Xu Jiaying, Zhou Junwei, Li Man-Shan, Ng Chor-Fung, Ng Yuen-Keng, Lai Paul Bo-San, Tsui Stephen Kwok-Wing
School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
PLoS One. 2014 Aug 14;9(8):e99359. doi: 10.1371/journal.pone.0099359. eCollection 2014.
Four and a Half LIM protein 2 (FHL2) is a LIM domain only protein that is able to form various protein complexes and regulate gene transcription. Recent findings showed that FHL2 is a potential tumor suppressor gene that was down-regulated in hepatocellular carcinoma (HCC). Moreover, FHL2 can bind to and activate the TP53 promoter in hepatic cells. In this study, the activity of the two promoters of FHL2, 1a and 1b, were determined in the human embryonic kidney cell line HEK293 and the activation of these two promoters by p53 was investigated. Our results showed that the 1b promoter has a higher activity than the 1a promoter in HEK 293 cells but the 1a promoter is more responsive to the activation by p53 when compared with the 1b promoter. The regulation of FHL2 by p53 was further confirmed in liver cells by the overexpression of p53 in Hep3B cells and the knockdown of p53 in HepG2 cells. Combining promoter activity results of truncated mutants and predictions by bioinformatics tools, a putative p53 binding site was found in the exon 1a of FHL2 from +213 to +232. The binding between the p53 protein and the putative p53 binding site was then validated by the ChIP assay. Furthermore, the expression of FHL2 and TP53 were down-regulated in majority of HCC tumour samples (n = 41) and significantly correlated (P = 0.026). Finally, we found that the somatic mutation 747 (G→T), a hot spot mutation of the TP53 gene, is potentially associated with a higher expression of FHL2 in HCC tumour samples. Taken together, this is the first in-depth study about the transcriptional regulation of FHL2 by p53.
四半LIM蛋白2（FHL2）是一种仅含LIM结构域的蛋白，能够形成多种蛋白复合物并调节基因转录。最近的研究结果表明，FHL2是一种潜在的肿瘤抑制基因，在肝细胞癌（HCC）中表达下调。此外，FHL2可在肝细胞中结合并激活TP53启动子。在本研究中，我们测定了FHL2的两个启动子1a和1b在人胚肾细胞系HEK293中的活性，并研究了p53对这两个启动子的激活作用。我们的结果显示，在HEK 293细胞中，1b启动子的活性高于1a启动子，但与1b启动子相比，1a启动子对p53激活的反应更敏感。通过在Hep3B细胞中过表达p53以及在HepG2细胞中敲低p53，进一步证实了p53对肝细胞中FHL2的调控作用。结合截短突变体的启动子活性结果和生物信息学工具的预测，在FHL2的外显子1a中从+213至+232发现了一个假定的p53结合位点。然后通过染色质免疫沉淀（ChIP）试验验证了p53蛋白与假定的p53结合位点之间的结合。此外，在大多数HCC肿瘤样本（n = 41）中，FHL2和TP53的表达下调，且两者显著相关（P = 0.026）。最后，我们发现TP53基因的热点突变体747（G→T）体细胞突变可能与HCC肿瘤样本中FHL2的高表达相关。综上所述，这是关于p53对FHL2转录调控的首次深入研究。
