Plasma specimens were obtained from 30 patients with MM and 10 healthy volunteers from Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (Shanghai, China). Plasma cells from patients with MM and normal controls were purified from bone marrow aspirates using CD138 MicroBeads (cat. no. 130-051-301; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), as described previously (15). All patients provided written informed consent to participate in the study. This study was approved by the Ethics Committee of Shanghai University of Traditional Chinese Medicine.

The U266 and RPMI-8226 MM plasma cell myeloma cell lines, and the 293 cell line, used in the present study were purchased from the American Type Culture Collection (Manassas, VA, USA). The U266 and RPMI-8226 cells were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.). The 293 cells were cultured in Dulbecco's minimum essential medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂.

Total RNA was isolated from MM tissues, adjacent non-tumor tissues and MM cell lines using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Purified mRNA and miRNA were detected using an RT-qPCR assay with the All-in-One miRNA RT-qPCR Detectionkit (GeneCopoeia, Inc., Rockville, MD, USA), according to the manufacturer's protocol. All primer sequences are listed in Table I. U6 small nuclear RNA was used as an internal control for the normalization and quantification of miRNA expression. β-actin was used as the internal control for the normalization and quantification of CABIN1 mRNA expression as described previously (16).

The luciferase reporter construct was created by cloningthehuman CABIN1 mRNA sequence into a pMIR-Report construct (Ambion; Thermo Fisher Scientific, Inc.). CABIN1 mRNA fragment (from 5178 to 5240) was amplified and cloned into the luciferase reporter via SpeI and HindIII sites using Ready-to-Use Seamless Cloning kit (Songon Biotech, Shanghai, China), according to the manufacturer's protocol. All primers are listed in Table I. 293 cells (1×10⁴) were plated in a 96-well plate and co-transfected with 50 nM miRNA mimics or negative control oligonucleotides RNA oligonucleotides were chemically synthesized and purified by Shanghai GenePharma Co., Ltd. (Shanghai, China). A total of 30 ng firefly luciferase reporter and 10 ng pRL-TK (Promega Corporation, Madison, WI, USA) were transfected into the cells using the Interferin^® reagent (Polyplus-transfection SA, Illkirch, France). Cells were collected 48 h after the last transfection and analyzed using the Dual-Luciferase Reporter Assay system (Promega Corporation), according to the manufacturer's protocol. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity.

RNA oligonucleotides were chemically synthesized and purified by Shanghai GenePharma Co., Ltd. The sequence of human miR-15a was 5′-UAGCAGCACAUAAUGGUUUGUG-3′, and that of human miR-16 was 5′-UAGCAGCACGUAAAUAUUGGCG-3′. The negative-control oligonucleotide sequence was 5′-CAGUACUUUUGUGUAGUACAA-3′. The small interfering RNA (siRNA) sequences were as follows: CABIN1 sense, 5′-CUACAUUGAGGGAACUUCATT-3′ and antisense, 5′-UGAAGUUCCCUCAAUGUAGTT-3′; control sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGUAGAATT-3′. Transfections were performed using Interferin^® reagent (Polyplus-transfection SA). The final concentration of miRNA was 50 nM; the final concentration of siRNAs was 20 nM.

Cell proliferation was measured using a CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), according to the manufacturer's protocol. A total of 1.0×10⁴ cells were plated into each well of a 96-well plate and transfected with miRNA or CABIN1 siRNA to a final concentration of 50 or 20 nM, respectively using Interferin^® reagent (Polyplus-transfection SA), according to the manufacturer's protocol. Once the cells were harvested, 10 µl CCK-8 reagent was added to 90 µl RPMI-1640 medium. The cells were subsequently incubated for 2 h at 37°C and the optical density was measured at 450 nm. Three independent experiments were performed.

Total cellular extracts (20 µg) were separated by SDS-PAGE (4–20% Tris-glycine gel) then transferred onto a polyvinylidene fluoride membrane (Immobilon-P transfer membranes; EMD Millipore, Billerica, MA, USA). Following transfer, the blots were blocked with 5% non-fat dry milk in PBS and 0.1% Tween-20 for 2 h at 4°C and washed three times with PBS and 0.1% Tween-20 at 4°C. The blots were then probed with the primary anti-CABIN1 antibody (cat. no. ab3349; dilution, 1:200; Abcam, Cambridge, MA, USA), CDKN1A (cat. no. sc-6246; dilution, 1:200) and β-actin (cat. no. c-11;1: 200; Santa Cruz Biotechnology, Inc.). The blots were then probed with the horseradish peroxidase-conjugated goat anti-rabbit IgG H&L secondary antibody, (cat. no. ab6721, dilution, 1:750; Abcam) for 1 h at 4°C, followed by washes in PBS + 0.1% Tween-20, and then detected using an Odyssey Scanning system (Gene Company, Ltd., Hong Kong, China).

All statistical analyses were performed using the SPSS 16.0 statistical software package (SPSS, Inc., Chicago, IL, USA). Quantitative data is expressed as mean ± standard deviation. Data of mRNA expression levels in MM specimens compared to healthy volunteers were subjected to Student's t-test analysis. Data from dual-luciferase reporter assays and CCK-8 assay were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls post-test. P<0.05 was considered to indicate a statistically significant difference. Pearson's correlation coefficient and Spearman's correlation coefficient was used forthe comparison of mRNA expression of miRNA and its target gene.

miR-15a and miR-16, whose expression is decreased or absent in MM, have similar sequences and are considered to have the same target genes. To investigate the biological significance of these miRNAs and the underlying mechanisms of their downregulation in MM, the potential target genes of miR-15a/16 that have functions in MM pathogenesis were further analyzed.

Previously published crosslinking, ligation, and sequencing of hybrids (CLASH) data in 293 cells provided direct evidence for the miRNA-mRNA pairing (17). The CLASH data revealed that four genes, HIST1H2K, BTRC, CNN3 and CABIN1, aretargeted by miR-15a and miR-16 (Fig. 1A). All genes targeted by miR-15aor miR-16 in the CLASH dataset (17) are listed in Table II. CABIN1 is involved in several cellular processes including cell proliferation and the restraint of p53 activity on chromatin (18); therefore, CABIN1 was selected for further investigation. Alignment results indicated that the potential targeting sequence for miR-15a/16 was within the protein-coding region of CABIN1 mRNA between nucleotides 5,178 and 5,240 (Fig. 1B).

Previous studies have reported that the expression of miR-15 and miR-16 was downregulated in MM tissues (8,19). To determine whether miR-15a and miR-16 are capable of targeting and regulating CABIN1 expression in MM, a gain-of-function study was performed.

Luciferase reporter plasmids were created with targeting sequences of CABIN1 mRNA (Fig. 2A). miR-15a and miR-16 were transfected into 293 cells and a dual-luciferase reporter assay was used to assess the miRNA regulation of CABIN1. The results revealed that the upregulation of miR-15a or miR-16 decreased luciferase activity, whereas a reporter plasmid without the presence of CABIN1 sequence produced no change in luciferase activity (Fig. 2B). These results indicated miR-15a and miR-16 negatively regulated CABIN1 expression.

Whether the endogenous CABIN1 in MM cells was similarly regulated was subsequently investigated. The MMU266 and RPMI-8226 cells lines were transfected with miR-15a and miR-16, and CABIN1 mRNA and protein levels were examined by RT-qPCR and western blotting, respectively. The levels of CABIN1 mRNA (Fig. 2C) and protein (Fig. 2D) were consistently and substantially downregulated by miR-15a and miR-16 in MM cells. The results of the present study indicated that miR-15a and miR-16 directly targeted CABIN1 mRNA and negatively regulated expression of CABIN1 at the mRNA and protein level.

Overexpression of CABIN1 increased the proliferation of cancer cells by physically interacting with p53 and repressing p53 transcriptional activity on target genes, including CDKN1A (18). Given that CABIN1 is a direct target of miR-15a/16, it was hypothesized that miR-15a and miR-16 may affect MM cell viability through CABIN1. miR-15aand miR-16a or CABIN1 siRNA was transiently transfected into U266 and RPMI-8226 cells. The results of western blotting revealed that transfection with miR-15a and miR-16or CABIN1-targeted siRNA decreased the expression of CABIN1 and p53 downstream gene CDKN1A in U266 and RPMI-8226 cells (Fig. 3A).

Using a cell proliferation assay, the overexpression of miR-15a and miR-16 in U266 and RPMI-8226 cells was determined to result in the significant suppression of cell proliferation, similar to CABIN1 siRNA (Fig. 3B). The results of the present study demonstrated that the downregulation of CABIN1 expression by miR-15a and miR-16 contributes, partially, to the suppression of MM cell growth.

Previous studies have reported that miR-15a and miR-16 downregulated in several types of cancer, including MM (8,19). Overexpression of CABIN1 is associated with poorer patient outcomes in a number of cancer types (18,20,21). The results of the present study demonstrated that CABIN1 is negatively regulated by miR-15a and miR-16 in MM cells. Next, the correlation between miR-15a and miR-16 expression levels and CABIN1 mRNA levels was assessed in MM specimens.

To determine the levels of miR-15a and miR-16 in MM samples, total RNA was extracted from MM specimens (n=30), following which the expression levels of miR-15a and miR-16 were analyzed using RT-qPCR and normalized to an endogenous control (U6 RNA). miR-15a and miR-16levels were significantly decreased in MM specimens compared with in healthy volunteers (n=10; Fig. 4A; P<0.001), which was concordant with the results of certain previous studies (8,19). RT-qPCR analysis was performed to detect the mRNA expression of CABIN1 in the same MM specimens and normalized to β-actin. As presented in Fig. 4B, CABIN1 mRNA levels were significantly increased in MM specimens compared with healthy volunteers. RNA levels of miR-15a and miR-16, and mRNA levels of CABIN1 in MM tissues were analyzed by Pearson's correlation coefficient analysis. CABIN1 mRNA expression levels were negatively correlated with those of miR-15a (R=−0.736; P<0.001) and miR-16 (R=−0.641; P<0.001) in MM tissues (Fig. 4C). Collectively, these results suggested that high endogenous miR-15a and miR-16levels may target and downregulate the expression of CABIN1 in MM.