ABSTRACT
Objective: To investigate the effects of nephroblastoma overexpressed (NOV) gene on proliferation, apoptosis and migration of human osteosarcoma 143B cells in vitro, and to explore its underlying mechanism. Methods: The osteosarcoma cell line with low expression of NOV was screened out by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The 143B cells were infected with recombinant adenoviruses AdNOV and AdGFP (as a negative control) and cultured for 24 h. Then the infection efficiencies of recombinant adenoviruses AdNOV and AdGFP in 143B cells were observed under a fluorescence microscope. The expression levels of NOV mRNA and protein were detected by semi-quantitative RT-PCR and Western blotting, respectively. The effects of NOV overexpression on proliferation, apoptosis and migration of 143B cells were measured by MTT, flow cytometry (FCM) and Transwell migration test, respectively. The expression levels of NOV, B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) in 143B cells were examined by semi-quantitative RT-PCR and Western blotting. The expression levels of phospho-p38 (p-p38), total p38, phospho-c-Jun N-terminal kinase (p-JNK) and total JNK were detected by Western blotting. Results: The expression level of NOV was lowest in 143B cells (P 0/G1 phase (P < 0.05). As compared with the control group, NOV overexpression increased the expession level of Bax (P < 0.001) and down-regulated the expression level of Bcl-2 (P < 0.05) in 143B cells. At the same time, the expression levels of p-p38 and p-JNK were elevated significantly in 143B cells overexpressing NOV (P < 0.001). Conclusion: NOV overexpression induced by adenovirus can inhibit the proliferation of 143B cells and promote the apoptosis and migration of 143B cells in vitro. The p38/mitogenactivated protein kinase (MAPK) and JNK/MAPK signaling pathways may be involved in this process.
ABSTRACT
Objective To explore the effect of dendrobium nobile polysaccharides on the expression of the WT 1 gene in my-eloid leukemia cells.Methods The CCK8 assay was used to detect the half maximal inhibitory concentration(IC50 )of dendrobium nobile polysaccharides in 3 kinds of leukemia cells;the each kind of leukemia cells were divided into the treatment group and the control group.The cells in the control group maintained the normal growth,while which in the treatment group were given the den-drobium nobile polysaccharides stimulation.The Hoechst33258 staining was used to detect the apoptosis situation of the cells in the two groups.The WT l gene expression level was detected by the real-time PCR and the protein expression levels of WT1,53 and BAX were detect the Western blot.Results Dendrobium nobile polysaccharides had the similar IC50 values in 3 kinds of myeloid leukemia cells,which were (110.71±6.49),(104±48.50),(96.66±5.10)mg/mL respectively,the difference among them had no statistical significance (P >0.05);the apoptosis rate of the treatment group was higher than that of the control group (P <0.05);the expression levels of WT1 gene and protein in the treatment group were decreased compared with the control group(P <0.05), while the expression of P53 and BAX protein was increased.Conclusion Dendrobium nobile polysaccharides can obviously decrease the expression level of WT1 protein,and has a certain killing effect on myeloid leukemia cells.
ABSTRACT
Objective To construct an eukaryotic expression vector for shRNA targeting WT1 gene.Methods Firstly, one site targeting human WT1 gene in gene bank was selected according to the guidelines for the selection of highly effective siRNA sequence and then BLAST test was performed. Secondly,a pair of oligonucleotides fragments were synthesized and annealed to double strand. Thirdly, the double strand was cloned into plasmid vector pGenesil-1. Finally, the recombinant plasmid was identified by restriction enzyme digestion and sequencing analysis. Results Enzyme cutting, and sequencing analysis have confirmed that the construction was successful. Conclusion WT1shRNA is constructed which is the fundament to further research of WT1 gene.
