AP-2α抑制胃癌细胞BGC-823增殖并诱导凋亡的作用机制研究

Study on the mechanism of AP-2α involved in the proliferation inhibition and apoptosis induction of human gastric cancer cell line BGC-823

目的研究核转录因子AP-2α抑制胃癌细胞株BGC-823增殖活性及诱导凋亡的作用和机制。方法收集临床胃癌及其癌旁组织标本30对,免疫印迹及荧光定量法检测AP-2α及Krüppel样因子4(Krüppel-like factor 4,KLF4)基因mRNA含量;生物软件预测AP-2α蛋白与KLF4基因启动子上的转录因子结合位点并进行验证。分别转染pc DH1-AP-2α及pshRNAKLF4到BGC-823细胞,转染后48 h,确定细胞转染效率,免疫印迹法检测胞内AP-2α、KLF4、P53及BCL-2蛋白表达;噻唑蓝(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-Htetrazolium bromide,MTT)检测基因干预后各组细胞增殖活性;流式法检测各组细胞凋亡情况。结果所有检测的30对组织标本中,肿瘤中AP-2α表达和KLF4基因mRNA含量均明显低于癌旁组织,差异有统计学意义(P <0. 01,vs癌旁);生物软件预测显示,AP-2α在KLF4基因启动子上存在9个碱基"5'-GCCGCCGGC-3'"的理论结合位点,位点位于转录起始位点前52碱基处,荧光素酶活性检测证实AP-2α可以结合于预测位点并对下游基因的转录进行正调控;细胞转染后48 h,GFP计数显示,细胞瞬时转染效率约为85%,免疫印迹检测数据表明,AP-2α过表达导致KLF4和P53蛋白表达上调,BCL-2蛋白表达下调,而KLF4基因沉默则能够明显减弱AP-2α对KLF4或者P53蛋白表达的影响;细胞转染后24～72 h,pc DH1-AP-2α转染能够明显抑制BGC-823细胞对数期增殖活性,pshRNA-KLF4转染则能够明显减弱AP-2α基因过表达对细胞增殖活性的抑制作用; pc DH1-AP-2α转染细胞后48 h,能够明显导致BGC-823细胞产生凋亡,pshRNA-KLF4转染则能够明显减弱AP-2α过表达产生的诱导细胞凋亡的能力。结论核转录因子AP-2α通过KLF4基因的转录调控对BGC-823细胞的增殖和凋亡产生影响。

Objective To study the mechanism of transcription factor AP-2α involved in the proliferation inhibition and apop- tosis induction of human gastric cancer cell line BGC-823. Methods Thir）y pairs of clinical gastric cancer and adjacent carcinoma tissues were harvested for the study, and the levels of AP-2α and Kuippel-like factor 4（ KLF4 ） mRNA were quantitatively detected by Western blotting and fluorescence method. The theoretical binding sites of AP-2α in KLF4 gene promoter were predicted by bioi＊ffomatits, and validated by luciferase repor） assay, pcDH1-AP-2α and pshRNA-KLF4 were respectively transfected into the BGC-823 cells, and transfection efficiency was detemined 48 hours later. Protein levels of AP-2α, KLF4, P53 and BCL-2 were detected by Western blotting, and the proliferation and apoptosis of BGC-823 ceils in various groups after genetic intervention were assayed by the MTT method or flow cytometry. Results In all the 30 pairs of samples detected, the expression levels of AP-2α and KLF4 mRNA in tumor weresignificantly higher than those in the adjacent tumor tissue （P 〈 0.01 vs adjacent tumor tissue）. Bioinfommtics analysis showed that there was a theoretical binding site of a 9-nucleotide （5＇ -GCCGCCGGC-3＇） for AP-2α in KLF4＇s promoter, and was located at the 52＂d base at the transcription start site. Luciferase activity assay demonstrated that AP-2α could be bound to the promoter of KLF4 gene and positively regulate gene transcription. Foru-eight hours after cell transfection, GFP analysis indicated that instant cell transfection efficiency was about 85%. Data of Western blotting showed that the over-expression of AP-2α could respectively enhance the up-regulation of KLF4 and P53 and down-regulation of BCL-2, while KLF4 gene silencing could significantly weaken the effect of AP-2α on the expression levels of KLF4 or P53. TwenU-four to 72 hours after cell transfection, the transfection of pcDH1-AP-2α could obviously inhibit the proliferation of BGC-823 cell at the logarithmic phase, and the transfection of pshIINA-KLF4 could markedly weaken the effect of AP-2α gene over-expression on the inhibition of cell proliferation activity-. Foru-eight hours after pcDH1-AP-2α cell transfection, ap- optosis of BGC-823 cell was obviously induced, while the transfection of pshRNA-KLF4 could markedly weaken cell apoptosis induced by the over-expression of AP-2α Conclusion Through the regulation of KLF4 gene transfection, AP-2α could exert its effects on the proliferation and apoptosis of BGC-823 cell.