Lentivirus-mediated RNA Interference Of MIC-1Expression In RPMI-8226Cells Inhibits Human Osteoclasts Maturation

[Backgroud]Multiple myeloma(MM) is malignant disease characterized by uncontrolled expansionof malignant plasma cells and accounts for13%of hematological malignancies. MMis still an incurable disease nowadays. The main clinical manifestations of the diseaseare bone pain and lytic bony lesions, namely myeloma bone disease (MBD). Theinitiator of MBD is osteoclast (OC). OC, a multinucleated giant cell with the functionof bone resorption is derived from the monocytes-macrophages and reside in bonemarrow. In MM, changes occur in bone marrow microenvironment because of theinfiltration of myeloma cells. Under the stimulation of abnormal signal, OC precursorsexcessively differentiate into mature OCs, leading to bone destruction. MacrophageInhibitory Cytokine-1(MIC-1) is one of the divergent member of human transforminggrowth factor β (TGF-β) superfamily and has been found of complex biologicalfunctions. In MM bone marrow, high expression of the MIC-1was found. Studiesshowed MIC-1might be a new facilitating factor of OC and involoed in thepathogenesis of MBD.[Objective]To evaluate the effect of target inhibition of MIC-1in RPMI-8226cells on thematuration of OCs which were co-culture with RPMI-8226cells.[Methods]1. Human peripheral blood mononuclear cells (PBMNCs) were isolated fromperipheral blood by density gradient centrifugation. Under the treatment ofreceptor activator of nuclear factor (NF)-кB ligand（RANKL）and macrophagecolony-stimulating factor （M-CSF），the human PBMNCs were incubated inlower compartment of Transwell insert, and then co-cultured with RPMI-8226cells which were in upper compartment. As control, PBMNCs were cultured in thesame medium without RPMI-8226cells co-culture. OC formation and their function were investigated by tartrate-resistant acid phosphatase (TRAP) staining,lacunar resorption assag on dentine slices, and CD51/CD61flow cytometryanalysis.2. The expression of MIC-1in several cell lines were measured by real-time RT-PCR.The human MIC-1cDNA was amplified by reverse transcription-polymerasechain reaction (RT-PCR) technique, and then inserted into the expression vectorGV115. PCR and sequencing were used to identify the recombinant plasmidEGFP-MIC-1-FLAG. The recombinant plasmid was transfected into293T cell,and its expression was identified by Western blot and fluorescence microscope.3. Five shRNA oligonucleotides of MIC-1were designed and synthesized. Each wasthen inserted into iRNA lentivirus vector GV143which had been digested byendonuclease. Each connection product was transfected into the prepared bacterialcompetent cells. Then they were identified by PCR and sequencing. Each of themwas transfected into293T cells, together with the recombinant plasmidEGFP-MIC-1-FLAG. The optimal shRNA was selected by Western blot. Thelentivirus vector with optimal shRNA was transfected into293T cells togetherwith packaging vector and envelope protein vector. After being cultured for48h,lentivirus was concentrated and then the viral titer was determined. The lentiviruswith different multiplicity of infection (MOI=0,5,10) was transfected intoRPMI-8226, and the knocking down efficiency was tested by real-time RT-PCR.4. The human PBMNCs were isolated from peripheral blood by density gradientcentrifugation. The human PBMNC were incubated in lower compartment ofTranswell insert and RPMI-8226in upper compartment. OC formation andfunction were estimated by TRAP staining, lacunar resorption on dentine slicesand CD51/CD61flow cytometry analysis.[Results]1. Compared with control group, human PBMNCs formed more multinucleated giantcells after co-culture with RPMI-8226cells. Many purplish red vesicles in thecytoplasm and numerous nuclei with prominent nucleoli can be observed in TRAPstaining. Toluidine blue staining showed obvious lacunar resorption on dentine slices. The expression rate of CD51/CD61was53.88%, which was higher thancontrol group (p＜0.05).2. Real-time RT-PCR showed that high expression of MIC-1was found inRPMI-8226cells. The full length of MIC-1was amplified successfully usingRT-PCR in vitro. The length of amplified product was946bp which wasconsistent to anticipated size. PCR and Sequencing of the expression vectorindicated that MIC-1gene was correctly inserted. After the eukaryotic expressionvector containing MIC-1gene was successfully transfected into293T cells,obvious fluorescence was seen in the cells and63kDa fusion proteinEGFP-MIC-1-FLAG was found to exist in Western blot. MIC-1gene can bestably expressed in eukaryotic cells.3. PCR and sequencing confirmed that five synthesized shRNAs were successfullyinserted into the lentivirus vector. Western blot showed MIC-1gene werespecifically inhibited after the lentivirus vector and fusion gene expression vectorwere simultaneously transfected into293T cells. When the ratio of lentivirusvector and fusion gene expression vector was4:1or2:1, MIC-1-RNAi(shRNA1)was the best powerful inhibitor. ShRNA lentivirus particles were successfullypacked and concentrated, and the Virus titer was1×109TU/ml. When thelentivirus were transfected into RPMI-8226cells, CCK8asssy showed that cellviability was unaffected, the infection efficiency was more than80%underfluorescence microscope. Real-time RT-PCR confirmed that the expression ofMIC-1in RPMI-8226cells was decrease obviously, and the knockdown efficiencywas more than75%. Obviously decreased MIC-1protein expression was alsoconfirmed in Western blot.4. When PBMNC were co-culture with RPMI-8226cells which were transfectedwith lentivirus, fewer multinucleated giant cells were formed, compared withthose PBMNCs transfected with control group (Lv-NC). In the meantime, TRAPmultinucleated cells, TRAP positive activity and lacunar resorption size intoluidine blue staining on dentine slices, the expression rate of CD51/CD61flow cytometry were significantly decreased, comparing with Lv-NC group (p＜0.05）.[Conclusions]1. Co-culture of RPMI-8226with PBMNCs can promote PBMNCs differentiatinginto mature OC, indicating the co-culture system was successfully established.This system can simulate myeloma microenvironment in vitro and provide anefficient method to study the mechanism of OC differentiation and regulatingfactor.2. MIC-1fusion gene expression vector have been successfully constructed andexpressed in eukaryotic cells after tranfection.3. Five shRNA oligonucleotides of MIC-1has been sucessfully constructed andsynthesized and the optimal shRNA targeting MIC-1selected. High titer ofshRNA lentivirus was successfully produced. After RPMI-8226cells weretransfected with shRNA lentivirus, the expression of MIC-1was blockedconstantly, effectively and exclusively.4. When PBMNCs were co-culture with RPMI-8226cells where the MIC-1genewas exclusively blocked, the capacity of PBMNCs differentiating into mature OCdecreased. We concluded that MIC-1might promote the mature of OC and couldbe a new treatment target for MBD.