从转录水平研究SATB1表达调控的分子机制

Molecular Mechanism of Regulating Expression of SATB1

目的:研究SATB15’转录调控区及3’UTR的调控作用,以阐明SATB1在各肿瘤细胞系中的表达和调控机制。方法:采用半定量RT-PCR分析人乳腺癌细胞系BT549和MCF7、人肺癌细胞系NCI-H-446、QG56和SPC-A1中SATB1的转录水平,并利用Western Blot方法检测各肿瘤细胞系中蛋白表达水平。分别构建SATB1两个转录本5’上游序列驱动的报告基因载体,将载体瞬时转染QG56及SPC-A1。构建含SATB1基因3’非翻译区(3’UTR)的报告载体,瞬时转染NCI-H-446、QG56及SPC-A1。运用双通道荧光素酶报告系统检测荧光素酶活性。结果:在BT549、NCI-H-446、QG56和SPC-A1中检测到SATB1的转录本2,仅在BT549及QG56有转录本1表达;但在NCI-H-446、QG56及SPC-A1中,未检测到蛋白水平的表达。在QG56中,转录本1上游-638～+404序列段荧光素酶活性最高,而在SPC-A1中转录本2上游-1218～+48序列段的荧光素酶活性最高。运用生物信息学分析-638～+404和-1218～+48两个序列段的转录因子结合位点。在NCI-H-446中,含有SATB13’非翻译区(3’UTR)报告载体的荧光素酶活性显著低于PGL3control的活性(P<0.05)。结论:在肺癌细胞中,SATB1的表达与细胞转移能力的高低无关。RT-PCR、荧光素酶活性及生物信息学分析结果的一致表明SATB1的两个转录本分别受其5’上游序列调控。在NCI-H-446中,SATB1的表达受其3’UTR的调控。

Objective：To investigate the expression and regulation mechanism of SATB1 in various cancer cells through assaying 5＇ upstream sequence and 3＇untranslated region of SATB1.Methods：mRNA of SATB1 is assayed by semi-quantitative PCR in six cell lines including BT549,MCF7,NCI-H-446,SPC-A1,QG56,HL60.Western blot is used for detecting SATB1.Reporter vectors composed of truncated 5＇upstream sequence of SATB1 were constructed.Then these vectors were transected into QG56 and SPC-A1 cells.In addition,3＇UTR of SATB1 was inserted into PGL3 control.PGL3-control-3＇UTR was transfected into three lung cancer cell lines.Dual-luciferase reporter assay is used to detect the activity of luciferase.Bioinformatics analysis is used to predict transcription factors binding-site of two sequence segment including-638～＋404 and-1218～＋48.Resaults：T1 exists in BT549,NCI-H-446,SPC-A1,and QG56;T2 exists in BT549 and QG56.For QG56,PGL3-T1-638～＋404 has highest luciferase activity,while for SPC-A1,transfected PGL3-T2-1218～＋48 has highest luciferase activity.In NCI-H-446,luciferase activity of PGL3-control-3＇UTR is markedly lower than that of PGL3-control（P〈0.05）.Conclusions：Metastasis of lung cancer isn＇t related with SATB1.RT-PCR,luciferase activity and bioinformatics analysis show that two transcription of SATB1 are regulated by respective 5＇upstream sequence.In NCI-H-446,expression of SATB1 is inhibited by 3＇UTR.