利用寡核苷酸微阵列芯片分析ERRα过度表达对子宫内膜癌HEC-1A细胞转录因子活性的影响

Analysis on the effect of over-expression of estrogen receptor-related receptor-α on the activities of transcriptional factors in endometrial cancer Hec-1A cells by oligonucleotide microarray

目的:讨论过度表达雌激素受体相关受体(ERRα)对子宫内膜癌细胞HEC-1A中不同转录因子转录活性的影响。方法:构建带有绿色荧光蛋白和G418耐药基因筛选标记的真核表达质粒pEGFP-N1-3FLAG-ERRα,将质粒转染子宫内膜癌细胞株HEC-1A,荧光显微镜下检测并通过G418高浓度筛选、低浓度维持建立ERRα稳定高表达的子宫内膜癌细胞株HEC-1A/ERRα。分别抽提子宫内膜癌细胞HEC-1A、转染质粒空载体的HEC-1A/空载体细胞,ERRα过度表达的HEC-1A/ERRα细胞的核蛋白。将核蛋白与CY3、CY5双色标记的326检测信号通道的转录因子芯片(寡核苷酸微阵列芯片)杂交并通过双色共聚焦激光扫描仪分析。结果:转染ERRα真核表达质粒后,与HEC-1A细胞和转染载体的HEC-1A/空载体细胞对比,子宫内膜癌细胞HEC-1A/ERRα中ERRα的mRNA和蛋白表达明显增加。HEC-1A-ERRα细胞株与HEC-1A/空载体细胞株转录因子活性比较显示7个转录位点的活性发生显著改变,其中调控SREBP,AP-1,c-Myc,Usf-1/2转录因子的结合位点活性下调,而调控RFX-1,PPAR-α,PPAR-γ的转录因子的结合位点活性上调。而在HEC-1A/空载体细胞株和HEC-1A细胞株中未检出上述7种转录因子的差异表达。结论:①寡核苷酸微阵列芯片是一种有效的筛选转录因子的工具;②ERRα过度表达下调子宫内膜癌细胞HEC-1A中转录因子SREBP,AP-1,c-Myc,Usf-1/2的转录活性,上调RFX,PPARα,PPARγ的转录活性。

Objective： To explore the effect of over - expression of estrogen receptor - related receptor - α （ERRoL） on the activi- ties of different transcriptional factors in endometrial cancer Hec - 1A cells by oligonucleotide microarray. Methods： The eukaryotic expres- sion plasmid pEGFP - N1 - 3FLAG - ERRα with green fluorescent protein and G418 drug resistance gene selection marker was constructed, then the plasmid was transfected into endometrial cancer Hec -IA cell strain, the expression was detected under fluorescence microscope, endometrial cancer Hec - ! A/ERRct cell strain with stable and high expression of ERRα was established by screening with high concentration G418 and maintenance with low concentration G418. The nucleoproteins were abstracted from endometrial cancer Hec - 1A cells, Hec - 1A/ empty vector cells transfected by empty vector, and Hec - 1 A/ERRc~ ceils with over - expression of ERRcL, respectively. The nucleoproteins were hybridized with transcription factor chips （oligonucleotide microarray） in 326 detection signal channel labeled by CY3 and CY5, then they were analyzed by double -color confocal laser scanner. Results： After transfected by ERRor eukaryotic expression plasmid, compared with Hec - 1A cells and Hec - 1A/empty vector ceils transfected by empty vector, the expressions of ERRor mRNA and protein in endometri- al cancer Hec - 1A/ERRor cells increased significantly. Compared with the activities of transcription factors in Hec - 1A/empty vector cell strain, the activities of seven transcription factors in HEC - 1A - ERRor cell strain changed significantly, the activities of binding sites regu- lating SREBP, AP - 1, c - Myc, and Usf - I/2 transcription factors were down - regulated, while the activities of binding sites regulating RFX - 1, PPAR - c~, and PPAR - ~/transcription factors were up - regulated. The differential expressions of above - mentioned seven tran- scription factors were not detected in Hec - 1 A/empty vector cell strain and Hec - 1A cell strain. Conclusion： Oligonucleotide microarray is an effective method to screen transcription factors; over - expression of ERRα down - regulates the activities of binding sites regulating SREBP, AP - 1, c - Myc, and Usf - 1/2 transcription factors, and it up - regulates the activities of binding sites regulating RFX - 1, PPAR - ct, and PPAR - ~/transcription factors in endometrial cancer Hec - 1A cells.