Abstract
Purpose
Hepatocyte nuclear factor 6 (HNF6) is a liver-enriched transcription factor and highly expressed in mature bile duct epithelial cells. This study sought to investigate the role of HNF6, particularly the molecular mechanisms for how HNF6 is involved in the growth and metastasis of cholangiocarcinoma (CCA) cells.
Methods
The expression of HNF6, miR-122 and key molecules was examined by Western blot analysis and real-time RT-PCR. Stable transfectants, HCCC-HNFlow and RBE-HNFhigh, were generated from human CCA HCCC-9810 and RBE cells, respectively. The regulatory effect of HNF6 on miR-122 was evaluated by luciferase reporter assay. Cell proliferation, cycle distribution, migration and invasion were analyzed. The xenograft model was used to assess the effects of HNF6 overexpression on tumorigenesis, growth, metastasis and therapeutic potentials.
Results
Human CCA tissues and cells expressed lower levels of HNF6, which positively correlated with miR-122. HNF6 regulated the expression of miR-122 by stimulating its promoter. HNF6 overexpression inhibited cell proliferation by inducing cell cycle arrest at G1 phase through regulating miR-122, cyclin G1 and insulin-like growth factor-1 receptor. HNF6 inhibited the migration and invasion of CCA cells by regulating matrix metalloproteinase-2 and metalloproteinase-9, reversion-inducing-cysteine-rich protein with kazal motifs, E-cadherin and N-cadherin. Co-transfection of anti-miR-122 abrogated the effects of HNF6. HNF6 overexpression inhibited the ability of cells to form tumors and to metastasize to the lungs of mice, and the growth of established tumors.
Conclusions
The results indicate that HNF6 may serve as a tumor suppressor by regulating miR-122, and its overexpression may represent a mechanism-based therapy for CCA.
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Acknowledgments
This study was supported by grants from the National Natural Scientific Foundation of China (81272467 and 81472321), Shandong Provincial Natural Scientific Research Foundation (BS2015YY024), Shandong Provincial Scientific and Technology Development Program (2014GGH218039) and Heilongjiang Provincial Scientific Fund for Youths (QC2011C089). Huaqiang Zhu and Yuetang Mi contributed equally to this work.
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The study has been approved by the ethic committees of Provincial Hospital Affiliated to Shandong University and Liaocheng People’s Hospital in China. Preoperative informed consent was obtained from each patient registered in this study in accordance with institutional guidance. The pathological samples were taken from the surgical resection specimens, which would not result in any disadvantages to health and prognosis of patients. We are committed to maintain the privacy of patients’ information. The animal study has been approved (permit SYXK20020009) by the Animal Ethics Committee of Harbin Medical University, in compliance with the Experimental Animal Regulations by the National Science and Technology Commission, China.
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Zhu, H., Mi, Y., Jiang, X. et al. Hepatocyte nuclear factor 6 inhibits the growth and metastasis of cholangiocarcinoma cells by regulating miR-122. J Cancer Res Clin Oncol 142, 969–980 (2016). https://doi.org/10.1007/s00432-016-2121-8
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DOI: https://doi.org/10.1007/s00432-016-2121-8