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-β-actin antibodies (1:1000, Lianke Biotechnology Co., Ltd, Hangzhou, China) were used for the Western blot assays. Total protein, nucleoprotein and cytoplasm protein were extracted from the myeloma cells using the extract kit and cell lysis buffer (Beyotime Biotechnology Co., Ltd., Haimen, China). The protein purity and concentration were analyzed by a NanoPhotometer (Implen, Inc., CA, USA). After the PAGE, the proteins were transferred into PVDF membranes and then incubated with the primary antibodies. After blocking, the blots were incubated with secondary antibodies. Western blot bands were visualized in the gel imaging system machine using enhanced chemiluminescence (ECL) kit (Beyotime Biotechnology Co., Ltd., Haimen, China).
NF-κB P65 nuclear translocation assay
The NF-κB nuclear translocation assay was performed as previously described14. Briefly, the myeloma cells that were directly adherent to BMSCs were fixed, washed and then blocked with BSA for one hour at room temperature. The myeloma cells were subsequently incubated with anti-NF-κB p65 antibodies and Alexa Fluor488-conjugated secondary antibodies successively. On the other hand, the cell nucleus was stained with hoechst 33342. Images were evaluated by the leica dm500 fluorescence microscope (scale bar=50μm).
Detection of apoptosis using flow cytometry
We co-cultured the myeloma cells directly or indirectly with primary BMSCs, with or without bortezomib for 24 hours. The myeloma cells were transfected with miR-30a-5p mimics, inhibitor, BCL2L11 overexpression plasmid, BCL2L11 shRNA or negative controls. The myeloma cells were harvested and stained with annexin V-conjugated fluorescein isothiocyanate (FITC) and propidium iodide (PI) (BD Biosciences, Franklin Lakes, NJ, USA) following the manufacturer’s instructions. Cell apoptosis was then detected by the FACScan flow cytometer (BD Biosciences).
Chromatin immunoprecipitation (ChIP) assay
Briefly, the myeloma cells were fixed and then crosslinking stopped with glycine, followed by isolation of the nuclei and cracking of the chromatin with a Branson Digital Sonifier D250 ultrasonic instrument. Afterwards, we incubated the diluted chromatin with anti-NF-κB p65 antibody at 4°C overnight and then eluted the chromatin from magnetic beads for quantitative PCR analysis. We used IgG as the negative control.
Statistical analysis
Statistical analysis was performed using SPSS Statistics 17.0 software (SPSS Inc., Chicago, IL, USA) and then related graphs were drawn by GraphPad Prism v 5.0 software (GraphPad Software Inc, La Jolla, CA, USA). Kolmogorov-Smirnov test was used to determine the normality of the distribution of the data in each group. Normally distributed variables were expressed as a mean ± standard deviation (SD), while non-normally distributed variables were expressed as median (25th, 75th percentiles). The experiments were repeated three times. The statistical significance of the differences between related experimental groups was determined by one-way analysis of variance (ANOVA). A p<0.05 was considered as statistically significant.