ABSTRACT
OBJECTIVES@#To study the role and mechanism of platelet-derived growth factor BB (PDGF-BB) on platelet production in Kawasaki disease (KD) mice and human megakaryocytic Dami cells through in vitro and invivo experiments.@*METHODS@#ELISA was used to measure the expression of PDGF in the serum of 40 children with KD and 40 healthy children. C57BL/6 mice were used to establish a model of KD and were then randomly divided into a normal group, a KD group, and an imatinib group (30 mice in each group). Routine blood test was performed for each group, and the expression of PDGF-BB, megakaryocyte colony forming unit (CFU-MK), and the megakaryocyte marker CD41 were measured. CCK-8, flow cytometry, quantitative real-time PCR, and Western blot were used to analyze the role and mechanism of PDGF-BB in platelet production in Dami cells.@*RESULTS@#PDGF-BB was highly expressed in the serum of KD children (P<0.001). The KD group had a higher expression level of PDGF-BB in serum (P<0.05) and significant increases in the expression of CFU-MK and CD41 (P<0.001), and the imatinib group had significant reductions in the expression of CFU-MK and CD41 (P<0.001). In vitro experiments showed that PDGF-BB promoted Dami cell proliferation, platelet production, mRNA expression of PDGFR-β, and protein expression of p-Akt (P<0.05). Compared with the PDGF-BB group, the combination group (PDGF-BB 25 ng/mL + imatinib 20 μmol/L) had significantly lower levels of platelet production, mRNA expression of PDGFR-β, and protein expression of p-Akt (P<0.05).@*CONCLUSIONS@#PDGF-BB may promote megakaryocyte proliferation, differentiation, and platelet production by binding to PDGFR-β and activating the PI3K/Akt pathway, and the PDGFR-β inhibitor imatinib can reduce platelet production, which provides a new strategy for the treatment of thrombocytosis in KD.
Subject(s)
Child , Humans , Animals , Mice , Mice, Inbred C57BL , Becaplermin , Imatinib Mesylate/therapeutic use , Mucocutaneous Lymph Node Syndrome/drug therapy , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Thrombocytosis/etiology , RNA, MessengerABSTRACT
<p><b>OBJECTIVE</b>To explore the regulating effect of miR-202 on B cell-activating factor, and check whether the regulation influences the growth of multiple myeloma cells.</p><p><b>METHODS</b>The potential binding sites of BAFF for miR-202 were predicted using bioinformatics software. Luciferase reporter gene analysis was used to evaluate the regulatory effect of miR-202 on BAFF. Human multiple myeloma U266 cells were transfected with has-miR-202-mimics, has-miR-202-inhibitor, siBAFF and their negative controls, respectively. After above treatments, BAFF mRNA and protein levels were detected by real-time PCR and Western blot analysis, and the proliferation and apoptosis in the multiple myeloma (MM) cells were examined by WST-1 and annexin V-FLUOS assay, respectively.</p><p><b>RESULTS</b>The BAFF mRNA expression levels in the untransfected group, has-miR-202-3P-mimics transfected group, has-miR-202-3P-inhibitor transfected group and siBAFF transfected group were 1.040 ± 0.057, 0.573 ± 0.073, 1.205 ± 0.097 and 0.368 ± 0.052, respectively. BAFF mRNA expressions in U266 cells transfected with has-miR-202-3P-mimics and siBAFF were significantly decreased compared with that in the untransfected group (P < 0.05). The BAFF protein expression level of each group was consistent with the mRNA assay result. The absorbance value in 450 nm of the untransfected group, has-miR-202-3P-mimics transfected group, has-miR-202-3P-inhibitor transfected group and siBAFF transfected group were 1.063 ± 0.052, 0.714 ± 0.045, 0.936 ± 0.066 and 0.764 ± 0.053, respectively. In comparison with the untransfected group, the absorbance value at 450 nm of has-miR-202-3P-mimics and siBAFF transfected groups was significantly reduced (P < 0.05). The cell apoptosis rates of untransfected group, has-miR-202-3P-mimics transfected group, has-miR-202-3P-inhibitor transfected group and siBAFF transfected group were 26.2%, 49.6%, 21.1% and 30.7%, respectively. Therefore, the cell apoptosis rate of has-miR-202-3P-mimics transfected group was significantly increased than that of the untransfected group (P < 0.05). p-JNK protein expression level was decreased in the has-miR-202-3P-mimics transfected cells.</p><p><b>CONCLUSIONS</b>MiR-202 can inhibit the proliferation and induce apoptosis in MM cells via regulating BAFF. JNK/SAPK signaling pathway is involved in the regulation of BAFF by miR-202.</p>