Interaction of human genes WT1 and CML28 in leukemic cells.

The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms' tumor 1 suppressor gene (WT1) and CML28 abnormally expressed in malignant diseases of the hematopoietic system and WT1 played an important role in leukemogenesis. However, the relationship between molecular CML28 and WT1 has not been reported. Here we described the use of small interfering RNA (siRNA) against WT1 and CML28 in leukemic cell line K562 to examine the interaction between CML28 and WT1. WT1 and CML28 gene expression in transfected K562 cells was detected by using RQ-PCR and Western blotting. K562 cells transfected with WT1-siRNA could greatly decrease both mRNA and protein expression levels of WT1 and CML28. In contrast, CML28-siRNA did not exert effect on WT1. Further, subcellular co-localization assay showed that the two proteins could co-localize in the cytoplasm of K562 cells, but WT1/CML28 complexes were not detected by using immunoprecipitation. It was suggested that there exists the relationship between CML28 and WT1. CML28 may be a downstream target molecule of WT1 and regulated by WT1, which will provide important clues for further study on the role of CML28 and WT1 in leukemic cells.

Genistein inhibits angiogenesis developed during rheumatoid arthritis through the IL-6/JAK2/STAT3/VEGF signalling pathway.

BACKGROUND: Angiogenesis plays an important role in the development of rheumatoid arthritis (RA), which increases the supply of nutrients, cytokines, and inflammatory cells to the synovial membrane. Genistein (GEN), a soy-derived isoflavone, has been validated that can effectively inhibit the angiogenesis of several tumours. We thus carried out a study in vitro to investigate the effect of GEN in vascular endothelial growth factor (VEGF) expression and angiogenesis induced by the inflammatory environment of RA. METHODS: MH7A cells were used to verify whether GEN can inhibit the expression of VEGF in MH7A cells under inflammatory conditions and demonstrate the mechanism. EA.hy926 â€‹cells were used to verify whether GEN can inhibit the migration and tube formation of vascular endothelial cells in inflammatory environment. RESULTS: GEN dose-dependently inhibited the expression and secretion of interleukin (IL)-6 and VEGF, as well as the nucleus translocation of Signal transducer and activator of transcription 3 (STAT3) in MH7A. Furthermore, GEN inhibited IL-6-induced vascular endothelial cell migration and tube formation in vitro. CONCLUSION: GEN inhibits IL-6-induced VEGF expression and angiogenesis partially through the Janus kinase 2 (JAK2)/STAT3 pathway in RA, which has provided a novel insight into the antiangiogenic activity of GEN in RA. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Our study provides scientific guidance for the clinical translational research of GEN in the RA treatment.

[Human osteoblasts response to different magnitudes of mechanical stimulation in vitro].

OBJECTIVE: To investigate the effect of different magnitudes of tensile strain on human osteoblasts differentiation. METHODS: According to the strain amplification mechanism at cellular level and a data calculated by finite element analysis, the cellular level strain of 0.8%, 1.6%, 2.4% and 3.2% was respectively applied to human osteoblasts for 48 h at a frequency of 1 Hz. Alkaline phosphatase activity and the expression of osteoblast-related genes were detected by Semi-Quantitative RT-PCR and densitometric analysis. RESULTS: Alkaline phosphatase activity significantly increased at 0.8% and 1.6%. The level of osteocalcin mRNA increased at 2.4% and 3.2%. Cbfa1/Runx2 gene expression only increased at 3.2%. Comparing to static control, mRNA level of type I collagen increased at every magnitude. The mRNA level decreased at 0.8% and increased at 3.2% when compared to the group with 1.6% elongation. CONCLUSIONS: Higher magnitudes of strain enhance expression of osteocalcin, type I collagen gene and Cbfa1/Runx2 in human osteoblasts, but lost the ability to increase ALP activity which is remained by lower magnitudes of strain. Type I collagen gene expression increases in a strain magnitude dependent manner.

[Expression and purification of GST-CML28 fusion protein and preparation of its polyclonal antibody].

This study was aimed to investigate the expression of GST-CML28 in Escherichia Coli and to prepare its antibody. The constructed recombinant expression vectors CML28-pGEX-3X were transformed into Escherichia Coli BL21 under IPTG induction. The protein was abstracted from the transformers, and purified by a GSTrap FF column. The rabbits were immunized by the purified fusion protein to produce serum with anti-CML28 antibody. The serum was purified by chromatographic column stuffed with glutathione Sephamse 4B to get the antibody. The specific antibody against CML28 was further identified by ELISA, Western blot, immunohistochemistry and quantum dot luminescence. The results indicated that GST-CML28 fusion protein was expressed in Escherichia coli and its specific polyclonal antibody was obtained. It is concluded that the anti-CML28 polyclonal antibodies with high titer and specificity are successfully prepared. These antibodies provide an useful experimental tool to profoundly research the physiological significance and biological function of the CML28 gene.

[Comparison of bicyclol therapy for patients with genotype B and C of hepatitis B virus].

OBJECTIVE: To compare the efficacy of bicyclol tablets on patients infected with hepatitis B virus between genotype B and C. METHODS: 70 patients with chronic viral hepatitis B were selected. The patients divide into two groups: HBV genotypes B (26 cases) and HBV genotypes C (other 44 cases). All patients received bicyclol tablets orally 150 mg daily (50mg, tid, po) for 24 weeks. The efficacy were observed after 12 weeks and 24 weeks. RESULTS: After treatment for 24 weeks, the serum aminotransferase were decreased obviously, and HBV DNA levels turn to be negative with 19.2 percent (genotype B group) and 15.9 percent (genotype C group), respectively. The difference was not statistically significant between HBV genotype B and C. CONCLUSION: Bicyclol not only has hepatoprotective activity but also inhibited virus replication in patients infected with HBV. The difference of the response to bicyclol therapy between HBV genotypes B and C was not statistically significant.