| Multiple myeloma (MM) is a kind of hematological malignancy originated from B-cell lines, and MM has monoclonal plasma cells in bone marrow hyperplasia and their malignant proliferation as main characteristics. MM patients often clinically have the typical manifestations of osteodynia, anemia and renal insufficiency and repeated infections, as well as complications such as osteolytic bone reabsorption and bone dyspoiesis. Epidemiological data reveals that the incidence of MM is about 1/100,000, which occupies 10% of hematologic malignancies, showing an increasing trend in recent years. The median survival time of MM patients in progressive stage is only 6 months if no effective treatments. Patients received conventional chemotherapy is less than 3 years. Only 25% patients struggle for 5 years or more. Although autologous and allogeneic hematopoietic stem cell transplantation therapy have achieved significant effects, but MM still can't be cured. One of reasons is there are cancer stem cells(CSCs) that drive tumor initiation, exhibit resistance to treatment, and promote recurrence and metastasis. Therefore, targed CSCs may be one of an effective treatment methods for MM patients.MicroRNA (miRNA) is a variety of endogenous non-coding single-strand RNA found in eukaryotes and viruses, which plays an important role in cell proliferation, apoptosis, aging and differentiation processes. Studies have confirmed that miRNAs have close relationship with the tumors, and play roles of oncogenes, tumor suppressor, signaling molecules and other characteristics involved in the regulation of tumor biology, and that there exists miRNAs'abnormal expression in multi CSCs, and miRNAs was involved in maintenance of the malignant phenotype of CSCs.As one of the members of miRNA-34 family, miR-34a is thought to be a potential tumor growth inhibitor due to resulting in tumor cell's G1 arrest and apoptosis. Accumulating evidence indicates that there are abnormal miR-34a expression in pancreatic carcinoma, kidney carcinoma and glioblastoma tumor tissues, and it is true of in MM patients. A recent study indicated that miR-34a can also block secretion of osteoclast-activating factor from MM cells by inhibitive expression of its target gene TGIF2(Transforming growth interaction factor 2, also known as TG-interaction factor 2), that led to preventing the formation of osteoclasts, reducing bone resorption and osteoporosis symptoms, as well as rate of bone metastases. However, the role miR-34a in MM and MM CSCs needs to be explored.In view of above mentioned research background, we chose human MM cell line RPMI8226 as research object in this study, constructing recombinant containing miR-34a that was transfected into MM cells by using Pullulan-spermine (Ps) lipopolysaccharide-nanoparticle, and the recombinant stably up-regulating expressing miR-34a was acquired. Then we discussed and proved that up-regulating expression of miR-34a in MM cells or MM CSCs may be a new method for treatment of MM by means of in vivo and in vitro experiments as well as dual-luciferase report assay.Objective:To explore the effects of over-expression of miR-34a on biology properties of MM and MM CSCs, and provide a new experimental evidence for treatment of MM by targeted up-regulating the miR-34a expression in MM cells and MM CSCs.Methods and contents:1. Detection of miR-34a expression in RPMI8226 cells and construction of the recombinant plasmid containing miR-34a. Extracting total RNA from human MM cell line RPMI8226 and detecting expression of miR-34a by RT-PCR. Constructing and identifying the recombinant expressing miR-34a.2. To explore effects of miR-34a on RPMI8226 cells by in vitro and in vivo experiments, we transfected the recombinant vector into RPMI8226 cells by Ps lps-nanomaterials, and selected cell clones that stably expressed miR-34a by G418 screening. Effects of miR-34a on cell proliferation, ability of resistance of apoptosis, and clonal formation were separately detected in wild type of RPMI8226 cells and RPMI8226 cells transfected with recombinant containing miR-34a by CCK8 experiment, soft agar assay and flow cytometry (FCM). The CD138-CD34-MM CSCs were isolated from RPMI 8226 cell line using magnetic activated cell sorting system according to CD molecular on surface of human MM CSCs. Effects of up-regulating the miR-34a expression on the oncogenicity, bone mineral density and tumor formation times, tumor sizes, and tumor bearing mouse survival were respectively observed by subcutaneous injection of ST and WT-RPMI8226 cells, as well as ST and WT-RPMI8226 CSCs in NOD/SCID mice and bone CT scan assay. Pathology change of tumor cells was analyzed by H&E staining assay.3.MiR-34a might prevent the formation of osteoclasts by inhibiting the expression of TGIF2. Using bioinformatics tools (Miranda) to forecast TGIF2, a miR-34a downstream target, then verifying it by the dual-luciferase reporter system. Expression of miR-34a and TGIF2 in RPMI8226 cells transfected with miR-34a mimics or miR-control mimics as well as in the tumor tissues of NOD/SCID mice were respectively detected by q-PCR and Western blot.Results:1. The result of RT-PCR confirmed that there was miR-34a expression in MM RPMI8226 cells. We successfully constructed and identificated a recombinant vector that stably expressed miR-34a, and named it for pIRES-miR-34a.We transfected the recombinant vector into RPMI8226 cells by Ps lps-nanomaterials and selected cell clones which stably expressed miR-34a by G418, named ST-RPMI8226. Expression of miR-34a in RPMI8226 cells was increased significantly 48h after transfection. The differences were statistically significant (p<0.05 or p<0.01).2. Compared with control group, miR-34a-overexpressed clones inhibited the cellular proliferation, colony formation, and apoptosis rate was increased in vitro. The tumorigenicity was significantly decreased but the bone density was increased in NOD/SCID mice injected with miR-34a-overexprcssed cells and CD138- CD34-MM CSCs compared with the control groups. The differences were statistically significant (p<0.05 or p<0.01). And H&E staining results in tumor tissues was also true.3. The results of both bioinformatics prediction and dual-luciferase reporter system showed that TGIF2 was target point that was regulated by miR-34a. QRT-PCR results showed that miR-34a expression was significantly increased whereas the expression of TGIF2 was markedly decreased in RPMI8226 cells transfected with miR-34a mimics compared with the control group in vitro. The analogous results were found in tumor tissues of NOD/SCID mice injected with miR-34a mimics or miR-34a control.Conclusions:1. Over-expression of miR-34a reduced the clonality, cellular proliferation and oncogenic capability; meanwhile increased apoptosis rate of MM RPMI8226 cells, and inhibited the development of MM in NOD/SCID mice.2. Over-expression of miR-34a reduced the tumorigenicity of MM CD138-CD34-CSCs,and decreased the TGIF2 expression and the lytic bone lesions compared with the control group, suggesting that miR-34a or its downstream target point of TGIF2 might be new targets for the treatment of MM. |
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