摘要目的 探讨肿瘤转移抑制基因1(TMSG-1)的基因转录调控机制.方法 PCR扩增TMSG-1转录起始位点上游-271 bP与下游+303 bp之间不同长度片段,连入荧光素酶报告基因质粒pGL3-basic,各重组质粒转染后对表达的荧光素酶活性进行检测;对重组质粒pGL3-237插入DNA序列的潜在转录因子结合位点进行定点突变,然后检测其荧光素酶活性的变化;运用凝胶迁移阻滞和染色质免疫共沉淀验证转录因子KLF6和Sp1与TMSG-1DNA调控区的相互作用;通过免疫共沉淀实验分析转录因子KLF6和Sp1之间的相互作用;对人前列腺癌PC-3M不同转移潜能亚系PC-3M-1E8、PC-3M-2B4,人肺巨细胞癌PG不同转移潜能亚系PG-BE1、PG-LH7通过荧光定量PCR分析TMSG-1和野生型KLF6 mRNA水平;将KLF6真核表达质粒转染PC-3M-2B4后检测TMSG-1蛋白水平及细胞侵袭能力的变化.结果 通过荧光素酶报告基因实验鉴定出TMSG-1基因第1号外显子内存在明显促进基因转录的调控区域+59～+123 bp.对该区域序列定点突变后重组荧光素酶报告基因表达的荧光素酶活性下降,而共转染未突变的荧光素酶报告基因质粒和KLF6或Sp1的真核表达质粒则荧光素酶活性上升,差异具有统计学意义(P＜0.01).凝胶迁移阻滞和染色质免疫共沉淀结果显示,转录因子KLF6和Sp1与该区域特定序列存在相互作用.免疫共沉淀结果验证转录因子KLF6与Sp1之间存在相互作用.荧光定量PCR结果显示前列腺癌细胞低转移亚系PC-3M-2B4中TMSG-1和野生型KLF6 mRNA水平均高于高转移亚系PC-3M-1E8(P＜0.05,P＜0.01),同样肺巨细胞癌低转移亚系PG-LH7中TMSG-1和野生型KLF6 mRNA水平均高于高转移亚系PG-BE1(均P＜0.01).PC-3M-2B4细胞转染KLF6真核表达质粒后检测到TMSG-1蛋白的增加和细胞侵袭能力的下降.结论 前列腺癌细胞中转录因子KLF6与Sp1共同作用促进肿瘤转移抑制相关基因TMSG-1的转录激活;不同转移潜能前列腺癌和肺巨细胞癌细胞亚系之间TMSG-1的差异表达与其野生型KLF6差异表达相关.

abstractsObjective To investigate the regulatory mechanism of the transcription of tumor metastasis suppressor gene TMSG-1. Methods Luciferase reporter assay and site-directed mutagenesis were used to analyze the regulatory region of TMSG-1. Electrophoretic mobility shift assay(EMSA) and chromatin immunoprecipitation(CHIP) were carried out to verify the interaction of KLF6 and Sp1 with the regulatory region of TMSG-1. Co-immunoprecipitation(ColP) was performed to analyze the interaction between KLF6 and Spl. TMSG-1 and wt-KLF6 mRNA expressions in cells with different metastatic capacities were quantitated by real-time PCR. Cell invasive capability was determined by Matrigel invasion assay. Results A 63 bp inducible regulatory region ( +59 bp - + 123 bp) in exon 1 was identified by luciferase assay using reporter plasmids with a series of TMSG-1 regulatory region deletions. Mutations in KLF6/Spl binding sites of this region resulted in a decrease of luciferase activity, while cotransfection with KLF6 or Sp1 expressing plasmids led to a remarkable increase of luciferase activity. EMSA and ChIP demonstrated that KLF6 as well as Spl interacted with this region. CoIP also indicated a possible interaction between KLF6 and Spl proteins. In the highly metastatic cell sublines, a low level of wild type KLF6 was associated synchronously with a low TMSG-1 level. Prostate carcinoma cells overexpressing KLF6 exhibited a higher TMSG-1 level and a lower invasive capability. Conclusions Transcription factor complex of KLF6 and Spl may participate in the inducible transcriptional regulation of TMSG-1, and a decreased wild type KLF6 expression is likely associated with a low TMSG-1 level in the highly metastatic cell sublines.