Wound healing assay was performed to evaluate the effect of the overexpression and knockdown of NELF-B on cell migration. malignancy HepG2 and SNU449 cell lines. Functional assays were performed to examine the effects on gene and protein expression, viability, migration and invasion of cells. Overexpression of NELF-B did not alter the proliferation and migration of HepG2 cells, or the expression of tested genes, indicating that overexpression alone may not be sufficient for altering these features in HepG2 cells. By contrast, knockdown of NELF-B in SNU449 cells resulted in decreased cell proliferation, together with induction of apoptosis and decreased expression levels of Ki-67 and survivin, which are markers of proliferation and inhibition of apoptosis, respectively. Additionally, silencing of NELF-B resulted in a significant decrease in the hallmarks of epithelial-mesenchymal transition (EMT), including cell migration and invasion, and decreased the expression levels of EMT markers, such as N-cadherin, vimentin and -catenin. Decreased expression levels of forkhead box F2 transcription factor and increased mRNA levels of trefoil factor 1, a putative N-Desethyl amodiaquine tumor suppressor, were also detected following the silencing of NELF-B. The current N-Desethyl amodiaquine results exhibited that NELF-B enhanced the manifestation of most hallmarks of cancer, including cell proliferation, migration, invasion and inhibition of apoptosis, indicating its crucial role in the progression of HCC. experiments (20C22) and was further confirmed by the low levels observed in breast carcinoma tissues (17). Conversely, NELF-B was found to act as an oncogene in upper gastrointestinal adenocarcinoma, as well as prostate and liver malignancy (23C25). The diverse effects of NELF-B among different types of cancer are suggestive of the tissue/context-dependent functions of NELF-B. We have previously exhibited the upregulation of NELF-B in HCC tissue samples compared with its expression in adjacent non-cancerous liver tissues, which is consistent with the analysis of the Oncomine HCC microarray database (25). Subsequent experiments have exhibited the role of NELF-B in cell proliferation and migration through loss-of-function analyses in HepG2 cells (25), which represents an early stage of liver malignancy. In continuation of our previous work, and to gain further insights into the mechanism underlying the role of NELF-B, the present study involved a gain-of-function analysis in HepG2 cells. To further elucidate the involvement of NELF-B in HCC progression, a loss-of-function analysis was also performed in an intermediate-stage HCC cell line (SNU449) to elucidate the involvement of NELF-B in the progression of HCC. Materials and methods Cell culture HepG2 and SNU449 cells were generously provided by Dr Mehmet Ozturk, Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey. HepG2 cells represent an early stage of liver cancer and are derived from a well-differentiated tumor from a 15-year-old Caucasian male. HepG2 is usually classically described as an HCC cell line, but has also been suggested to have originated from hepatoblastoma (26). SNU449 represents an intermediate stage of HCC and is derived from a grade IICIII/IV HCC from a 52-year-old Asian male, positive for HBV DNA. The cells were cultured in RPMI-1640 basal medium (Lonza Group, Ltd.) supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were incubated in a humidified incubator at 37C with 5% CO2. Images were captured using an inverted fluorescence phase contrast microscope at 100, 200 or 400 magnifications (Olympus IX70; Olympus Corporation); some images were converted to grey-scale and were adjusted for brightness. Plasmid constructs The NELF-B overexpression plasmid, pCMV5-HCOBRA1, was a nice gift from Dr Rong Li, University of Texas Health Science Center, San Antonio TX, USA. An empty pCMV5 plasmid was used as a negative control. Briefly, pCMV5-HCOBRA1 was digested with % is the percentage of wound closure, is the wound area at 0 h, and is the wound area at 24 h. Transwell invasion assay Transwell Boyden 24-well chambers ( em ThinCert /em ?; Greiner Bio-One) were used to determine the effect on the invasive capacity of cells. Cells were harvested 96 h post-transfection, and 2105 cells were suspended in 100 l RPMI-1640 medium (supplemented with 1% FBS) and seeded in the upper chamber of the Transwell N-Desethyl amodiaquine cell culture insert (8-m pore size). The cell culture insert was coated with 40 g collagen I (SERVA Electrophoresis GmbH; cat. no. 47254), allowed to dry overnight in an incubator at 37C, and rehydrated with 1% FBS-containing medium, 30 min before the seeding of cells. The lower chamber was filled with 600 l RPMI-1640 medium supplemented with 10% FBS. Cells were placed inside the upper chamber and incubated for 22 h at 37C. Subsequently, cotton swabs were used to remove cells around the Mouse monoclonal to DPPA2 upper side of the membrane, and the cells that invaded.