Yang Tian, Li Hong, Thakur Asmitananda, Chen Tianjun, Xue Jing, Li Dan, Chen Mingwei
Department of Respiratory Medicine, The First Affiliated Hospital, Medical College of Xi'an Jiaotong University, 277 West Yanta Road, Xian, 710061, China.
Tumour Biol. 2015 Sep;36(10):8185-91. doi: 10.1007/s13277-015-3498-8. Epub 2015 May 21.
Family of forkhead box transcription factors, including forkhead box P4 (FOXP4), plays an important role in oncogenesis. The current study is to evaluate the role of FOXP4 in regulating human non-small cell lung cancer (NSCLC). Quantitative RT-PCR and Western blot were performed to evaluate the gene and protein expressions of FOXP4 in six NSCLC cell lines and 55 NSCLC patients. Lentivirus of small hairpin RNA (FOXP4-shRNA) was used to downregulate FOXP4 in NSCLC cell lines A549 and H1703 cells. Its effect on NSCLC growth, invasion, and cell cycle were evaluated by cell proliferation assay, migration assay, and cell cycle assay, respectively. Dual luciferase assay and Western blot were used to examine whether microRNA-138 (miR-138) was an upstream regulator of FOXP4. The dependence of FOXP4 on miR-138 associated signaling pathway was evaluated by ectopically overexpressing enhancer of zeste homolog 2 (EZH2), a known miR-138 target in NSCLC. FOXP4 was highly expressed in both NSCLC cell lines and NSCLC patients. FOXP4 downregulation by FOXP4-shRNA markedly reduced cancer cell growth and invasion, as well as induced cell cycle arrest in A549 and H1703 cells. MiR-138 was confirmed to be an upstream regulator of FOXP4 and directly regulated FOXP4 expression in A549 and H1703 cells. FOXP4 downregulation-mediated inhibition on cancer cell growth and invasion was independent on overexpressing EZH2, another direct target of miR-138 in NSCLC. Our data demonstrated that FOXP4 was a critical regulator in NSCLC and independently associated with miR-138 regulation.
叉头框转录因子家族，包括叉头框P4（FOXP4），在肿瘤发生过程中发挥重要作用。当前研究旨在评估FOXP4在调控人类非小细胞肺癌（NSCLC）中的作用。采用定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测6种NSCLC细胞系及55例NSCLC患者中FOXP4的基因和蛋白表达。利用小发夹RNA慢病毒（FOXP4-shRNA）下调NSCLC细胞系A549和H1703细胞中的FOXP4。分别通过细胞增殖实验、迁移实验和细胞周期实验评估其对NSCLC生长、侵袭及细胞周期的影响。采用双荧光素酶报告基因检测法和蛋白质免疫印迹法检测微小RNA-138（miR-138）是否为FOXP4的上游调节因子。通过异位过表达已知的NSCLC中miR-138靶标——zeste同源物2增强子（EZH2），评估FOXP4对miR-138相关信号通路的依赖性。FOXP4在NSCLC细胞系和NSCLC患者中均高表达。FOXP4-shRNA下调FOXP4可显著降低A549和H1703细胞的癌细胞生长和侵袭能力，并诱导细胞周期停滞。证实miR-138是FOXP4的上游调节因子，并可直接调控A549和H1703细胞中FOXP4的表达。FOXP4下调介导的对癌细胞生长和侵袭的抑制作用不依赖于过表达EZH2，EZH2是NSCLC中miR-138的另一个直接靶标。我们的数据表明，FOXP4是NSCLC中的关键调节因子，且独立于miR-138调控。
