Role of NELF‑B in supporting epithelial‑mesenchymal transition and cell proliferation during hepatocellular carcinoma progression
- Authors:
- Published online on: September 6, 2021 https://doi.org/10.3892/ol.2021.13022
- Article Number: 761
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Copyright: © Ghouraba et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Liver cancer ranks sixth among the most frequently diagnosed types of cancer and fourth among the leading causes of cancer-associated deaths globally, with ~841,000 newly diagnosed cases and 782,000 deaths reported annually (1). Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer, accounting for 85–90% of all liver cancer cases (2). HCC has a poor prognosis, with an average 5-year survival rate of 19.6%, that can be as low as 2.5% in patients with advanced stages in the United States (3). These low survival rates are attributed to the late diagnosis and limited effectiveness of the current treatment options. Screening of high-risk patients involves the use of non-specific and less-sensitive tools, such as determination of a-fetoprotein serum levels, ultrasound (4,5), expensive imaging techniques, such as computed tomography and magnetic resonance imaging (6,7), and invasive techniques including guided biopsies (8).
Risk factors predisposing individuals to HCC include hepatitis B virus (HBV) infection, hepatitis C virus infection, aflatoxin and alcohol consumption, which mediate the pathogenesis of HCC through different mechanisms (9). Several molecular alterations have been identified in HCC; these include genetic and epigenetic alterations, which make it a complex and heterogeneous disease (10). The current knowledge of molecular biomarkers that would aid the diagnosis, prognosis and therapy monitoring is insufficient. Therefore, investigations on the mechanisms underlying the development of HCC should provide improved diagnostic and prognostic markers, and should promote the development of targeted therapy.
Cofactor of BRCA1 (COBRA1) was first identified through yeast two-hybrid screening as a novel BRCA1-interacting protein (11). It was later found to be the same protein identified as negative elongation factor-B (NELF-B), which is one of the four subunits of the NELF complex (12). The NELF complex interacts with other factors to negatively regulate the elongation step in transcription by pausing RNA polymerase II (RNAPII) (13,14). The NELF complex consists of four subunits, namely NELF-A, NELF-B, NELF-C/D and NELF-E (15). NELF-C is similar to NELF-D in structure; both are believed to be translational variants of the same mRNA transcript, and either of them can be involved in the formation of the complex at a given point (12). The complex core is composed of NELF-B and NELF-C/D, which brings the other two subunits together (12). NELF-A includes an RNAPII-binding domain, and NELF-E has an RNA-binding domain (15,16). Along with the NELF-B subunit, all the subunits are required for the assembly of a functional NELF complex (12). In fact, the interdependent manner in which the NELF subunits are regulated has been identified in several studies, revealing that depletion of one of these subunits results in dampening of the protein levels of the remaining subunits (17).
Lacking a DNA-binding domain, NELF-B interacts with other transcription complexes, such as steroid hormone receptors and activator protein-1, to mediate their regulatory functions on gene expression (18,19). Some of these interactions occur through the NELF complex, whereas the mechanisms of others remain unclear.
The involvement of NELF-B in the essential transcription regulation machinery governing several cellular processes suggests its potential role in a disease like cancer. Nonetheless, the role of NELF-B in cancer has been studied only in a few types of cancer. NELF-B was first studied in breast cancer; its tumor suppressor role was demonstrated through in vitro experiments (20–22) 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 cancer (23–25). The diverse effects of NELF-B among different types of cancer are suggestive of the tissue/context-dependent roles of NELF-B. We have previously demonstrated the upregulation of NELF-B in HCC tissue samples compared with its expression in adjacent non-cancerous liver tissues, which is consistent with the in silico analysis of the Oncomine HCC microarray database (25). Subsequent experiments have demonstrated 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 cancer.
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 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 II–III/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 37°C 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 generous 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 EcoRI and SalI (New England Biolabs, Inc.). The empty vector (4.7 Kb) was purified using the QIAquick Gel Extraction kit (Qiagen Sciences, Inc.), following the manufacturer's protocol. The blunting of the 5-overhangs resulting from the digestion was performed by filling the ends with the Klenow fragment of DNA polymerase I (New England BioLabs, Inc.). For each µg of DNA, 1 unit of Klenow was added, followed by incubation at 25°C for 15 min and heating at 67°C for 20 min. T4 DNA ligase (Promega Corporation) was used to re-ligate the blunted vector. The pEGFP-N1 expression plasmid was used to assess the transfection efficiency. The plasmid was provided by Dr Ahmed Osman, Ain Shams University, Cairo, Egypt, and was initially purchased from Clontech Laboratories, Inc.
NELF-B overexpression
HepG2 cells were transfected using Lipofectamine® 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, HepG2 cells were cultured in 6-well plates and incubated for 24 h until they were 70–90% confluent. Before transfection, 3.75 µl Lipofectamine 3000 was added to 125 µl Opti-MEM reduced serum medium (Gibco; Thermo Fisher Scientific, Inc.) and the mixture was incubated for 5 min at room temperature. Meanwhile, 2.5 µg of either the NELF-B overexpression plasmid pCMV5-HCOBRA1 or pCMV5 empty plasmid was diluted in a mixture of 125 µl Opti-MEM and 5 µl Lipofectamine 3000. The two dilutions were mixed and incubated for 20 min to allow for complex formation. The DNA-Lipofectamine complex (250 µl) was then added dropwise to each well containing cells in 2 ml of antibiotic-free medium (RPMI + 10% FBS). Overexpression efficiency was assessed based on the percentage of green fluorescent cells, as well as the protein and mRNA expression levels of NELF-B following transfection. Transfection efficiency was measured 24 and 48 h post-transfection, and was found to be 45 and 60%, respectively using an inverted fluorescence phase contrast microscope at ×100 magnification (Fig. S1). At 48 h, NELF-B expression in HepG2 cells transfected with the NELF-B overexpression plasmid (pCMV5-HCOBRA1) was increased 4-fold compared with that in cells transfected with pCMV5 empty plasmid (P≤0.0001; Fig. 1A). The cells were collected 48 h post-transfection for downstream analysis.
Gene silencing
siGENOME SMARTPool siRNA (cat. no. M-015839-02; GE Healthcare Dharmacon, Inc.), a pool of four siRNAs targeting different exons of the NELF-B mRNA, was used for the knockdown of NELF-B. Allstars' negative control siRNA (cat. no. SI03650318; Qiagen, Inc.) was used as a control. Approximately 1.5×105 cells suspended in antibiotic-free medium (RPMI + 10% FBS) were mixed with the transfection complex prior to seeding (reverse transfection). The complex was prepared by adding 40 nM siRNA and 3.75 µl Lipofectamine 3000 to 500 µl Opti-MEM medium, followed by incubation for 15–20 min at room temperature. The medium was changed 24 h after transfection, and fresh antibiotic-free medium (RPMI + 10% FBS) was added. The optimum knockdown conditions were determined by performing the transfection using different siRNA concentrations (25 and 40 nM) and post-transfection incubation time points (48, 72 and 96 h) (data not shown). The use of 40 nM siRNA, and an incubation of 96 h after transfection, resulted in a decrease in the protein levels of NELF-B by an average of 96% (P≤0.001; Fig. 1C), which was considered to be the optimal knockdown. The cells were collected 96 h following transfection for downstream analysis.
Trypan blue exclusion test
Cell viability was determined using the trypan blue exclusion assay (27). HepG2 cells were harvested and counted 48 h post-transfection to monitor the effect of NELF-B overexpression on cell proliferation. For monitoring the effect of NELF-B silencing on cell proliferation, the number of SNU449 cells was counted 96 h post-transfection. Cells were mixed with 0.4% trypan blue in PBS at a ratio of 1:1, and viable cells were counted using a hemocytometer (Hausser Scientific) and an inverted fluorescence phase contrast microscope at ×200 magnification.
Reverse transcription-PCR (RT-PCR)
TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for extraction of total RNA, according to the manufacturer's recommendations. RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Inc.) was used for cDNA synthesis, following the manufacturer's protocol. Random primers were used for reverse transcription of 0.5 µg of the total RNA in a final volume of 20 µl. MyTaq DNA Polymerase kit (Bioline; Meridian Bioscience) was used for semi-quantitative RT-PCR using 1 µl cDNA, according to the manufacturer's recommendations. All the genes were analyzed under similar PCR conditions, except for the cycle number and annealing temperature (Table I). Initial denaturation was performed at 94°C for 3 min, followed by cycles of 94°C for 30 sec, annealing temperature for 30 sec and 72°C for 45 sec, and a final extension at 72°C for 7 min. Amplicons were electrophoresed on a 2–2.5% agarose gel and visualized using the Gel Doc EZ System (Bio-Rad Laboratories, Inc.). Intensities of bands generated in RT-PCR were quantified using ImageJ software version 1.51j8 (National Institutes of Health) (28). The band intensities were normalized to those of respective loading controls (GAPDH or β-tubulin). Images were converted to grey scale.
 | Table I.Reverse transcription-semi-quantitative PCR primer sequences, PCR conditions and amplicon sizes. |
Quantitative PCR (qPCR)
qPCR amplification was performed using SYBR-Green as a DNA-specific binding dye, and continuous monitoring of fluorescence was done. Each PCR reaction (10 µl) consisted of 2X SYBR-Green I Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), 1 µl cDNA and 0.5 µl each primer. All the primers used for the selected genes are listed in Table II. The thermal cycler was set up for 40 cycles of the following amplification program: 50°C for 2 min, 95°C for 2 min, 95°C for 15 sec and 60°C for 1 min. The instrument was set to perform the default dissociation step under the following conditions: 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. All experiments were performed three times using the 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Data analysis was performed using the 2−ΔΔCq method (29) with GAPDH used as the reference gene.
Western blot analysis
Cells were washed with ice-cold PBS before being lysed in 1X ice-cold Laemmli Lysis Buffer (50 mM Tris pH 6.8, 2% SDS and 10% glycerol) supplemented with 100X Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Inc.; 10 µl protease inhibitor per ml of Laemmli Lysis buffer was used). Protein concentrations were determined using the BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.), following the manufacturer's protocol. Whole-cell lysates (20–30 µg/lane) were separated via 10% SDS-PAGE and the proteins were blotted onto a nitrocellulose membrane. The membranes were blocked in 5% non-fat dry milk, and then incubated overnight with primary antibodies at 4°C. Subsequently, the membranes were incubated for 1 h with the secondary antibody at room temperature. Colorimetric detection of the tested proteins was performed using BCIP/NBT Phosphatase colorimetric substrate (KPL). Primary antibodies used were as follows: Mouse monoclonal anti-GAPDH (Abcam; cat. no. ab8245; 1:10,000 in 5% non-fat dry milk), mouse monoclonal anti-β-tubulin (Sigma-Aldrich; Merck KGaA; cat. no. T7816; 1:20,000 in 5% non-fat dry milk), rabbit monoclonal anti-NELF-B (anti-COBRA1; Abcam; cat. no. ab167401; 1:1,000 in 3% non-fat dry milk) and rabbit monoclonal anti-NELF-E (Abcam; cat. no. ab170104; 1:1,000 in 5% non-fat dry milk). Secondary antibodies used were polyclonal goat anti-mouse (KPL; cat. no. 4751-1806; 1:10,000 in 5% non-fat dry milk) and polyclonal goat anti-rabbit (KPL; cat. no. 4751-1516; 1:10,000 in 5% non-fat dry milk), both have alkaline phosphatase conjugates. For all purposes, non-fat dry milk used in western blot analysis was dissolved in 1X TBS-0.1% Tween-20. Intensities of bands generated were quantified using ImageJ software version 1.51j8 (National Institutes of Health) (28). The band intensities were normalized to those of respective loading controls (GAPDH or β-tubulin).
Wound healing assay
Classic wound healing assay (30) was performed to assess the effect of the overexpression/knockdown of NELF-B on the cell migration rate. After 48 h from NELF-B overexpression, and 96 h post-NELF-B silencing, the spent medium was discarded, a scratch was made in the confluent (~90%) cell monolayer using a sterile 20 µl yellow tip. The cells were gently washed twice with PBS and incubated for 24 h in fresh 10% FBS RPMI medium. The migration of cells was monitored using an inverted fluorescence phase contrast microscope at ×100 magnification (Olympus IX70; Olympus Corporation); images were converted to greyscale. The wound area was measured using the TScratch software version 1.0 (31), and the percentage of wound closure was calculated using the following equation (32):
 |
where WC % is the percentage of wound closure, WA 0 h is the wound area at 0 h, and WA 24 h is the wound area at 24 h.
Transwell invasion assay
Transwell Boyden 24-well chambers (ThinCert™; Greiner Bio-One) were used to determine the effect on the invasive capacity of cells. Cells were harvested 96 h post-transfection, and 2×105 cells were suspended in 100 µl RPMI-1640 medium (supplemented with 1% FBS) and seeded in the upper chamber of the Transwell cell culture insert (8-µm pore size). The cell culture insert was coated with 40 µg collagen I (SERVA Electrophoresis GmbH; cat. no. 47254), allowed to dry overnight in an incubator at 37°C, and rehydrated with 1% FBS-containing medium, 30 min before the seeding of cells. The lower chamber was filled with 600 µl RPMI-1640 medium supplemented with 10% FBS. Cells were placed inside the upper chamber and incubated for 22 h at 37°C. Subsequently, cotton swabs were used to remove cells on the upper side of the membrane, and the cells that invaded to the lower side of the membrane were fixed using 4% formaldehyde for 10 min at room temperature, and subsequently stained with DAPI for 10 min at room temperature in the dark (KPL, Inc.; cat. no. 71-03-01; 1:1,000 in PBS). The images were captured with a fluorescence microscope at ×200 magnification in five random fields for each condition, and the average of cell counts was compared.
Statistical analysis
Relative gene expression was calculated in reference to the negative control siRNA or pCMV5-empty and represented as fold-change. Data are presented as the mean ± SD of two to four independent experiments. Statistical significance for comparison between two groups was performed using unpaired Student's t-test (two-tailed). GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla California USA) was used to generate graphs. P<0.05 was considered to indicate a statistically significant difference.
Results
NELF-B-knockdown significantly decreases NELF-A expression in SNU449 cells
Downregulation of NELF-B in the early-stage liver cancer HepG2 cell line has been previously shown to inhibit the migration and proliferation of the cells (25). To investigate whether ectopic expression of NELF-B in the same cell line would lead to an opposite effect to that induced by its downregulation, NELF-B was overexpressed in HepG2 cells. To further investigate the role of NELF-B in the progression of liver cancer, loss of function analysis was performed on the intermediate-stage HCC cell line, SNU449. The efficiency of overexpression and knockdown was assessed by measuring the protein expression levels by western blot analysis. HepG2 cells transfected with the NELF-B overexpression plasmid (pCMV5-HCOBRA1) exhibited a 4-fold increase in NELF-B expression compared with the pCMV5-empty-transfected control cells (P≤0.0001; Fig. 1A); however, NELF-E expression was not significantly altered upon overexpression of NELF-B in HepG2 cells (P>0.05; Fig. 1B). SNU449 cells transfected with NELF-B siRNA showed a significant decrease in protein expression by an average of 96% compared with that in the negative siRNA-transfected control cells (P≤0.001; Fig. 1C). Consistent with the protein expression levels, the mRNA expression levels of NELF-B assessed by RT-PCR exhibited a 2.2-fold increase in HepG2 cells following NELF-B overexpression and an average of 93.5% decrease in SNU449 cells following NELF-B-knockdown compared with their respective controls (P≤0.0001 and P≤0.001; Fig. 1D and E).
The effect of overexpression and knockdown of NELF-B on the expression levels of other NELF subunits was assessed using RT-PCR. The expression levels of NELF-A, NELF-C/D and NELF-E were not significantly affected following the overexpression of NELF-B (P>0.05, Fig. 1D). In SNU449 cells, the expression levels of NELF-C/D and NELF-E were not significantly altered, while NELF-A expression was significantly decreased by an average of 30% upon knockdown of NELF-B (P≤0.001; Fig. 1E).
NELF-B-knockdown suppresses cell proliferation and expression of the proliferation marker, Ki67, in SNU449 cells
No morphological changes were observed after the overexpression or knockdown of NELF-B compared with the control cells (Fig. 2A and B). However, cells in which NELF-B was knocked down appeared less dense than their negative controls, suggesting a decrease in the proliferation rate and/or survival of cells (Fig. 2B). To further examine the effect of deregulation of NELF-B on cell proliferation, cells were counted at time points when optimum overexpression/knockdown was observed post-transfection (48 and 96 h for HepG2 and SNU449 cells, respectively). The count of NELF-B-overexpressing HepG2 cells was similar to that of cells transfected with the empty vector, and no significant difference in Ki-67 mRNA expression was observed (P>0.05; Fig. 2C). By contrast, the cell count was significantly decreased upon silencing of NELF-B in SNU449 by an average of 58% compared with the negative control (P≤0.001; Fig. 2D). Consistent with these results, a significant decrease in the expression levels of the proliferation marker, Ki67 (by an average of 51%), compared with the negative control was also observed (P≤0.05; Fig. 2D).
NELF-B-knockdown significantly inhibits the migration and invasion of SNU449 cells
Cancer progression involves epithelial-mesenchymal transition (EMT), a process during which the migratory and invasive abilities of cells increase (33). The present study investigated the association between NELF-B and EMT in liver cancer. Wound healing assay was used to examine the effect of overexpression and knockdown of NELF-B on cell migration. Scratches were made in cell monolayers, 48 and 96 h post-transfection in HepG2 and SNU449 cells, respectively, and wound closure was monitored 24 h later. The overexpression of NELF-B in HepG2 cells resulted in a 1.4-fold higher migration rate compared with that in the negative control group; however, the difference was not statistically significant (P>0.05; Fig. 3A). In SNU449 cells, knockdown of NELF-B significantly decreased the cell migration rate by an average of 49% compared with that in the negative control group (P≤0.001; Fig. 3B).
Transwell invasion assay was performed to assess the effect of NELF-B-knockdown on the invasive capacity of cells through the extracellular matrix equivalent collagen. The knockdown of NELF-B significantly decreased the number of cells that invaded through collagen by an average of 30% compared with the control (P≤0.01; Fig. 3C).
NELF-B-knockdown decreases the expression levels of FOXF2 and the EMT markers N-cadherin, vimentin and β-catenin, in SNU449 cells
RT-qPCR was used to assess some of the primary EMT markers, including N-cadherin, vimentin and β-catenin (34), in SNU449 cells following NELF-B-knockdown. In line with the results of invasion and migration assays, the expression levels of N-cadherin and vimentin were significantly decreased by an average of 75 and 70%, respectively, compared with the negative control (P≤0.01; Fig. 3D). Another critical gene involved in EMT is the signal transducer molecule, β-catenin. β-catenin expression is usually elevated in cancer and the protein is localized in the nucleus, where it drives the expression of downstream genes involved in the EMT process (35–37). A significant decrease was observed in the expression levels of β-catenin post-NELF-B-knockdown by an average of 85% (P≤0.05; Fig. 3D). The expression levels of the transcription factor FOXF2 were also analyzed, which is reported to enhance the invasion and migration of HCC cells (38). FOXF2 expression was significantly decreased by an average of 60% following NELF-B-knockdown (P≤0.05; Fig. 3D).
NELF-B-knockdown decreases survivin gene expression and induces apoptosis in SNU449 cells
To examine whether NELF-B affected apoptosis in liver cancer, the mRNA expression levels of survivin were measured, which is known to be deregulated in cancer and serves critical roles in the survival and proliferation of cancer cells (39). The expression levels of wild-type survivin have been previously shown to be decreased upon knockdown of NELF-B in HepG2 cells (25). In the present study, the mRNA expression levels of survivin were examined following the overexpression and knockdown of NELF-B. No significant difference in survivin expression was detected upon overexpression of NELF-B in HepG2 cells (P>0.05; Fig. 4A). By contrast, survivin expression was decreased by an average of 43% following silencing of NELF-B in SNU449 cells (P≤0.05; Fig. 4B).
DAPI staining revealed fragmented nuclei in cells in which NELF-B was silenced (Fig. 4D). Fragmented nuclei are considered a marker for apoptosis (40), which is in agreement with the decreased survivin expression and decrease in cell counts.
NELF-B silencing decreases trefoil factor 1 (TFF1) expression in SNU449 cells
Several types of cancer, such as breast, gastric, prostate and colorectal cancers exhibit aberrant expression levels of trefoil factor family members, such as TFF1 and TFF3 (41–45). In HCC, the downregulation of TFF1 and the upregulation of TFF3 are frequently observed (46,47). In the present study, the mRNA expression levels of these genes were assessed after knockdown of NELF-B in SNU449 cells. A significant increase of 1.67 fold was observed in the expression levels of TFF1 (P≤0.01; Fig. 4C), while TFF3 expression was decreased by an average of 21% (P>0.05, Fig. 4C) compared with the negative control group. Notably, the overexpression of NELF-B in HepG2 cells did not significantly alter the expression levels of either of these genes (data not shown).
Discussion
The potential role of NELF-B in the pathogenesis of liver cancer has been previously shown (25,48). NELF-B expression has been reported to be upregulated in HCC tissue samples compared with that in paired non-neoplastic tissues (25). In addition, NELF-B has been shown to aid the proliferation and migration in the early stage liver cancer cell line, HepG2 (25). Furthermore, the differential expression of NELF-B has been examined among different liver cancer cell lines, representing various stages of liver cancer (48). The results have revealed that NELF-B expression is the highest in the early stages, and decreases gradually with the advance in stage of liver cancer, suggesting a possible involvement of NELF-B in the initiation of liver cancer rather than in its progression (48). The present study aimed to gain further insights into the role of NELF-B through its ectopic expression in HepG2 cells and tried to unravel the underlying mechanism. To investigate the involvement of NELF-B in the maintenance and progression of HCC, a loss-of-function analysis was also performed in SNU449 cells, which represents an intermediate-stage HCC cell line.
Sustenance of cell proliferation is the most fundamental trait of cancer cells (49). In the present study, overexpression of NELF-B did not induce the proliferation of HepG2 cells, as evidenced by cell counting and Ki-67 mRNA expression assays. The migratory ability of NELF-B-overexpressing cells was similar to that of empty plasmid-transfected cells; however, this assay may have been affected by the low initial confluence of the cells. Additionally, NELF-B-overexpressing cells exhibited a non-significant effect on the apoptotic marker, survivin. The current results indicated that overexpression of NELF-B in HepG2 cells did not have an effect opposite to that of its knockdown, which significantly decreased the proliferation and migration potential of HepG2 cells and decreased Ki-67 and survivin expression (25).
In prostate cancer, ectopic expression of NELF-B has been found to support the viability, proliferation and anchorage-independent growth of cells (24). This effect was opposite to that observed following the knockdown of NELF-B (24). In breast cancer, ectopic expression of NELF-B decreased the proliferation of cells, whereas its knockdown led to the enhancement of proliferation (20). The latter study has suggested that NELF-B may act through the NELF complex to repress estrogen receptor α (ERα)-mediated transcription and that NELF-B may be a rate-limiting step for enhanced NELF activity in the examined cell line, since its ectopic expression increased NELF-E promoter binding, and the expression of a subset of ER-α responsive genes was subsequently repressed (20). On the contrary, the current results indicated that overexpression of NELF-B was inadequate at promoting the proliferation and migration of cells. These results suggest that NELF-B may not be the rate-limiting factor for exerting an effect on the tested cellular features in HepG2 cells, and that it may be acting through the NELF complex, which requires the abundance of other subunits to form a functional complex.
The present study detected similar mRNA expression levels of the NELF subunits and NELF-E protein following the overexpression of NELF-B in HepG2 cells. These results are consistent with previous findings (17), and indicate a tight control on the abundance of the NELF complex in the regular cellular context. The interdependence of the NELF subunits has been shown in different cell types, where the knockdown of one subunit did not affect the mRNA expression levels of other subunits, but resulted in instability of the NELF complex and co-depletion of other subunits at the protein level (17,50). Notably, in the present study, a significant decrease in the mRNA expression levels of NELF-A was detected in NELF-B-silenced SNU449 cells, which has not been previously reported to the best of our knowledge. It would be interesting to further examine the mechanism involved in this regulation.
NELF-B silencing resulted in a significant decrease in cell proliferation, as well as in the mRNA expression levels of Ki-67, which is the most predominantly used proliferation marker (51). These results are consistent with the effect of NELF-B-knockdown in HepG2 cells (25). Inhibition of apoptotic pathways is another main hallmark of cancer, promoting the survival of defective cancer cells (49). BIRC5, also known as survivin, is a member of the inhibitor of apoptosis proteins family, which inhibit apoptosis by binding to caspases (52). The expression of survivin is undetectable or low in normal adult tissues, but the protein is overexpressed in most types of cancer, such as liver, blood and gastric cancers (52–55). Additionally, survivin supports cell cycle progression, counteracts the tumor suppressor retinoblastoma protein and induces angiogenesis (56–58). In the present study, a significant decrease in survivin expression was observed upon knockdown of NELF-B, which is in concordance with the decrease in cell count and the detection of nuclear fragments, which signifies apoptosis (59). This association between survivin expression and NELF-B has been shown in different cancer cell lines, including SNU449, HepG2 (25), cervical cancer HeLa cells (experiments are ongoing) and breast cancer T47D cells (21). This consistent association over different stages and different types of cancer suggests the possibility of a direct regulation that requires to be further examined.
To further understand the involvement of NELF-B in cancer progression, EMT markers were investigated. EMT occurs during cancer progression, allowing metastasis through enhanced migration and invasion of cancer cells (33). Silencing of NELF-B in SNU449 cells resulted in a significant decrease in cell migration, consistent with the findings reported for HepG2 cells (25). Additionally, NELF-B-silenced cells exhibited a decrease in the invasive ability of cells. To the best of our knowledge, the present study demonstrated for the first time that NELF-B supported invasion, in addition to migration, in HCC. Consistently, decreased expression levels of the mesenchymal markers N-cadherin, vimentin and β-catenin were observed. Vimentin is an intermediate filament that is central to the cytoskeletal structure and cell integrity (60), whereas N-cadherin promotes collective cell migration and modulates the expression of several cancer-associated genes (61). There are contradictory studies regarding the effect of N-cadherin on metastases in HCC, with one study linking its downregulation to enhanced metastasis (62) and another linking overexpression to metastasis (63). β-catenin, an EMT marker, is a central signal transducer of the canonical Wnt/β-catenin signaling pathway (64,65). Although this pathway is vital for the development of normal liver, its aberrant activation is frequently implicated in HCC (66). In the absence of Wnt signaling molecules, the β-catenin destruction complex is activated, ultimately leading to both phosphorylation and ubiquitination of β-catenin (67). Upon deactivation of the destruction complex, β-catenin is accumulated in the cytosol and is eventually translocated to the nucleus, where it binds to LEF/TCF factors to regulate the expression of target genes (68–70). It is suggested that β-catenin, via the Wnt/β-catenin signaling pathway, activates the expression of several downstream genes, such as vimentin (71), matrix metalloproteinases (72) and fibronectin (73), which are associated with a mesenchymal phenotype. Additionally, β-catenin has been reported to be involved in the induction of survivin expression (74).
FOXF2 is another interesting protein associated with EMT. It is a transcription factor with a dual role of promoting or inhibiting proliferation, invasion and metastasis in tumors, depending on the tumor type and subtype (75). In HCC, the downregulation of FOFX2 induces mesenchymal-epithelial transition and inhibits the invasion and migration of HCC cells (38), which is in accordance with the concomitant decrease of FOXF2 expression, invasion and migration in NELF-B-silenced cells observed in the present study. The downregulation of FOXF2 promotes the proliferation of Huh7 cells, which represent well-differentiated HCC (76,77). Nonetheless, in SNU449 cells, decreased FOXF2 expression was accompanied by the inhibition of proliferation, which suggests that the effect of FOXF2 on proliferation is dependent on the context.
NELF is a critical regulator of clustered genes, including those of the trefoil family such as TFF1 and TFF3 (22). The knockdown of NELF-B did not significantly affect the steady-state expression of TFF3 mRNA in SNU449 cells; however, the steady-state expression of TFF1 mRNA was significantly increased. The current results suggested that NELF-B may be implicated in repressing TFF1 expression. TFF1 is encoded by the TFF1 gene and belongs to the trefoil family; this family of secretory proteins characteristically contains at least one trefoil motif (46). Although mainly secreted by gastric epithelial cells, TFF1 is also secreted by hepatic cells, albeit to a lower degree (46). Studies performed in breast cancer and upper gastrointestinal adenocarcinoma have revealed that NELF-B negatively regulates TFF1 (20,23). TFF1 has been shown to function as a protectant and restorer of the gastrointestinal tract, an inflammatory suppressor and a possible regulator of tissue regeneration (78). In HCC, the putative tumor suppressor, TFF1, has been shown to suppress proliferation in HCC cell lines, whereas its deficiency in a TFF knock-out mouse model promoted HCC progression (46). Additionally, TFF1 expression has been found to affect the localization of β-catenin and to negatively regulate its transcriptional activity and downstream targets in both gastric cancer and HCC (46,78). The current results suggested that NELF-B may promote tumorigenesis in SNU449 cells by negatively regulating TFF1 expression. It has been reported that NELF-B represses the expression of specific estrogen-responsive genes, including TFF1, in breast cancer (22). Nevertheless, NELF-B has also been shown to modulate gene expression, independent of Erα (23). Therefore, NELF-B may regulate TFF1 expression in either an estrogen-dependent or estrogen-independent manner; however, the exact mechanism by which NELF-B regulates TFF1 in SNU449 cells remains to be determined.
A previous study investigating the role of NELF-E in HCC has shown that NELF-E supports cell proliferation, colony formation, oncosphere formation and cell migration through loss- and gain-of-function analysis (79). The similar effects of NELF-E- and NELF-B-knockdown suggest that NELF-B may mediate its action in HCC predominantly through the NELF complex. Nonetheless, it should be considered that these subunits do not act through NELF exclusively, and further elucidation of additional mechanisms and overlap of their functions is required.
The present study provided evidence that NELF-B served a critical role in the progression of HCC. It supported some of the significant cancer hallmarks, including cell proliferation, apoptosis inhibition and EMT, which is essential for cancer metastasis. The lack of effect on proliferation and migration following ectopic expression in HepG2 cells suggested the insufficiency of NELF-B overexpression in promoting an increase in these processes. NELF-B may have a great potential for use as a prognostic factor and a therapeutic target for liver cancer. Further examination of different etiologies among HCC and more advanced stages is recommended. Additionally, an elucidation of the mechanisms of NELF-B action and its effect on the entire transcriptome would provide valuable insights.
Supplementary Material
Supporting Data
Acknowledgements
The authors would like to thank Mr. Amged Ouf (The American University in Cairo, New Cairo, Egypt) for his technical support in performing the plasmid transfection experiments, and Mr. Tarek Saleh for his help in generating the figures (Cannonball VFX LLC, San Diego, CA, USA).
Funding
The present study was supported by the American University in Cairo Internal Faculty Research Grant and Graduate Student Research Grants.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Authors' contributions
AA conceived and designed the study. AA supervised and contributed to the analysis and troubleshooting of all the experiments and results. RJM performed the overexpression and subsequent functional assays in HepG2. MHG performed the knockdown and subsequent functional assays in SNU449. MHG, RJM, EZM, RZ and AMA performed and interpreted RT-PCR results. OMA performed and interpreted qPCR results. MHG, RJM, EZM and OMA contributed to data analysis. MHG drafted the initial manuscript with outstanding contributions from RJM, EZM and OMA. All authors have read and approved the final manuscript. RJM and AA confirmed the authenticity of the raw data related to NELF-B overexpression. MHG and AA confirmed the authenticity of the raw data associated with the NELF-B knockdown.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
|
COBRA1 |
cofactor of BRCA1 |
|
EMT |
epithelial-mesenchymal transition |
|
ERα |
estrogen receptor α |
|
FOXF2 |
forkhead box F2 |
|
HBV |
hepatitis B virus |
|
HCC |
hepatocellular carcinoma |
|
NELF |
negative elongation factor |
|
RT-qPCR |
reverse transcription-quantitative PCR |
|
TFF1/3 |
trefoil factor 1/3 |
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