MiRNA-21 Regulation Of Growth And Drug Resistance In Multiple Myeloma Cells

Multiple myeloma (MM) is characterized by the growth and accumulation of malignant plasma cells in the bone marrow (BM) milieu. Studies showed that progression to MM is a multiple step model, in which those who suffered from MM stared with monoclonal gammopathy of undetermined significance (MGUS) in an early stage and some patients developed extramedullary myeloma at last stage.A mass of malignant plasma cells localize within the BM through the interaction of adhesion molecules with BM stromal cells and extracellular matrix proteins, by which multiple pathway signaling are trigged and confer MM cell growth, survival and drug resistance. Signaling pathway induced by adhesion in MM cells and /or growth factors include the MAPK/ERK pathway, the JAK/Stat3 pathway, the NF-kB pathway, and the PI3K/Akt pathway. An increasing number of evidences suggested that MAPK/ERK pathway is involved in the growth and survival of MM cells. IL-6 and IGF-1 mediate MM cell growth, survival and drug resistance through activating MAPK/ERK pathway. In addition, MAPK/ERK pathway can also be trigged by VEGF and BAFF that secreted as autocrine and paracrine manner in MM cells and bone marrow cells. Some investigations showed that Sprouty 2 (SPRY2), a negative regulator of MAPK/ERK pathway signaling, is increasingly recognized for their functions in blocking pro-growth signaling responses that could inhibit malignant transformation or progression. Repression of SPRY2 genes are involved in some solid tumors including the glioma, breast cancer and prostate carcinoma. MicroRNAs(miRNA)are a group of endogenous, small, non-coding RNAs approximately 22 nucleotides in length, which regulate gene expression by translational repression and mRNA cleavage. Recently, miR-21 was found to be related with malignant characteristic including tumorigenesis, invasion, and apoptosis resistance. Function studies have disclosed that SPRY2, a negative regulator of MAPK/ERK pathway signaling, was one of the targets of miR-21. Expression of miR-21 in myeloma cells increased obviously, by which myeloma cells was resistant to apoptosis induced by IL-6 starvation. Up to now, circulating miR-21 has not been reported in MM and SPRY2 inhibiting MAPK/ERK signaling in MM cells was modulated by miR-21 is not clear. In addition, we found that NF-κB, a transcription factor, possiblely regulating expression of miR-21 by using a bioinformatic tool TFSEARCH. In order to confirm our hypothesis based on upper mentioned knowledge, three sections were included in our investigations: 1) To measure the expression levels of circulating miR-21 in serum of MM patients and observe its relationship with some clinical parameters; 2) To explore the role of miR-21 in growth and drug resistance of MM cells;3) To identify the putative NF-κB binding site in promoter region of miR-21 gene in myeloma cells.Part 1 Expression of circulating miR-21 in patients with multiple myeloma and its clinical significanceMiR-21 is one of the oncogene miRNAs that up-regulated in most types of human malignancy. A close relation has been found between miR-21 and tumorigenesis, invasion, metastasis and drug resistance in several solid tumors. Recently, up-regulation of miR-21 has been determined in myeloma cells compared to normal plasma cells. However, if miR-21 can be released into blood circulation and serve as a biomarker for multiple myeloma (MM) is not clear.In this part, we prepare to investigate the expression of circulating miR-21 in serum from 21 MM patients by and evaluate significance by its correlation with some clinical parameters. Circulating miR-21 in serum from patients of MM group, MGUS group and normal control (NC) group was determined by real-time PCR. In addition, lambda and kappa light chain, IgG, IgA, IgM,β2-MG, LDH, ALB, GLB,Hb and Ca2+ were measured by routine methods and their relation with miR-21 was analyzed by SPSS 13.0.Our results show that circulating miR-21 expression level was significantly elevated in patients with MM. Expression of miR-21 in serum was increased in patients in MM group (1.98±0.32) compared with MGUS group (0.76±0.20, p=0.014) and NC group (0.43±0.11, p=0.001), and there was no significance between circulating miR-21 of MGUS group and NC group(p=0.119). Circulating miR-21 in patients with MM was positively correlated with Stages of ISS, miR-21 level in patients of Stage I was 0.86+0.19, which was lower significantly than in patients of Stage II (2.77+0.64) and Stage III (2.86+0.53) (P<0.05). However, there was no significant difference between Stage II and Stage III. Further analysis disclosed that miR-21 positively correlated with serum kappa light chain (r=0.532, p=0.013), sum of kappa and lambda light chain (r=0.615, p=0.003), IgG (r=0.542, p=0.011) and sum of IgG, IgA and IgM (r=0.625, p=0.002),β2-MG(r=0.491, p=0.024) and negtively correlated with ALB (r=-0.585, p=0.002), but no correlation with GLB (r=-0.357, p=0.112),LDH (r=0.250, p=0.280),Hb (r=-0.286, p=0.209) and Ca2+ (r=-0.227, p=0.225). These data indicate that a close relation between miR-21 and MM and possible role of miR-21 involved in progression of MM.Part 2 Mechanism of growth and drug resistance of myeloma cells regulated by miR-21Our previous studies have shown that circulating miR-21 in MM patients increased significantly than NC group and a positive correlation with level of kappa light chain in serum, these facts suggested that the key role of miR-21 in the progression of MM. However, how miR-21 was involved in the development of MM? Some investigation declared that SPRY2, a negative regulator of MAPK/ERK pathway signaling, can be regulated by miR-21. Therefore, we supposed that high level of miR-21 in MM cells modulate MAPK/ERK pathway signaling by suppression of SPRY2 and promote survival and drug resistance of MM, the sensitivity of myeloma cells to drug was influenced by suppression of miR-21.In this section, we detected miR-21 expression and SPRY2 protein in bone marrow samples of MM by real-time PCR and Western blot. To observe the effects of apoptosis, migration and proliferation on myeloma cells by down-regulation of miR-21, we transfected miR-21 mimics and inhibitor into myeloma cells in vitro. In addition, we used lentivirus mediated suppression of miR-21 in myeloma cells to study the proliferation role in xenograft model, and clarify the possible role of miR-21 in MM.As a result, we found that high expression of miR-21 and lower expression of SPRY2 protein in bone marrow mononuclear cells of MM patients, SPRY2 was regulated by miR-21 in myeloma cells, and cell growth and MAPK/ERK activity was blocked by ectopic expression of SPRY2 in myeloma cells. Furthermore, we found that apoptosis resistance of myeloma cells was strengthened after transfection of miR-21 mimics, sensitivity to drugs was increased and migration was retarded after transfection of miR-21 inhibitor. Moreover, xenograft tumor of nude mice was blocked by lentivirus mediated down-regulation of miR-21 in myeloma cells.These data indicated that MAPK/ERK pathway signaling was activated by miR-21 mediated SPRY2 silence, by which myeloma cells suffer from uncontrollable growth and drug resistance partly.Part 3 Transcription regulation of miR-21 gene by NF-κB in myeloma cells Adhesion of myeloma cells to BMSCs activate multiple survival pathways signaling including NF-κB. Up-regulation of miR-21 expression in myeloma and other tumors has been reported in documents and our up-mentioned study. Some transcription factors including AP-1 and STAT3 was found to regulate transcription of miR-21 gene. Our study found miR-21 expression can be repressed by selective NF-κB inhibitor BAY 11-7082, hereby we supposed that miR-21 transcription may be modulated by NF-κB.In this section, we detected changes in miR-21 expression of myeloma cells that adhered to BMSCs, then we synthesized probe marked by biotin to perform EMSA with nuclear protein according analysis of sequence in miR-21 gene promoter by TFSEARCH.Our results showed that miR-21 was up-regulated after adhesion of myeloma cells to BMSCs and repressed by selective NF-κB inhibitor BAY 11-7082,both standard probe and synthesized probe of NF-κB combined with nuclear extracts from myeloma cells.These data suggested that miR-21 transcription can be directly regulated by NF-κB.In conclusion, our investigations showed that expression level of miR-21 increased in both serum and myeloma cells in MM patients, expression of SPRY2 in BMMCs was lower or silenced. Repression of SPRY2 by miR-21 caused MAPK/ERK signaling pathway activating overly, which helped growth and drug resistance of myeloma cells. Therefore, miR-21 was a promising target in MM therapy.