[Effect of a recombinant lentiviral vector carrying hepatocyte nuclear factor 6 gene on migration and invasion abilities of SW620 cells].

OBJECTIVE: To construct a recombinant lentiviral vector that stably express hepatocyte nuclear factor 6 (HNF6) in colorectal cancer cell line and examine its effects on the invasive ability of SW620 cells. METHODS: The lentiviral vector pLeno-DCE-HA-HNF6 was constructed and transfected into 293T cells. The supernatant containing the lentivirus particles was harvested to determine the virus titer. A stable cell line was established by infecting SW620 cells with the lentivirus particles, and the transfection efficiency was examined by fluorescence microscopy and flow cytometry. The invasion ability of the transfected SW620-HNF6 cells was assessed by wound healing and transwell assays. RESULTS: The recombinant lentiviral vector was correctly constructed and verified by sequencing. SW620-HNF6 cell line with stable HNF6 expression was established successfully, and the transfection efficiency reached 82.3%. Western blotting and quantitative PCR demonstrated significantly upregulated HNF6 expression in SW620-HNF6 cells, which showed obviously suppressed invasive ability in wound healing and transwell assays. CONCLUSION: We have successfully established a colorectal cancer cell line SW620-HNF6 stably expressing HNF6, which shows a lowered migration activity in vitro.

Dynamic expression of the Onecut transcription factors HNF-6, OC-2 and OC-3 during spinal motor neuron development.

The Onecut (OC) transcription factors, namely Hepatocyte nuclear factor 6 (HNF-6), OC-2 and OC-3, are transcriptional activators expressed in liver, pancreas and nervous system during development. Although their expression and roles in endoderm-derived tissues and in the trigeminal ganglia have been investigated, their expression in the CNS has not been described yet. In this study, we report a qualitative and quantitative expression profile of the OC factors in the developing spinal motor neurons (MN). We provide evidence that OC expression is initiated in newly-born MN. At later stages, they are differentially and dynamically expressed in subsets of differentiating motor neuron within the four motor columns. We also show that the expression profile of HNF-6 in spinal MN is conserved in chick embryos. Together, our data unveil a complex and dynamic expression profile of the OC proteins in spinal MN, which suggests that these factors may participate in regulatory networks that control different steps of motor neuron development.

Cloning and characterization of liver progenitor cells from the scattered cell clusters in primary culture of porcine livers.

The scattered cell clusters that can differentiate into hepatocytes or biliary epithelial cells have been isolated from primary cultures of adult porcine livers. We have generated 11 clonal cell lines from this system and identified liver progenitor cells (LPCs) among the clonal lines. These clonal lines expressed c-kit, HNF-1, HNF-6, and/or CK19 mRNA. An immunocytochemical study of the clonal lines indicated that clonal line CL-11 expressed liver epithelial cell markers CK14, vimentin, CK18, and BD-1. The expression of albumin and alpha1-antitrypsin (alpha1-AT) mRNA was only upregulated in CL-11 among the clonal lines when they were grown as aggregates. Under these conditions, CL-11 also exhibited ammonia metabolic activity and several indicators that suggest hepatocytic differentiation, including the upregulation of liver-specific genes such as dipeptidyl peptidase IV, CYP1A1, and CYP3A4 mRNA, and the downregulation of biliary cell markers such as gamma-glutamyltrans-peptidase (GGT), CK19, and HNF6 mRNA. After culturing CL-11 in Matrigel, the expression of GGT and HNF6 mRNA was upregulated. These results indicate that CL-11 has dual potential: the ability to differentiate as hepatocytes or as bile duct cells. The isolation of scattered cells could provide a simple method to generate LPC lines from adult livers.

Sustained expression of hepatocyte nuclear factor-6 leads to loss of pancreatic beta-cells by apoptosis.

Hepatocyte nuclear factor-6 (HNF-6) is the ONECUT-homeodomain transcription factor that is enriched in liver and also present in pancreas and central nervous system. It is expressed in the pancreatic bud at E10.5. In adult pancreas, its expression is restricted to the exocrine pancreas and duct cells. Since duct cells are thought to be precursors of endocrine cells and HNF-6 is involved in the regulation of the expression of HNF-4alpha and -1beta, genes that cause maturity onset diabetes of the young (MODY), we hypothesized that the sustained expression of HNF-6 would affect beta-cell function. We generated transgenic mice over-expressing human HNF-6 using the mouse insulin I promoter (MIP). We obtained one female founder in which the transgene had been incorporated into two sites; the chromosome (Ch) 14 and the X chromosome. The integration site of the latter was within centromeric heterochromatin and the transgene was inactivated. Studies on mice in which the transgene was integrated into Ch14 showed beta-cell specific defects functionally and pathologically. The insulin secretory response to glucose and arginine in the in situ-perfused pancreas was also significantly impaired in these mice. Immunohistochemical analysis revealed that the islets were smaller and had an abnormal architecture with an inverted ratio of alpha- and beta-cells resulting from beta-cell loss to 30% by 6-wk of age. The decreased number of beta-cells was quantified first time by fluorescent activated cell sorting using entire pancreata from the transgenic mice crossed with MIP-green fluorescent protein (GFP) mice. This severe loss of beta-cells involved programmed cell death.

[Expression of HNF4alpha and HNF6 mRNA during the process of mouse liver development].

OBJECTIVE: To investigate the function of HNF4a and HNF6 during liver development. METHODS: The expression levels of HNF4alpha and HNF6 at E8, 9, 13, 15, 17, P1 and in adult mouse liver were detected by RT-PCR and in situ hybridization. RESULTS: RT-PCR results showed that HNF4alpha first expressed at E9, the time of liver bud formation, and lasted through all gestation and existed in adult liver. In situ hybridization showed that the expression of HNF4alpha was detected at the cells of liver cords during various stages of mouse liver development, and there were still a few HNF4alpha positive hepatocytes in adult liver. The cells of bile duct plate and biliary epithelial cells, endothelial cells, hematopoietic cells of liver were negative for HNF4alpha. The expression of HNF6 mRNA was detected in the liver at E9, the time of liver formation onset. Then, HNF6 mRNA disappeared transiently at E13, but it appeared again at E15. Its expression lasted until adult. In situ hybridization studies showed that most liver cord cells were positive for HNF6 at E9 and E15. At E17 and P1, the expression levels of liver cord cells declined, and HNF6 strongly expressed in the cells of bile duct plate and biliary epithelial cells. CONCLUSION: HNF4alpha could modulate the formation of liver bud, trigger the differentiation of hepatic stem cell towards hepatocytes, and keep the shape of hepatocytes. HNF6 might play a role at the onset of liver development, in the differentiation of hepatic stem cell towards biliary epithelial cells, and in maintaining the morphological characteristic of biliary epithelial cells.