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Objective To investigate whether miR-200a suppresses cell proliferation by targeting AP-2γexpression, and reveal molecular mechanism that miR-200a functions as a tumor-suppressor in neuroblastoma cells. Methods Dual-luciferase reporter gene assay was employed to examine the effect of miR-200a on AP-2γpromotor luciferase activity. Neu-roblastoma cells were transfected with miR-200a mimics, and the expressions of AP-2γmRNA and protein were detected by RT-PCR and Western blot assay. The effects of AP-2γdown-regulation on cell proliferation were observed after AP-2γshRNA was transfected into neuroblastoma cells. Neuroblastoma cell proliferation was detected by MTS assay after being co-transfected with miR-200a mimics and AP-2γplasmid. Results Results showed that miR-200a could inhibit proliferation of neuroblastoma cells at cell viability (66.33 ± 5.13) compared with that of control group (100 ± 0), and also miR-200a can bind to the 3'untranslated region of AP-2γpromotor and inhibit its luciferase activity with an inhibit ratio at (0.624±0.051). AP-2γmRNA and protein expressions were significantly down-regulated when miR-200a was over-expressed in neuroblas-toma cells. Furthermore, results showed that shRNA-mediated down-regulation of AP-2γthat suppressed the cell prolifera-tion of neuroblastoma at (62.5±2.4) by comparing with the control group (100±0). Moreover, restoring AP-2γexpression re-versed the effect of miR-200a with a cell viability suppression at (92.4±1.4). Conclusion miR-200a suppresses cell prolif-eration by targeting AP-2γexpression in neuroblastoma cells.
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Objective To explore the expression and clinical signiifcance of microRNA-200a in childhood B-cell acute lymphoblastic leukemia (ALL). Methods Bone marrow samples were collected from 45 children with B-cell ALL and 18 chil-dren without hematology disease as control. Total RNA was acquired from bone marrow. qRT-PCR was performed to detect the expression level of miR-200a. Results The relative expression level of miR-200a in B-cell ALL group was signiifcantly lower than that in control group (P<0.05);the expression of miR-200a in children over 10 years old was signiifcantly lower than those in children under 10 years old (P<0.05);the expression of miR-200a in newly diagnosed samples was lower than those in those samples taken on Day 33 and at Week 12, respectively (P<0.05, P<0.01). In addition, the expression of miR-200a in low-risk group was higher than those in mid-risk and high-risk group, respectively (P<0.05). Conclusions Low level of miR-200a had a close correlation with the development and prognosis of childhood B cell ALL, which could be used as a potential target of thera-py and a biomarker of childhood B cell ALL in the future.
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Objective To explore the expression of interleukin-17 (IL-17) in the murine myocardium with viral myocarditis (VMC) ,and the influence of astragaloside intervention on its expression .Methods 60 male 4-week-old Balb/c mice were randomly divided into four groups ,namely normal control group ,model control group ,low-dose and high-dose intervention groups ,15 mice in each group .Mice in the latter three groups were inoculated with 0 .1 mL coxsackie B3 virus intraperitoneally .Then ,mice in low-dose and high-dose intervention groups were treated with 0 .01 g/L and 0 .09 g/L astragaloside solution ,respectively .All mice were killed on 15 days .The mortality and heart weight/body weighty (HW/BW ) were calculated .Histological cross sections of heart were stained with hematoxylin-eosin and histopathologic scores of inflammatory cells infiltration and myocardial necrosis were eval-uated under optical microscope .The expression levels of myocardial IL-17 mRNA and protein were detected through real-time quan-titative PCR and Western blot .The contents of IL-6 and tumor necrosis factor-α(TNF-α) in the myocardium were examined by ELISA .Results The mortality and histopathologic scores of inflammatory cells infiltration and myocardial necrosis in high-dose in-tervention group were significantly lower than those in model controlgroup (P<0 .05) .Compared with normal control group ,the HW/BW ,the expression levels of myocardial IL-17 mRNA and protein as well as the contents of IL-6 and TNF-αin the myocardi-um were markedly increased in model control group(P<0 .01) ,whereas these parameters were significantly decreased in high-dose intervention group as compared to model group (P<0 .05) .Conclusion IL-17 may be involved in the pathogenesis of VMC .The therapeutic effect of astragaloside on VMC may be associated with inhibiting IL-17-mediated inflammatory response .
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Objective To explore the role of interleukin-23 (IL-23)/interleukin-17 (IL-17) signaling pathway in viral myocarditis (VMC) and evaluate the intervention effect of Aastragaloside. Methods Seventy-five male BALB/c mice were randomly divided into 4 groups, control group (n=15), model group (n=20), low-dose intervention group (n=20) and high-dose intervention group (n=20). Mice in control group were inoculated with 0.1 ml virus cultivation solution intraperitoneally while mice in the other 3 groups were treated with 0.1ml virus cultivation solution containing 1×102 TCID50 coxsackievirus B3 (CVB3) to establish VMC model. On the day of inoculation, mice in low- and high-dose intervention groups were intra-gastrically administered with 0.1 ml of 1% and 9%Astragaloside solution respectively, whereas those in control and model groups were treated with 0.1 ml carboxymethycellulose solution. Astragaloside or carboxymethycellulose was given once a day and continued 15 days. The number of mice death and the performance of mice were recorded in experimental period. All mice were sacrificed on day 15. The heart and blood sample were obtained. Histological cross sections of heart were stained with hematoxylin-eosin and scored for myocardial histopathologic changes under optical microscope. Th17 cells were analyzed by flow cytometry. The mRNA and protein expression levels of myocardial IL-23 and IL-17 were detected by real-time quantitative PCR and Western blotting, respectively. Results The mortality was statistically significant differ-ences among the four groups (P= 0.013), which was the lowest in the control group. The myocardial histopathologic scores, the percen-tage of Th17 cells, as well as expression levels of myocardial IL-23 and IL-17 mRNA and protein were significantly lower in high-dose intervention group than those in model group and low-dose intervention group, but higher than those in control group (P 0.05). Conclusions Astragaloside may dose-dependently protect against VMC by in-hibiting IL-23/IL-17 signaling pathway.
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Background and purpose:MicroRNAs are 19 to 25-nucleotide noncoding RNA molecules. The aim of this article was to investigate the expression and clinical signiifcance of microRNA-141 in childhood B-cell acute lymphoblastic leukemia (ALL). Methods:Bone marrow samples were collected from 35 children with B-cell ALL and 15 children with non hematologic disease. Total RNA was acquired from bone marrow. Real-time PCR was performed to detect the expression level of miR-141. Results:The relative expression level of miR-141 in B-cell ALL group was remarkably lower than those in the control (P<0.05). The expression of miR-141 in newly diagnosed samples was lower than those in Day 30 and Week 12 samples respectively (P<0.05). Besides, the expression of miR-141 in Day 30 samples was lower than those in week 12 samples (P<0.05). The expression of miR-141 in children over 10 years old was signiifcantly lower than those in children under 10 years old (P<0.05). The expression of miR-141 in low-risk group was higher than those in mid-risk and high-risk group respectively (P<0.05), and there was signiifcant difference between mid-risk and high-risk groups (P<0.05). Conclusion:MiR-141 is likely to have tumor suppressor effect and to be a potential prognostic biomarker in childhood B cell ALL.
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Objective To determine the immunogenic and adhesive abilities of a segment (P1C protein) that located at the carboxy terminal region of P1 protein (1125 to 1395 amino acids).Methods A recombinant prokaryotic vector (pGEX6p-2/p1c) was constructed for P1C protein expression in E.coli BL21DE3.The expressed target recombinant protein (rP1C) was identified using SDS-PAGE and Western blot assay,and then extracted by GST-based affinity chromatography.The purified rP1C was used to immunize BALB/c mice to obtain rP1C-antiserum and titer of the antiserum was determined by ELISA.Immunoreactivity of the rP1C to the sera form M.pneumoniae-infected patients was detected using Western blot assay,while activity of the rP1C adhering to HeLa cells as well as adhesion blockage of the rP1C antiserum were detected using indirect immunofluorescence assays.Results The constructed prokaryotic expression system could efficiently express soluble rP1C with a relative molecular weight of 66×103.The antiserum from rP1Cimmunized mice showed an ELISA titer as high as 1:64 000.Both the M.pneumoniae-infected patients' sera and the mouse antiserum against rP1C could recognize as well as combine with the rP1C.rP1C could adhere to HeLa cells and the adhesion could be blocked by the mouse antiserum with an antiserum concentration-dependent manner.Conclusion P1C,a segment of M.pneumoniae P1 protein,possesses powerful immunogenicity and immunoreactivity and cell-adhered activity,indicating the protein segment can be used as an antigen candidate for developing vaccines and serological diagnostic methods of M.pneumoniae-induced diseases.