Study On The Effects And Mechanism Of Brain-derived Neurotrophic Factor On Bone Destruction In Multiple Myeloma

Part1Study of the effects and mechanism of BDNF on RANKL expression in human BMSCsObjective To evaluate the effects of BDNF on RANKL in BMSCs and the relative signaling pathwayMethods Culture and identify human BMSCs; Use human recombinant BDNF to stimulate BMSCs, and then detect RANKL mRNA and protein expression in BMSCs by RT-PCR and Western blot; Add K252a to the BMSCs culture system, and then detect RANKL secretion levels by BMSCs using ELISA; Detect the phasphorylation of ERK, AKT and IkB level in BMSCs pretreated with BDNF, and detect the nuclear transformation of p65by immunofloresense; BMSCs were pre-incubated with Then MEK/ERK inhibitor U0126and or AKT inhibitor LY204002were added to BMSCs cultures to detect their effects on RANKL expression; Construct MM-BMSCs and MM-BMSCs-preOC coculture system by Transwell with0.4um pores; Add K252a to the BMSCs culture system, and then detect osteoclast formation in these co-and triple cultures.Results Human BMSCs expressed CD44、CD105and CD29, but not CD14,CD45and characterized for osteocytes differentiation capacity; Human recombinant BDNF significantly increased RANKL expression by BMSCs in an dose-and time-depedent manner, and these effects could be reversed by K252a; Stimulation of BMSCs by25ng/ml BDNF led to significant increased phasphorelation of ERK and AKT, but had no effect on the phasphorelation of IkB levels. no obvious nuclear translocation of the NF-kB p65subunit was noted when compared to the untreated control BMSCs pre-treated with U0126showed a49.75%decrease in BDNF-stimulated RANKL secretion compared with BDNF stimulation alone (P<0.05). Treatment of BMSCs with LY204002partially inhibited BDNF-stimulated RANKL secretion but with no statistical significance (P=0.069). A marked increase of osteoclast number was detected when pre-OCs were co-cultured with MM cells or triple-cultured with human BMSCs and MM cells, these effects were partially reversed by neutralizing antibody to BDNF and completely abolished by OPGConclusion Our studies demonstrate that BDNF increases RANKL expression in human BMSCsthrough ERK pathway, which then leading to osteoclastogenesis in bone marrow microenvironment. Part2Gene silencing of BDNF in MM cells inhibits osteolytic bone destruction and tumor burden in SCID-rab miceObjective To evaluate the effects of MM derived BDNF on osteoclastogenesis and tumor burden in SCID-rab-MM model by gene silencing of BDNF by shRNAMethods Designed an construct an antisense structure to BDNF (short-hairpin RNA, shRNA), eGFP reporter gene was also used; ARH-77cells and RPMI8226cells were transfected with BDNF antisense shRNA/eGFP (AS-MM) or empty-vector shRNA/eGFP (EV-MM) by replication-incompetent lentiviral vectors. Then, the eGFP+MM cells were sorted using flow cytometer; Down-regulation of BDNF protein expression was confirmed by western blotting; Then rabbits bones were subcutaneously implanted into NOD/SCID mice, AS-ARH, EV-ARH, and wild-type ARH77were injected directly into the implanted bone. RPMI8226cells were intravenously injected into SCID mice. Blood samples were collectedand murine sera were analyzed for human IgG by ELISA; Radiographs were taken and changes in the bone mineral density (BMD) of the rabbit bone grafts were measured weekly. The fluorescence intensity of SCID-rab mice was monitored periodically; Rabbit bone sections were stained with Hematoxylin and eosin (H&E) staining or TRAP staining. Marrow plasma were collected for ELISA analysis; Then, RANKL levels in bone marrow plasma were measured by western blot and immunohistochemistry analysis;Results X ray detection shows that the osteolytic burden of rabbit bones from the AS group was significantly lower than that from the EV group; The BMD of bones injected with EV-ARH cells was reduced by68%±5%compared to pretreatment BMD, whereas in the AS-ARH group, BMD was decreased by15%±8%(EV-ARH vs. AS-ARH, n=12per group, P<0.05). Bones harboring AS-ARH cells had a significantly reduced level of RANKL compared to the EV-ARH group (P<0.01); TRAP-positive multinucleated OCLs in rabbit bone implants from the AS-ARH group were less numerous than those from the EV-ARH group; Significantly lower expression of RANKL and BDNF were found in AS-ARH-infiltrated bone; The number of RANKL-positive cells in bone sections from the AS-ARH group was markedly decreased compared to the EV-ARH group; The fluorescence intensity in the AS-ARH group was much weaker than EV-ARH group; Circulating hIgG in blood serum from the AS-ARH group was significantly lower than in the EV-ARH (P<0.05); Significantly prolonged overall survival of the mice were found in the AS group compared with EVgroup. Similar results were found in SCID-RPMI8226mice model. Mice from AS group hold a reduced bone destruction, decreased angiogenesis, and prolonged survival.Conclusion Stable knockdown of BDNF inhibits osteoclastogenesis and tumor growth in SCID mice, and BDNF may be a new target for the treatment of MM bone diseases.