【摘要】 目的 探讨Bmi-1是否可与Smad4和miR-128a形成Smad4-miR-128a-Bmi-1调控轴，并在人肺腺癌细胞株-A549中发挥调节作用，分析其作用机制。方法 通过生物信息学技术、测序、染色质免疫共沉淀实验和Western Blot技术确定筛选的转录因子与miR-128a之间的结合和调控关系，并通过5’Race实验明确miR-128a的转录起始位点；应用肿瘤免疫评估数据库和人类蛋白质图谱等数据库分析Smad4基因在肺腺癌中的表达情况，并在细胞水平验证Smad4、miR-128a和Bmi-1 mRNA和蛋白的表达情况；利用质粒构建过表达载体Smad4、敲减Bmi-1及化学合成miR-128a mimic,瞬时转染到A549细胞中，进一步验证转染效率；过表达Smad4、过表达miR-128a mimic和敲减Bmi-1后，验证Smad4、miR-128a和Bmi-1三者间的调控关系；分别共转染miR-128a mimic+Bmi-1及Smad4+miR-128a mimic,通过qRT-PCR实验反向验证Smad4、miR-128a和Bmi-1三者间的调控关系。结果 经筛选确定与miR-128a可能存在结合和调控关系的候选转录因子为Smad3和Smad4。与对照组相比，在A549细胞中，Smad4和miR-128a表达水平低(P<0.01),而Bmi-1表达水平高(P<0.01)。在A549细胞中分别过表达Smad4、miR-128a和敲减Bmi-1后，可发现Smad4、miR-128a和Bmi-1之间存在调控关系，经共转染反向验证也可得到相同结果。结论 转录因子Smad4可通过调节磷酸化Smad3进而调控miR-128a的表达，且miR-128a的转录起始位点位于其上游278bp的A碱基处；Smad4、miR-128a和Bmi-1可形成调控轴Smad4-miR-128a-Bmi-1,并在人肺腺癌细胞株-A549中发挥作用。更多还原

【Abstract】 Objective To investigate whether Bmi-1 can form a Smad4-miR-128a-Bmi-1 regulatory axis with Smad4 and miR-128a, and to play a regulatory role in human lung adenocarcinoma cell line-A549,and to study its mechanism of action.Methods The binding and regulatory relationships between the screened transcription factors and miR-128a were determined by bioinformatics techniques, sequencing, Chromatin immunoprecipitation and Western Blot, and the transcriptional start site of miR-128a was clarified by 5’Race experiments; Tumor IMmune Estimation Resource and The Human Protein Atlas databases were applied to analyze the expression of Smad4 in lung adenocarcinoma and the expression of Smad4,miR-128a and Bmi-1 mRNA and protein were verified at the cellular level; Overexpression vector Smad4,knockdown of Bmi-1 and chemical synthesis of miR-128a mimic were constructed using plasmids and transiently transfected into A549 cells to further validate the transfection efficiency; After overexpression of Smad4 and miR-128a mimic and knockdown of Bmi-1,the regulatory relationship among Smad4,miR-128a and Bmi-1 was verified; miR-128a mimic+Bmi-1 and Smad4~+miR-128a mimic were co-transfected respectively, and the regulatory relationship among Smad4,miR-128a and Bmi-1 was verified by qRT-PCR in reverse.Results Candidate transcription factors identified by screening for possible binding relationships with miR-128a were Smad3 and Smad4.Compared with the control group, in A549 cells, the expression levels of Smad4 and miR-128a were low(P<0.001),while the expression level of Bmi-1 was high(P<0.01).After overexpression of Smad4,miR-128a and knockdown of Bmi-1 in A549 cells, respectively, a regulatory relationship among Smad4,miR-128a and Bmi-1 could be found and the same results were obtained by reverse transfection of the co-transfected.Conclusion The transcription factor Smad4 can regulate the expression of miR-128a by regulating the phosphorylation of Smad3,and the transcriptional start site of miR-128a is located 278 bp upstream of the A base; Smad4,miR-128a and Bmi-1 can form the regulatory axis Smad4-miR-128a-Bmi-1 and play a role in lung adenocarcinoma A549 cells.更多还原