β-Catenin/LEF-1 transcription complex is responsible for the transcriptional activation of LINC01278
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Abstract Background Our previous study shows that LINC01278 inhibits the malignant proliferation and invasion of papillary thyroid carcinoma (PTC) cells by regulating the miR-376c-3p/DNM3 axis. However, the regulation mechanism of LINC01278 expression in PTC cells is still unclear. Methods The luciferase reporter and ChIP assays were used to confirm the binding of LEF-1 to the putative promoter site of LINC01278 gene. The RNA immunoprecipitation and RNA pulldown were used to determine the enrichment of LINC01278 in β-catenin protein. The proteasome inhibitors (MG132) was used for detecting the β-catenin ubiquitination-proteasome degradation. Wnt/β-catenin specific agonists (LiCI), inhibitors (WiKI4) and TOP/FOP-flash reporter assay were used for detecting the activation of Wnt/β-catenin signal. Western blot was used to detected the expression of target proteins. Results The online PROMO algorithm determines a putative LEF-1 binding site on LINC01278 promoter, the LEF-1 binds to the putative promoter site of LINC01278 gene, and β-catenin enhances the binding of LEF-1 to the LINC01278 gene promoter. Furthermore, LINC01278 negatively regulated the protein accumulation of β-catenin in the cytoplasm, into nucleus, and ultimately inhibited the transcription of downstream target genes activated by Wnt/β-catenin signal. The results of RNA immunoprecipitation and RNA pulldown proved the direct binding of LINC01278 to β-catenin protein. In addition, the combination of LINC01278 and β-catenin promotes the β-catenin ubiquitination-proteasome degradation. Conclusion In summary, we found the transcriptional activation of LINC01278 by the β-catenin/LEF-1 transcription factor, and the negative feedback regulation of LINC01278 onβ-catenin signal.Keywords:
MG132
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Objective To study the effect of proteasome inhibitor MG132 on expression of p53 in the apoptosis of cultured human umbilical vein endothelial cells induced.Methods Human umbilical vein endothelial cells were treated with MG132(2 μmol/L,5 μmol/L) for 24 hours.The apoptotic cells were determined by DNA fragment analysis and flow cytometric analysis.p53 protein expression was detected by immunohistochemistry.Results The results showed that the increase of the degree of human umbilical vein endothelial cells apoptosis was concentration dependent.MG132 could up-regulate the protein expression of p53.Conclusions The results implicated that proteasome inhibitor MG132 induced human umbilical vein endothelial cells apoptosis by inhibitting UPP and accumulation of p53.
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Abstract Sequential Chromatin Immunoprecipitation (SeqChIP) is a powerful technique for analyzing the simultaneous association of two different proteins with genomic DNA sequences in vivo. Cellular Protein‐DNA complexes are cross‐linked with formaldehyde ( UNIT ), and are purified via two successive immunoprecipitations, with each immunoprecipitation targeting a different protein. Protein‐DNA cross‐links are then reversed and DNA sequences of interest are analyzed by quantitative PCR. At each genomic region, calculated SeqChIP co‐occupancy values are compared to occupancy values of singly immunoprecipitated samples. The extent of enrichment brought about by the second immunoprecipitation relative to the singly immunoprecipitated sample is directly correlated with the degree of co‐occupancy between the two proteins at the genomic location assayed. In principle, the technique is not limited to Saccharomyces cerevisiae . Cells from a wide variety of organisms can be used.
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Chromatin immunoprecipitation
genomic DNA
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Chromatin immunoprecipitation
Immunoprecipitation
ChIP-on-chip
ChIP-sequencing
Methylated DNA immunoprecipitation
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MG132 (Z-Leu-Leu-Leu-CHO) is an inhibitor of proteasome, and it can enter the cell to reversibly inhibit the activation of proteasome, thereby influencing the course of cell cycle and inducing the apoptosis of cells. Currently, it has been reported that MG132 can induce apoptosis of all kinds of tumorous cells and can be used a anti-tumor drug. This article reviews the mechanisms of the apoptosis of the tumorous cells induced by MG132.
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To investigate the effect of different concentration proteasome inhibitor (MG132) on the apoptosis of human cervix cancer Hela cells,Hela cells was cultured with different concentrations of proteasome inhibitor (MG132),and the cell viability was determined by MTT assay. The cell apoptosis rate was determined by Annexin Ⅴ/PI staining and flow cytometry. Caspase-3 activity change was tested by Western blotting and ELISA Reader. The results indicate that high concentration of proteasome inhibitor (MG132) is more significant in injury and apoptosis of Hela cells than low concentration one. The different concentration of proteasome inhibitor (MG132) is obvious in inducing apoptosis of hela cells.
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Chromatin immunoprecipitation (ChIP) assays are widely used to investigate where chromatin-binding proteins bind to the genome. The standard assay is very time consuming. We have developed a rapid ChIP assay in which the immunoprecipitates serve directly as PCR templates. This assay eliminates the step to reverse the crosslinking, shortening the assay by 1 day. It also requires a less immunoprecipitating antibody, permits many samples to be tested simultaneously, and is more sensitive than the standard ChIP assay.
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Objective To study the effect of proteasome inhibitor MG132 on expression of caspase-3 and UPP associated genes in the apoptosis of human umbilical vein endothelial cells induced. Methods human umbilical vein endothelial cells were treated with MG132(2 μmol/L,5 μmol/L) for 24 hours.The apoptotic cells were detected by flow cytometric analysis.The level of E1,E2,E3 and caspase-3 mRNA expression were detected by reverse transcription-polymerase chain reaction(RT-PCR).Caspase-3 protein expression was detected by immunocytochemistry. Results The results showed that E1,E2,E3 genes expression were decreased in treatment group and apoptosis rate were increased obviously.MG132 could up-regulate the gene/protein expression of caspase-3. Conclusions Protesome inhibitor MG132 could induced human umbilical vein endothelial cells apoptosis by inhibitting UPP activity,up-regulating the gene/protein expression of caspase-3.
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Chromatin immunoprecipitation
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genomic DNA
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Aim To study the effect of proteasome inhibitor MG132 on the expression of caspase-3 and apoptosis in cultured human umbilical vein endothelial cells.Methods Human umbilical vein endothelial cells was treated with MG132 (2,5 μmol·L~(-1)) for 24 h. The apoptotic cells were determined by DNA fragment analysis and flow cytometric analysis. The level of caspase-3 mRNA was quantified by reverse transcription-polymerase chain reaction (RT-PCR). The protein contents of caspase-3 were analyzed by immunocytochemistry.Results The results showed that the increase of the degree of human umbilical vein endothelial cells apoptosis was concentration dependent. MG132 could up-regulate the gene/protein expression of caspase-3.Conclusions The results implicated that proteasome inhibitor MG132 induced human umbilical vein endothelial cells apoptosis by accumulation of caspase-3.
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