Suppressive Effect Of MiR-186 On The Proliferation Of Multiple Myeloma And Its Mechanisms

BackgroundMultiple myeloma (MM) is a plasma cell disorder with a relatively high incidence rate among hematological malignancies. The disease is characterized by clonal proliferation of malignant plasma cells in the bone marrow, monoclonal paraprotein in blood/urine, and related organ dysfunction. Clinical manifestations including bony damage, anemia, hypercalcemia, kidney damage and repeated infection, etc. Despite significant progress in elucidation of the biology and treatment options over the past few decades, myeloma remains incurable, highlighting an urgent requirement for the development of novel and more effective therapies.miRNAs are a class of small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level by directly binding to the 3’untranslated region (3’UTR) of target RNAs. miRNAs are reported to play essential roles in carcinogenesis and tumor progression. Acting as either tumor suppressors or oncogenes, miRNAs are involved in several aspects of cancer biology, including cell proliferation, apoptosis, migration and invasion. Due to their important functions in cancer, miRNAs present an attractive therapeutic target. Therapeutic modulation of miRNAs may therefore be successfully applied to achieve clinical benefits.A single miRNA can target multiple proteins and consequently regulate various physiological and pathological prosses. Aberrant miRNA expression is frequently observed in various human tumors, including MM, indicative of critical roles in tumorigenesis. Previous studies have shown that miR-186 acts as a tumor suppressor and is downregulated in many tumors, such as lung adenocarcinoma, esophageal and colorectal cancers, endometrial, prostate, and bladder cancers. However, the expression patterns and specific function of miR-186 in MM remain unclear. In this study, we determined the expression patterns and specific function of miR-186 in MM.ObjectiveThe aim of this study is to examine the expression of miR-186 in MM, explore the influence of miR-186 for MM cells in biological characteristics, identify its possible role in MM pathogenesis, and elucidate the molecular mechanisms of its suppressive or oncogenic activities on MM. We hope that this study will improve the better understanding of MM pathogenesis and the development of novel effective therapies for MM.Methods1. Patient samples and cell lines:Thirty MM and eighteen healthy control samples were collected, Plasma cells were isolated from bone marrow samples. Human MM cell lines (MM.1S, OPM-2, NCI-H929, U266, and RPMI-8226) and normal plasma cells (nPCs)were cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37℃ in a 5% CO2 atmosphere.2. Lentivirus transduction and oligonucleotide transfection:miR-186 Pri-miR sequences were amplified and cloned into hU6-MCS-PGK-EGFP vectorl. Lentiviral particles were generated by co-transfecting HEK293T cells. Virus particles were harvested 48h after transfection. U266 and RPMI-8226 were infected with recombinant lentivirus. H929 cells were transfected with antagomiR-186 and antagomiR-NC according to the manufacturer’s protocol. Expression of miR-186 in cells was validated via qRT-PCR.3. RNA extraction and quantitative RT-PCR:Total RNA was extracted from cells using TRIzol total RNA isolation reagent according to the manufacturer’s instructions. cDNA was synthesized from total RNA or purified small RNAs using gene-specific primers or random hexamers. To determine Jaggedl and miR-186 expression levels, real-time PCR was performed with SYBR Green,4. Plasmid construction and transfection:For rescue of the anti-proliferative effect of miR-186, we used a pcDNA-Jagged 1 open reading frame lacking the entire 3’UTR of endogenous Jagged 1. For luciferase assays, the Jagged 13’UTR sequence was inserted into the pGL3 luciferase reporter vector. Target sites were mutated using the QuikChange Site-Directed Mutagenesis kit. Transfection of plasmids was performed using the Lipofectamine 2000 reagent according to the manufacturer’s instructions.5. Cell proliferation and colony formation assays:For cell proliferation assays, proliferative ability determined using the MTT assay at 24,48, and 72 h after transfection. For colony formation assays, cells were incubated at 37℃ for 2 weeks. Next, cells were fixed in 100% methanol for 30 min, stained with 0.1% crystal violet, and images obtained under a microscope.6. Cell cycle analyses:At 48h post-transfection, cells were harvested via trypsinization, fixed in 70% ice-cold ethanol overnight, followed by staining with 50 mg/ml propidium iodide (PI) for 30 min. Cell cycle distribution was analyzed on the FACSCalibur flowcytometer.7. Tumor xenograft model:RPMI-8226/NC or RPMI-8226/miR-186 cells were injected subcutaneously into the right flanks of 6-8 week-old BALB/C nude mice. Tumor growth was monitored and calculated tumor volume. Thirty days after injection, tumors were removed and sectioned for immunohistochemical staining with Ki67 antibody to assess cell proliferation.8. Luciferase reporter assay:The pGL3-Jagged 1-3’UTR-WT/MUT vector was co-transfected with control plasmid or miR-186-expressing plasmid into U266 or RPMI-8226 cells using Lipofectamine 2000. Luciferase activity was measured after 48 h using the Dual luciferase reporter assay system.9. Western blotting:Cells were lysed in the RIPA buffer, Total protein was separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes. After blocking in 5% nonfat milk, the membrane was incubated with the primary Jagged 1 antibody and an anti-actin antibody as the loading control, prior to washing and exposure to peroxidase-conjugated secondary antibodies. Ultimately, blots were developed with enhanced chemiluminescence reagents.10. Statistical analysis:All data are presented as means±S.D. Student’s t test (two-tailed) was used for analysis of differences between groups. Statistical analyses were performed using SPSS 13.0 software, with P values less than 0.05 considered statistically significant.Results1. miR-186 expression is decreased in MM. We evaluated miR-186 expression via qRT-PCR in MM.1S, OPM-2, NCI-H929, U266 and RPMI-8226 MM cell lines, compared to normal healthy bone marrow-derived plasma cells (nPCs). miR-186 was downregulated in all the MM cell lines, with the lowest levels in U266 and RPMI-8226. Consistent with these data, miR-186 levels were downregulated in patient MM cells, compared to plasma cells from healthy donors. Our results support a potential role of miR-186 as a tumor suppressor in MM.2. miR-186 inhibits MM cell proliferation in vitro and induces cell cycle arrest at the G0/G1 phase. To explore the effect of miR-186 on cell growth, U266 and RPMI-8226 cells were transduced with lentivirus carrying miR-186 sequences or empty lentivirus vector, and qRT-PCR used to confirm miR-186 overexpression. Data from the MTT assay demonstrated significantly slower growth in cells infected with miR-186 than those infected with the negative control. Next, the effect of miR-186 on colony forming ability was determined. Overexpression of miR-186 significantly inhibited colony growth of U266 and RPMI-8226 cells, compared to the negative control. FACS analysis was further performed to examine the effects of miR-186 on cell cycle distribution. A higher percentage of cells infected with miR-186 were present in the G0/G1 phase and a lower percentage in the S phase, compared with those infected with the control. In contrast, inhibition of miR-186 by antagomiR-186 in H929 cells significantly increased cell proliferation and the percentage of cells at the S phase compared with the negative control. These findings collectively suggest that miR-186-induced inhibition of MM cell proliferation is attributable to G0/G1 cell cycle arrest.3. miR-186 inhibits tumor growth in nude mice. To further determine whether miR-186 has a negative effect on tumor growth in vivo, RPMI-8226 cells infected with lentivirus carrying miR-186 were injected subcutaneously into the right flanks of nude mice, tumors generated from RPMI-8226/miR-186 cells in nude mice grew more slowly than those from RPMI-8226/NC. Thirty days after inoculation, the weights of tumors induced from RPMI-8226/miR-186 cells were significantly lower, compared to those from control cells, a significant decrease in the number of Ki-67 positive cells in tumors formed from RPMI-8226/miR-186 cells, relative to those from control cells.4. Jaggedl is regulated by miR-186. To further investigate the molecular mechanisms underlying miR-186-mediated regulation of MM cell growth, we searched for potential targets of miR-186 using TargetScan algorithm. Among the identified genes, Jagged1 was selected as a candidate target of miR-186 in view of its involvement in the proliferation and survival of MM cells. To ascertain whether Jagged1 is the direct downstream target of miR-186, its 3’UTR region was cloned into the pGL3 luciferase reporter vector. U266 and RPMI-8226 cells were co-transfected with wt or mutant 3’UTR vector and miR-186-expressing plasmid. Overexpression of miR-186 significantly reduced the luciferase activity of cells transfected with wild-type Jaggedl-3’UTR, compared to the negative control, but had no effect on those with mutant 3’UTR. qRT-PCR results showed no effect of miR-186 on Jagged1 mRNA. However, Jaggedl protein levels were significantly reduced in U266 and RPMI-8226 cells infected with miR-186 and increased in H929 cells transfected with antagomiR-186, compared with the respective controls, as observed with western blot. Based on the collective results, we propose that miR-186 regulates protein expression of Jaggedl by directly targeting its 3’UTR region.5. Overexpression of Jaggedl reverses the effects of miR-186:To elucidate whether Jaggedl is a functional target of miR-186, we performed gain-of-function analyses by transfection of Jaggedl plasmids lacking 3’UTR into miR-186-expressing RPMI-8226 cells. Western blot analyses revealed a significant increase in Jagged 1 in cells transfected with Jagged 1 expressing plasmids, compared to RPMI-8226 or RPMI-8226/miR-186 cells. Importantly, ectopic expression of Jaggedl significantly reversed miR-186-induced cell growth inhibition, colony formation reduction and cell cycle arrest. The data collectively demonstrate that miR-186 inhibits cell growth through suppression of Jaggedl expression.Conclusions1. In our study, we observed downregulation of miR-186 in patient MM cells and MM cell lines. Our data revealed that miR-186 inhibits cell growth and colony formation, induces G0/G1 arrest in MM cells, and suppresses tumorigenesis in a murine model of MM xenograft, strongly supporting a tumor suppressor role in MM.2. Jaggedl is a direct target of miR-186 in MM cells, and miR-186 exerts tumor suppressor activity in MM through suppressing Jaggedl expression.