Study On Relativity Between MicroRNA15a And Biological Characteristics Of Multiple Myeloma

Background. Multiple myeloma (MM) is an incurable plasmatic neoplasm in hematological malignancies.Although chromosome 13q14 deletions are the most frequent genetic abnormalities in MM, the relationship between chromosome 13q14 deletions and biological features of MM is still less understood, particularly in the context of non-coding RNAs. MicroRNA15a, one of the non-coding RNAs located in chromosome 13q14, is recently reported to be a specific signature with differential expression in MM patients.Design and methods. Genetic characteristics of human myeloma U266 cell line were confirmed by fluorescence in situ hybridization (FISH). And then U266 was selected as the experimental model. Biological features, including drug resistance, proliferation and migration, before and after transient transfection of microRNA15a with Lipo2000, were compared. Besides, novel agents of Apo2L/TRAIL combined with thalidomide were employed to evaluate drug response. Moreover, microRNA15a expression was evaluated in CD138+ selected myeloma bone marrow of 10 patients affected with refractory/relapsed MM by RQ-PCR and respectively compared to genetic alterations by FISH and conventional karyotype.Results. PartⅠ. Biological characteristics of MM altered by Apo2L/TRAIL and thalidomide:①Haploidy deletions of chromosome 13q14 occurred in U266 cells.②The cell viability rates were 88.91±7.43%,85.14±10.26%,82.32±8.65%,78.10±6.78%, 76.64±3.68%,75.00±10.11% respectively when the cultured U266 cells were treated with thalidomide at 10,15,20,25,30,35μg/ml for 24 hours (P> 0.05). The cell viability rates were 86.43±7.35%,78.29±6.60%,70.79±9.80 respectively when the cultured U266 cells were treated with Apo2L/TRAIL at 1,10,100μg/ml for 24 hours (P > 0.05). These data indicated natural resistance of U266 cells to thalidomide and Apo2L/TRAIL. The respective percentages of apoptotic cells induced by lOμg/ml thalidomide and 1μg/ml TRAIL were 17.16±1.33%,11.43±1.48%. When combined treantment of both agents, the apoptotic rates were 28.95±2.35%, which presenting additive induction of apoptosis. In addition,24-hour incubation with recombinant Apo2L/TRAIL (1μg/ ml) significantly increased U266 cell migration almost 1.21 fold compared with the untreated cells (P< 0.05). Thalidomide (10μg/ml) significantly inhibited migration of these resistant cells to 67.10% when combined with Apo2L/TRAIL. Meanwhile, VEGF and MDR1 mRNA in U266 cells were significantly affected by the exposure to 1μg/ml Apo2L/TRAIL for 24 h, with a significant increase of 7.96±0.56 and 7.46±1.21-fold, respectively(P<0.05). Thalidomide, to some extent, can inhibit Apo2L/TRAIL-induced expression of VEGF mRNA to 2.04±0.28 folds (P< 0.05). Consistent alterations of VEGF and MDR1/GP170 were found at mRNA and protein levels. PartⅡ. Biological characteristics of MM altered by experimental upregulation of microRNA15a:①Pre-microRNA15a transfected and non-transfected U266 cells were cultured for 5 days ex vivo. CFSE staining showed that 87.94±2.58% of cells had proliferated into 2 subgenerations in transfected groups, significantly lower than 98.06±1.15% into 3 generations in control group (P< 0.05). Pre-microRNA15a transfected cells were mainly arrested in G2/M phase with increased numbers of cells in G1 phase (59.56±3.19% versus 48.84±1.66% in control, P<0.05). These data indicate changes in cell cycle regulatory proteins responsible for proliferation inhibition due to experimental upregulation of microRNA15a. Meanwhile, pre-microRNA15a transfected cells showed obvious inhibition in migration as 17.7±3.22% of the non-transfected group, with no significant difference when compared to 13.5±2.14% in no-SDF-1a chemotatic group (P>0.05). Besides, VEGF secretion was significantly decreased in pre-microRNA15a transfected U266 cells, as compared to nontransfected cells used as control (56.22±27.13 versus 165.01±29.66, P<0.05). On the other hand, microRNA15a up-regulation obviously inhibited GP170 protein/MDR1 mRNA expression. However, microRNA15a transfection can not reverse natural resistance of U266 cells to thalidomide and Apo2L/TRAIL. ②Among 10 cases affected with relapsed/refractory MM, downregulation of microRNA15a were found in 5/10 cases wih consistent to chromosome 13q14 deletion and 3/10 without 13q14 deletion.Conclusions. MicroRNA15a is a critical regulator of MM pathogenesis by modulating proliferation and migration of clonal plasma cells, and influencing drug resistance via altering the internal MDR-1/GP170-mediated pathways. Targeting the non-coding RNAs, and more specifically microRNA15a, may represent a novel potential strategy to inhibit MM progression.