BCL2 is a key regulator of apoptosis.Our previous work has demonstrated that special AT-rich sequence-binding protein 1 （SATB1） is positively correlated with BCL2 expression.In the present study,we report a new SATB...BCL2 is a key regulator of apoptosis.Our previous work has demonstrated that special AT-rich sequence-binding protein 1 （SATB1） is positively correlated with BCL2 expression.In the present study,we report a new SATB1 binding site located between P1 and P2 promoters of the BCL2 gene.The candidate SATB1 binding sequence predicted by bioinformatic analysis was investigated in vitro and in vivo by electrophoretic gel mobility shift assays （EMSA） and chromatin immunoprecipitation （ChIP）.One 25-bp sequence,named SB1,was confirmed to be SATB1 binding site.The regulatory function of SB1 and its relevance to SATB1 were further examed with dual-luciferase reporter assay system in Jurkat cells.We found that SB1 could negatively regulate reporter gene activity.Mutation of SATB1 binding site further repressed the activity.Knockdown of SATB1 also enhanced this negative effect of SB1.Our data indicate that the SB1 sequence possesses negative transcriptional regulatory function and this function can be antagonized by SATB1.展开更多
目的体外培养HepG2细胞观察川西獐牙菜醇提物(Swertia mussitii Franch)对多药耐药相关蛋白3(multidrug resistance protein 3,MRP3)、核转录因子SP1(specificity protein 1 transcription factor,SP1)及核受体孕烷X受体(pregnane X rec...目的体外培养HepG2细胞观察川西獐牙菜醇提物(Swertia mussitii Franch)对多药耐药相关蛋白3(multidrug resistance protein 3,MRP3)、核转录因子SP1(specificity protein 1 transcription factor,SP1)及核受体孕烷X受体(pregnane X receptor,PXR)、CYP7A启动子结合因子(CYP7A promoter-binding factor,CPF)表达的影响。方法用MTT比色法检测川西獐牙菜醇提物的最佳作用浓度,再分别于0、12、24、48、72h刺激HepG2细胞,抽提细胞的RNA、总蛋白和核蛋白,采用荧光定量PCR和蛋白免疫印迹技术检测膜转运蛋白MRP3、转录因子SP1及核受体PXR、CPF在转录与蛋白水平的表达变化。每个时间点设立阴性对照DMSO组和阳性对照熊去氧胆酸(ursodeoxycholic acid,UDCA)组。结果川西獐牙菜醇提物可显著诱导HepG2细胞膜转运蛋白MRP3的mRNA和蛋白水平的高表达,其作用强于UDCA(P<0.05)。川西獐牙菜醇提物也可明显上调核转录因子SP1及核受体PXR的mRNA和蛋白表达水平,作用强于UDCA。对CPF表达的上调作用与UDCA相当(P<0.05)。结论川西獐牙菜醇提物可刺激HepG2细胞膜转运蛋白MRP3上调,且有可能是通过核转录因子SP1及核受体PXR、CPF上调其表达。展开更多
基金supported by grants from the National Natural Science Foundation of China (No. 30772490)and Special Major National Natural Science Foundation of China (No. 90919051)
文摘BCL2 is a key regulator of apoptosis.Our previous work has demonstrated that special AT-rich sequence-binding protein 1 （SATB1） is positively correlated with BCL2 expression.In the present study,we report a new SATB1 binding site located between P1 and P2 promoters of the BCL2 gene.The candidate SATB1 binding sequence predicted by bioinformatic analysis was investigated in vitro and in vivo by electrophoretic gel mobility shift assays （EMSA） and chromatin immunoprecipitation （ChIP）.One 25-bp sequence,named SB1,was confirmed to be SATB1 binding site.The regulatory function of SB1 and its relevance to SATB1 were further examed with dual-luciferase reporter assay system in Jurkat cells.We found that SB1 could negatively regulate reporter gene activity.Mutation of SATB1 binding site further repressed the activity.Knockdown of SATB1 also enhanced this negative effect of SB1.Our data indicate that the SB1 sequence possesses negative transcriptional regulatory function and this function can be antagonized by SATB1.
文摘目的体外培养HepG2细胞观察川西獐牙菜醇提物(Swertia mussitii Franch)对多药耐药相关蛋白3(multidrug resistance protein 3,MRP3)、核转录因子SP1(specificity protein 1 transcription factor,SP1)及核受体孕烷X受体(pregnane X receptor,PXR)、CYP7A启动子结合因子(CYP7A promoter-binding factor,CPF)表达的影响。方法用MTT比色法检测川西獐牙菜醇提物的最佳作用浓度,再分别于0、12、24、48、72h刺激HepG2细胞,抽提细胞的RNA、总蛋白和核蛋白,采用荧光定量PCR和蛋白免疫印迹技术检测膜转运蛋白MRP3、转录因子SP1及核受体PXR、CPF在转录与蛋白水平的表达变化。每个时间点设立阴性对照DMSO组和阳性对照熊去氧胆酸(ursodeoxycholic acid,UDCA)组。结果川西獐牙菜醇提物可显著诱导HepG2细胞膜转运蛋白MRP3的mRNA和蛋白水平的高表达,其作用强于UDCA(P<0.05)。川西獐牙菜醇提物也可明显上调核转录因子SP1及核受体PXR的mRNA和蛋白表达水平,作用强于UDCA。对CPF表达的上调作用与UDCA相当(P<0.05)。结论川西獐牙菜醇提物可刺激HepG2细胞膜转运蛋白MRP3上调,且有可能是通过核转录因子SP1及核受体PXR、CPF上调其表达。
