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1.
Article in Chinese | WPRIM | ID: wpr-319464

ABSTRACT

<p><b>OBJECTIVE</b>To investigate AXIN1-related CSRNP1 gene expression and the mechanism of its transcriptional regulation in TGF-β1-induced tumor cells.</p><p><b>METHODS</b>Human lung carcinoma A549 cells or human prostate cancer PC3 cells were treated with TGF-β1 at different doses (0, 20, 40, and 80 ng/ml) or at 20 ng/ml for 0, 8, 12, or 24 h, and the dose and time effect of TGF-β1 on CSRNP1 mRNA expression in the tumor cells were evaluated with real-time RT-PCR. A549 cells were also treated with TGF-β1 and cycloheximide to clarify whether CSRNP1 expression induced by TGF-β1 required de novo protein synthesis. A549 cells transfected with pcDNA3.1, flag-SMAD3, or flag-SMAD3-mu, after serum starvation, were treated with or without TGF-β1 (20 ng/mL) for 24 h, and the overexpression of wild-type SMAD3 and dominant negative SMAD3-mu mutant were confirmed by Western blotting. The effect of SMAD3 or SMAD3-mu overexpression on CSRNP1 mRNA expression was also measured by real-time RT-PCR.</p><p><b>RESULTS</b>In both A549 and PC3 cells, TGF-β1 dose- and time-dependently stimulated CSRNP1 expression, which required de novo protein synthesis in A549 cells. Overexpression of wild-type SMAD3 significantly increased the expression of CSRNP1 mRNA induced by TGF-β1, while overexpression of dominant negative SMAD3 mutant remarkably reduced CSRNP1 mRNA expression in response to TGF-β1 in A549 cells.</p><p><b>CONCLUSION</b>TGF-β1 may contribute to CSRNP1 expression through SMAD3 activation and downstream signaling in tumor cells.</p>


Subject(s)
Apoptosis Regulatory Proteins , Genetics , Metabolism , Axin Protein , Genetics , Metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , RNA, Messenger , Genetics , Signal Transduction , Smad3 Protein , Genetics , Metabolism , Transfection , Transforming Growth Factor beta1 , Pharmacology
2.
Article in Chinese | WPRIM | ID: wpr-387576

ABSTRACT

The molecular and cellular mechanisms of bone metastasis in prostate cancer remain unclear. Current researches focus on chemotaxis of tumor cells in bone metastasis, interactions between tumor cells and bone micro-environment as well as the vicious circle among tumor cells, osteoclasts, osteoblasts and bone matrix.

3.
Article in Chinese | WPRIM | ID: wpr-554021

ABSTRACT

To construct the eukaryotic vector that expresses the fusion protein of Axud1 and influenza virus hemagglutin HA epitope tag, the total RNA was isolated from the peripheral blood lymphocytes, and reverse transcription reaction was used to amplify the full length of human Axud1 cDNA. PCR product of Axud1 was then amplified using specific primers containing HA epitope sequence, and inserted into eukaryotic expression plasmid pcDNA3.1(+)digested with BamH Ⅰand Xba Ⅰ. The recombinant plasmid was identified by polymerase chain reaction, restriction endonuclease mapping and sequencing, and then transfected into human lung adenocarcinoma SPC-A1 cell lines.The fusion HA-Axud1 protein expression in anti-G418 clones was verified by Western blot. This study might be instrumental in further study of the function of Axud1 protein in tumor cells.

4.
Article in Chinese | WPRIM | ID: wpr-529202

ABSTRACT

AIM: To determine the differences in phosphoproteome between LPS stimulated THP-1 cells with and without previous oxidative stress for screening of more potential regulators.METHODS: Differentiation of THP-1 cells into macrophages was induced by treatment with 100 ?g/L PMA for 36 h. Differentiated cells were rested for additional 36 h without PMA treatment, then treated with 100 ?mol/L H2O2 or medium for 1 h followed by LPS or medium treatment for 30 min. After desalted, phosphoproteins were enriched by phosphoprotein metal affinity column, and were run on 2-D electrophoresis, then the spots were analyzed to show the difference between LPS group (cells treated with LPS alone) and H2O2+LPS group (LPS stimulated cells also pretreated with H2O2). Finally, some of these spots were identified by MS and subsequent bioinformatic analysis was also conducted. RESULTS: Compared to LPS group, 29 reproducibly changed spots on the 2-D map in H2O2+LPS group were visualized and selected for MS analysis. Among these, 12 down-regulated spots (include those disappeared), 17 up-regulated spots (include those newly emerged) were selected. Up to now, 5 of these were identified, which were shown to be involved in various cellular processes such as proteolysis, signal transduction and protein folding. Among these, proteasome beta-4 subunit, which was dramatically down-regulated in H2O2+LPS group, was a major component of the proteasome complex and might participate in LPS signalling through various ways.CONCLUSION: With comparative phosphoprotein-affinity profiling, the interference brought by highly abundant house-keeping proteins is minimized, rendering us to detect less abundant signalling molecules. Aforementioned 5 proteins, especially proteasome beta-4 subunit, might be involved in LPS pathway reprogrammed by oxidative stress.

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