MicroRNA-451 regulates stemness of side population cells via PI3K/Akt/mTOR signaling pathway in multiple myeloma.

Side population (SP) cells are an enriched source of cancer-initiating cells with stemness characteristics, generated by increased ABC transporter activity, which has served as a unique hallmark for multiple myeloma (MM) stem cell studies. Here we isolated and identified MM SP cells via Hoechst 33342 staining. Furthermore, we demonstrate that SP cells possess abnormal cell cycle, clonogenicity, and high drug efflux characteristics-all of which are features commonly seen in stem cells. Interestingly, we found that bortezomib, As2O3, and melphalan all affected apoptosis and clonogenicity in SP cells. We followed by characterizing the miRNA signature of MM SP cells and validated the specific miR-451 target tuberous sclerosis 1 (TSC1) gene to reveal that it activates the PI3K/Akt/mTOR signaling in MM SP cells. Inhibition of miR-451 enhanced anti-myeloma novel agents' effectiveness, through increasing cells apoptosis, decreasing clonogenicity, and reducing MDR1 mRNA expression. Moreover, the novel specific PI3K/Akt/mTOR signaling inhibitor S14161 displayed its prowess as a potential therapeutic agent by targeting MM SP cells. Our findings offer insights into the mechanisms regulating MM SP cells and provide a novel strategy to overcome resistance to existing therapies against myeloma.


Flow cytometric analysis
For Hoechst 33342 staining and SP cells sorting: cells were washed with PBS and resuspended in above mentioned culture RPMI medium at 1X10 6 /mL. The cells were incubated with Hoechst 33342 (Sigma, St. Louis, MO) at 5µg/ml either alone or together with ABC transporter inhibitors verapamil (100µM, Sigma), or reserpine (50 mmol/L, Sigma), for 120 minutes at 37°C. After staining, the cells were centrifuged and resuspended in ice-cold PBS containing 1µg /mL propidium iodide (PI) and maintained at 4°C for flow cytometry analysis and sorting. Cell analysis and sorting were determined by a MoFlo cytometer (Beckman Coulter, Inc.). Hoechst dye was excited at 407 nm by trigon violet laser, and dual wavelengths were detected by 450/40 (Hoechst 33342-Blue) and 695/40 (Hoechst 33342-Red) filters.
Aldehyde dehydrogenase (ALDH) activity was tested by the Aldefluor reagent (Stem Cell Technologies, Vancouver, Canada) according to manufacturer's instructions, and diethylaminobenzaldehyde (DEAB) was used as control.
For apoptosis assay: cell apoptosis was detected by using annexin V staining. MM cells were cultured in media alone, or with media plus with various concentration agents treatment in culture medium for 24 and 48 hours. Cells were then washed twice with ice-cold PBS and resuspended (1 x 10 6 cells/mL) in binding buffer (10mmol/L HEPES, pH7.4, 140mmol/L NaCl, 2.5mmol/L CaCl 2 ). MM cells (1 x 10 5 ) were incubated with annexin V-FITC (5μL; Pharmingen, San Diego, CA, USA) and propidium iodide (PI, 5mg/mL) for 15 minutes at room temperature. AnnexinV+PI-apoptotic cells were enumerated by using the flow cytometer (Beckman Coulter).

MiRNA profiling and analysis
Total RNA was harvested using TRIzol (Invitrogen) and miRNeasy mini kit (QIAGEN) according to manufacturer's instructions. After having passed RNA quantity measurement using the NanoDrop 1000, the samples were labeled using the miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon) and hybridized on the miRCURY™ LNA Array (v.16.0). After hybridization, scanning was performed with the Axon GenePix 4000B microarray scanner (Molecular Devices, Downingtown, PA, USA). GenePix pro V6.0 (Molecular Devices) was used to read the raw intensity of the image. Background correction was performed using the "normexp + offset = 50" method, which provides variance stabilization and guaranties no negative values. Filtering was performed to identify miRNA up-or down-regulated at least 2.0-fold with a p-value of < 0.05 in SP cells compared with MP cells. The analysis was carried out using the limma package from Bioconductor according to the method (Smyth GK, Stat Appl Genet Mol Biol 3, 2004). The microarray data are deposited on the Gene Expression Omnibus (accession number GSE56163).
For pathway analysis: The network of the critical miRNAs and their targets was established according to the miRNA degree. The miRNA analysis involved generation of pathway networks using the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/). A two-sided Fisher's exact test and chi-square test were used to classify the enrichment of pathway category, and the false discovery rate (FDR) was calculated to correct the p-value. The pathway had a p-value of < 0.005 and an FDR of < 0.05, which was chosen for further analysis. The regulator pathway annotation was performed on the basis of scoring and visualization of the pathways collected in the KEGG database as well.

Quantitative RT-PCR assay (Q-RT-PCR)
All RNA samples, including small RNAs, were purified by miRNeasy Mini Kit (QIAGEN), and then cDNAs were synthesized using a reverse transcription kit (TaKaRa Bio Inc, Japan). After reverse transcription, qRT-PCR was performed using the quantitative SYBR Green PCR kit with gene-specific primers (TaKaRa Bio Inc, Japan) following the manufacturer's protocol. Real-time quantitative PCR studies used the LightCycler® 480 instrument (Roche Diagnostics). All quantitative the fold change was calculated by the 2-△△ Ct method. All experiments were done in triplicates. The U6 snRNA was used as a control to normalize miRNA quantitative, while GAPDH was used as a control to normalize mRNA quantitative in the Q-RT-PCR assay.

RNA oligonucleotide and cell transfection
MiRNA mimics, miRNA inhibitors, and their cognate control RNAs were purchased from Ambion (Austin, TX, USA) or Genepharma (Shanghai, China). Transfection was performed using SuperFection (Pufei, USA) transfection reagent according to the manufacturer's instructions. Transfection efficiency (>90%) was confirmed with the use of the Silencer 6-carboxy-fluo-rescine (FAM)-labeled Negative Control. Total RNA and protein were collected for assay 48 or 72 hours after transfection.

Cell-based assays
Cell viability was tested by colorimetric assay kit (CCK-8 assay kit; Dojindo Laboratories, Tokyo, Japan) based on the MTT assay, according to the manufacturer's instructions. Briefly, 1～5×10 3 cells were incubated in 96-well plates with different concentration treatments in culture medium for different time points depend on experiment design, and then 10μL of the CCK-8 solution was added to each well. After 2 hours incubation at room temperature, the optical density (OD) was measured using a spectrophotometer (Molecular Devices Co., Sunnyvale, CA) and the fold-increase in the OD compared to that of the control (proliferation index) was calculated.
Assays for colony-forming cell (CFC) activity were performed by plating the 4×10 2 or 2×10 3 sorted SP and MP cells in containing 1mL Iscove's MDM with 1% methylcellulose, 30% FBS, 10 -4 M 2-mercaptoethanol medium (Methocult; Stem Cell Technologies, Vancouver, BC, Canada) supplemented with a cocktail of growth factors (50ng/mL rh stem cell factor, 10ng/mL rh GM-CSF, 10ng/mL rh IL-3, and 3U/mL erythropoietin) with or without drugs in duplicate cultures. The plates were incubated at 37°C in a humidified incubator with 5% CO 2 for 14 days, and the number of colonies was counted using an inverted microscope with 4 × and 10 × planar objective.

Western blot analysis
Cell lysates and total protein concentration was measured with the BCA Protein Assay Kit (Pierce Biotechnology, Rockford IL, USA)．Equal amounts of protein were subjected to SDS-PAGE and proteins were transferred to nitrocellulose membranes (GE Healthcare, USA). The membrane was blocked in PBS containing 5% non-fat milk and 0.1% Tween-20, washed twice in PBS, and incubated with primary antibody at room temperature for 2 hours, followed by incubation with secondary antibody at room temperature for 45 minutes. Afterward, the proteins of interest were visualized using ECL chemiluminescence system (Santa Cruz Biotechnology, USA). ß-actin was used to normalize the amount of protein in each sample.

Luciferase assay
The hsa-miR-451 vector and pmirGLO, pmirGLO-tuberous sclerosis 1 (TSC1)-3'-UTR, or pmirGLO-TSC1 3'-UTR-mut were co-transfected into NCI-H929. To generate TSC1 3'UTR mutants containing mutations in the conserved miR-451 binding site, site-directed mutagenesis was performed using the wild-type 3'UTR as the template. In the 3'UTR mutant, the nucleotide sequence complementary to nt 2～5 of miR-451 was mutated to the same sequence as that in miR-451 (from CGG to TAA).Cell lysates were prepared at 24 hours post-transfection. The luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). The values obtained from hsa-miR-451 vector and pmirGLO were set as 100%.

Animal models
A total of 1Ｘ10 5 SP cells or MP cells from NCI-H929 cell lines were injected subcutaneously together with Matrigel basement membrane matrix (Becton Dickinson) into 7 or 8 weeks aged NOD/SCID mice, which were approved by the Second Military Medical University Institute Animal Care and Use Committee guidelines for the use of laboratory animals. Engraftment of SP or MP cells was monitored every 7 days and changes in tumor size were measured by calipers. The mice were sacrificed at day 100 in accordance with institutional guidelines. The tumors were surgically removed and pictures were performed using camera in Xenogen system, and then were immunostained with anti-CD138 and anti-CD38.