论文部分内容阅读
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 study the regulation of SATB15 ’transcriptional regulatory region and 3’UTR to elucidate the expression and regulation of SATB1 in various tumor cell lines. Methods: The transcription levels of SATB1 in human breast cancer cell lines BT549 and MCF7, human lung cancer cell lines NCI-H-446, QG56 and SPC-A1 were analyzed by semi-quantitative RT-PCR. Western Blot was used to detect the expression of SATB1 in various tumor cell lines Protein expression levels. The reporter gene vectors driven by the 5 ’upstream sequence of two transcripts of SATB1 were constructed, and the vectors were transiently transfected into QG56 and SPC-A1. A reporter vector containing 3 ’untranslated region (3’UTR) of SATB1 gene was constructed and transiently transfected into NCI-H-446, QG56 and SPC-A1. Luciferase activity was measured using a dual channel luciferase reporter system. Results: Transcript 2 of SATB1 was detected in BT549, NCI-H-446, QG56 and SPC-A1 and transcript 1 was only expressed in BT549 and QG56; however, in NCI-H-446, QG56 and SPC-A1 , No protein level expression was detected. In QG56, the luciferase activity was the highest at-638 ~ + 404 in transcript 1 upstream and the highest in luciferase at -1218 ~ +48 in transcript 2 upstream of transcript 2 in SPC-A1. Bioinformatics analysis was used to analyze the binding sites of transcription factors from -638 to +404 and -1218 to +48. In NCI-H-446, the luciferase activity of the vector containing SATB13 ’untranslated region (3’UTR) was significantly lower than that of PGL3control (P <0.05). Conclusion: The expression of SATB1 in lung cancer cells has nothing to do with the level of cell metastasis. The agreement of RT-PCR, luciferase activity and bioinformatics analysis indicated that both transcripts of SATB1 were regulated by their 5 ’upstream sequences, respectively. In NCI-H-446, SATB1 expression is regulated by its 3’UTR.