Inhibition of the liver enriched protein FOXA2 recovers HNF6 activity in human colon carcinoma and liver hepatoma cells.
Inhibition of the liver enriched protein FOXA2 recovers HNF6 activity in human colon carcinoma and liver hepatoma cells.
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DOI：
10.1371/journal.pone.0013344
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发表时间：
2010-10-13
期刊：
影响因子：
3.7
通讯作者：
Borlak J
中科院分区：
文献类型：
--
作者：
Lehner F;Kulik U;Klempnauer J;Borlak J
最近，我们证明了转录因子HNF 6和FOXA 2在人类结直肠肝转移中起关键调节作用。为了更好地理解他们提出的抑制性串扰，在人结肠Caco-2和HepG 2癌细胞系中研究了FOXA 2功能性敲低对HNF 6和C/EBPα活性的影响。具体而言，siRNA介导的FOXA 2基因沉默抑制了> 80%的转录物表达。这导致HNF 6和该转录因子靶向的基因的mRNA表达在统计学上显著增加6倍、3倍、4倍和8倍，例如，通过qRT-PCR测定HSP 105 B、CYP 51和C/EBPα。因此，FOXA 2的功能性敲除恢复了HNF 6活性。此外，在Caco-2细胞核提取物中未观察到HNF 6 DNA结合，但HNF 1 α、FOXA 2、FOXA 3和HNF 4 α蛋白表达丰富。因此，我们将编码HNF 6的质粒转染到Caco-2细胞中，但也采用逆转录病毒载体将HNF 6转染到HepG 2细胞中。这导致HNF 6蛋白表达与DNA结合活性被恢复，如通过EMSA带移测定所确定的。此外，通过流式细胞术研究了HNF 6表达对转染细胞中细胞周期调控的后果。本质上，HNF 6分别抑制Caco-2和HepG 2细胞系中G2/M和G1期的细胞周期进展。在此，如通过BrdU标记测定所确定的，Caco-2和HepG 2细胞中的增殖分别减少80%和50%。因此，FOXA 2的功能性敲除恢复了HNF 6的活性并抑制了肿瘤细胞的生长，并且可能代表原发性和继发性肝脏恶性肿瘤的新的治疗靶点。
Recently, we demonstrated that the transcription factors HNF6 and FOXA2 function as key regulators in human colorectal liver metastases. To better understand their proposed inhibitory crosstalk, the consequences of functional knockdown of FOXA2 on HNF6 and C/EBPα activity were investigated in the human colon Caco-2 and HepG2 carcinoma cell lines. Specifically, siRNA-mediated gene silencing of FOXA2 repressed transcript expression by >80%. This resulted in a statistically significant 6-, 3-, 4-, and 8-fold increase in mRNA expression of HNF6 and of genes targeted by this transcription factor, e.g., HSP105B, CYP51, and C/EBPα, as determined by qRT-PCR. Thus, functional knockdown of FOXA2 recovered HNF6 activity. Furthermore, with nuclear extracts of Caco-2 cells no HNF6 DNA binding was observed, but expression of HNF1α, FOXA2, FOXA3, and HNF4α protein was abundant. We therefore transfected a plasmid encoding HNF6 into Caco-2 cells but also employed a retroviral vector to transfect HNF6 into HepG2 cells. This resulted in HNF6 protein expression with DNA binding activity being recovered as determined by EMSA band shift assays. Furthermore, by flow cytometry the consequences of HNF6 expression on cell cycle regulation in transfected cells was studied. Essentially, HNF6 inhibited cell cycle progression in the G2/M and G1 phase in Caco-2 and HepG2 cell lines, respectively. Here, proliferation was reduced by 80% and 50% in Caco-2 and HepG2 cells, respectively, as determined by the BrdU labeling assay. Therefore functional knockdown of FOXA2 recovered HNF6 activity and inhibited growth of tumor-cells and may possibly represent a novel therapeutic target in primary and secondary liver malignancies.
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