Hepatocyte growth factor regulates HLX1 gene expression to modulate HTR-8/SVneo trophoblast cells

The human EVT cell line HTR-8/SVneo was generously provided by Dr. Charles H. Graham (Department of Anatomy & Cell Biology, Queen’s University at Kingston, Canada). Cells were cultured in RPMI-1640 medium (Hyclone, China) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sijiqing Biotec Co., China) in a sterile incubator at 37°C with 95% humidity and 5% CO₂.

For HGF treatment, cells were grown to log phase, released from the culture dish by trypsinization (0.25% trypsin; Sigma, USA), and seeded into 6-well plates at 2 × 10⁵ cells/well. Twenty-four hours later, cells were starved in RPMI-1640 medium containing 0.5% FBS for 12 h and then treated with HGF (PeproTech, USA) at different concentrations for a further 48 h.

Total RNA was extracted from cells using Trizol reagent (Invitrogen, USA), according to the manufacturer’s protocol. Following quantification by UV spectrophotometer, 2 μg of total RNA was reverse transcribed into cDNA by using a commercially-available reverse transcription kit (Fermentas, Lithuania). The cDNA was then used as a template for subsequent qPCR analysis with SYBR^® Premix Ex Taq™ and the following primers (TaKaRa, Japan): β-actin (internal control, amplicon size of 188 bp): upstream 5'-TGGCACCCAGCACAATGAA-3′ and downstream 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′; HLX1 (amplicon size of 175 bp): upstream 5′-CAGTTCAGCATCAGTTCCAAGACAC −3′ and downstream 5′-TCCGGCTTGGTCACGTACTTC-3′. Thermal cycling conditions for amplification were: one cycle of 95°C for 20 sec, followed by 45 cycles of 95°C for 5 sec, 60°C for 30 sec and 72°C for 10 sec. The relative gene expression was calculated using the 2^-ΔΔCt method, as previously described [16].

Cells were lysed in sodium dodecyl sulfate (SDS) lysis buffer (Beyotime, China) and the total protein concentration was determined using the BCA protein assay kit (Beyotime), following the manufacturer’s instructions. An 80 μg aliquot of total protein from each sample was separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred to a nitrocellulose membrane. After blocking with 5% skim milk at room temperature for 3 h, the membrane was incubated with one of the following primary antibodies at 4°C overnight: rabbit anti-HLX1 (0.25 μg/μL; Abcam, USA) or mouse anti-β-actin (0.1 μg/μL; Santa Cruz Biotech., USA). After washing, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:1000; Zymed, USA) at room temperature for 90 min and the signal was developed with enhanced chemiluminescent substrate and detected on a Kodak gel imaging station. Immunoreactive signals were quantified using JEDA imaging and analysis software (version 3.3; JEDA Science-Tech., China), and presented as the intensity ratio of HLX1 signal to β-actin signal.

At 24 h after transfection of HTR-8/SVneo cells with different concentrations of DY-547-tagged control siRNA, cells were trypsinized, resuspended in ice-cold sterile phosphate buffered saline (PBS), and analyzed by flow cytometry. The percentage of DY-547-positive cells was used as an indicator of transfection efficiency.

HTR-8/SVneo cells at log phase were seeded into 96-well plates at 5 × 10³ cells/well. After overnight incubation, cells were transfected with either control or HLX1 siRNA (time 0; five replicates for each condition). At 0, 24, 48, 72 and 96 h post-transfection, the cell viability was determined using MTT reagent (Gibco, USA) and reading absorbance (A) at 490 nm. The growth inhibition was calculated as [(1-A_Experiment/A_{Blank well}) × 100%].

Confluent cultures of trophoblast cells were starved for 24 h in RPMI-1640 medium supplemented with 0.5% FBS. Following starvation, the cells were stimulated with 20 ng/mL HGF in serum-free media for a further 24 h. The mock-stimulated control cells were treated with only serum-free medium, without HGF added. For experiments involving HLX1 down-regulation with HGF stimulation, cells were transfected with siRNA prior to serum starvation and HGF stimulation. Following the treatment, conditioned medium was harvested from the cells and a 10 μL aliquot was mixed with an equal volume of 2× Laemmli sample buffer and separated on an 8% SDS-PAGE gel containing 0.1% gelatin. After electrophoresis, gels were soaked and washed twice in 2.5% Triton X-100 for 45 min each at 37°C and then incubated in a buffer containing 50 mM Tris buffer, pH 7.5, 200 nM NaCl, 5 mM CaCl₂ 1 μM ZnCl₂, and 0.2% Brij 35 at 37°C overnight. After 12 h, the gels were stained for 4 h with 0.25% Coomassie Brilliant Blue G-250 in a solution of 10% acetic acid and 45% methanol, and then destained for 30 minutes in the same solution without Coomassie. The Coomassie-stained gel was imaged using the Kodak gel imaging station and analyzed by JEDA imaging and analysis software. Conditioned medium from human fibrosarcoma cells HT1080 was collected for use as the positive control for MMP2 and MMP9 activity.

The high concentration Matrigel matrix (BD Biosciences, USA) was thawed at 4°C, diluted 1:3 in RPMI-1640 medium, coated on the upperside of a transwell insert (8 μm pore size; Corning, USA) at 100 μL/insert and incubated at 37°C for 30 min. Meanwhile, cells were resuspended in serum-free RPMI-1640 medium at 1 × 10⁵ cells/mL and 400 μL aliquots were deposited on the top of the transwell insert. To the bottom well, 1200 μL of RPMI-1640 medium with 20% FBS was added. The invasion assay was allowed to proceed at 37°C for 12 h. After the invasion period, the cells remaining on top were gently removed by a cotton swab, and those that had transversed to the bottom side of the insert were fixed in methanol and stained with hematoxylin. The insert membrane was then cut out and mounted onto glass slides with the bottom side facing upward. The invasive cells were imaged under a light microscope (IX81; Olympus, Japan) at 400 × magnification. Five random fields were imaged for each membrane and the cell numbers detected in each were averaged.

In response to increasing concentrations of HGF, the steady-state mRNA level of HLX1 in HTR-8/SVneo cells showed a dose-dependent increase (Figure 1A), and significant difference was achieved with as little as 10 ng/mL HGF (P < 0.01). At 100 ng/mL HGF, the mRNA level of HLX1 increased to almost five-fold more than that of the endogenous level in HTR-8/SVneo cells. For subsequent experiments, we chose the intermediate dose of HGF (20 ng/mL) that showed the most robust dose response among all HGF concentrations tested. Similar changes were also observed at the protein level of HLX1 (Figure 1B). When HTR-8/SVneo cells were treated with 20 ng/mL HGF, the protein level of HLX1 was approximately 2.6-fold higher than that in cells without HGF treatment (P < 0.01).

Given the apparent HGF-inducible nature of HLX1, we investigated the significance of HLX1 in HGF-induced biological processes. A loss-of-function approach was applied by knocking down the expression of HLX1 with siRNA-mediated gene targeting. We first examined the efficiency of siRNA transfection by using the fluorescent probe DY-547-tagged non-targeting control siRNA (siCtrl). By transfecting 10, 50 and 100 nM siRNA, we found that the 100 nM concentration produced the highest transfection efficiency (86.3 ± 2.6%; Figure 2A). Therefore, this dosage was used for all subsequent experiments.

At 48 h after siRNA transfection, we examined the mRNA and protein levels of HLX1 by qPCR and Western blot, respectively. As shown in Figure 2B and 2C, transfection with the HLX1 siRNA pool (siHLX1) significantly reduced HLX1 expression on both mRNA and protein levels, as compared to the levels detected in siCtrl-transfected or non-transfected (NT) HTR-8/SVneo cells (P < 0.01). In addition to down-regulating endogenous HLX1, siHLX1 also inhibited its HGF-induced expression levels in both mRNA and protein (Figure 2D and 2E). HGF treatment of siCtrl cells led to an approximate 2.7-fold increase in HLX1 mRNA and 2.8-fold increase in HLX1 protein. In contrast, siHLX1 transfected cells presented reduced HLX1 mRNA (by 55%) and protein (by 52%), even in the presence of HGF (P < 0.01) suggesting that HLX1 siRNA has a more dominant effect over HGF in regulating HLX1 expression. These three treatments provide us a model where the intracellular HLX1 levels vary from each other, therefore allowing us to further observe the significance of HLX1 in cellular behaviors as detailed below.

HGF is an important mitogen and motogen for trophoblasts, essentially regulating their proliferation, apoptosis and migration/invasion capabilities [3, 17, 18]. To evaluate the significance of HLX1 in HGF-mediated biological processes, we first examined HGF-induced growth of cells transfected with either siCtrl or siHLX1 by using the MTT assay (Figure 3A). The same number of cells were transfected at time 0. Under basal conditions, namely without HGF treatment, both NT and siCtrl-transfected HTR-8/SVneo cells grew steadily within the 96 h post-transfection observation period. However, this growth was dramatically reduced in the siHLX1-transfected cells, and a significant difference was found to have occurred as early as 24 h after siRNA transfection. The largest difference was observed at 72 h after transfection, with growth inhibition of 58.1 ± 4.4% (P < 0.01).

In response to HGF treatment, siCtrl cells grew significantly slower than siCtrl + HGF cells, but faster than siHLX1 cells, over the 96 h observation period following siRNA transfection (P < 0.01; Figure 3B). The growth observed for siHLX1 + HGF cells, however, was not dramatically different from siCtrl cells at 24 and 48 h post-transfection, but was significantly reduced at 72 and 96 h post-transfection (P < 0.01).

Next, the effects of knocking down HLX1 were determined by the invasive properties of trophoblasts. By imaging and quantifying the cells invading through the matrigel, we found that siHLX1 transfection not only reduced the basal invasiveness of HTR-8/SVneo cells (vs. NT or siCtrl-transfected cells, P < 0.01; Figure 4A), but also significantly inhibited the HGF-induced invasion properties (vs. compared to siCtrl-transfected cells or siCtrl-transfected cells treated with HGF, P < 0.01; Figure 4B).

To understand the regulatory mechanism underlying HLX1 associated HGF-induced cell invasiveness, we focused on two matrix metalloproteinase molecules, MMP2 and MMP9, that are characterized as downstream targets for HGF and important regulators of cell migration/invasion [19]. By gelatin zymography analysis, we found that the MMP2 activity in the conditioned medium was significantly enhanced upon HGF treatment, but not affected by siCtrl transfection (siCtrl vs. siCtrl + HGF or NT vs. NT + HGF samples, P < 0.01; siCtrl vs. NT or siCtrl + HGF vs. NT + HGF samples, P > 0.05; Figure 4C). Transfection of siHLX1, however, significantly reduced the MMP2 activity in the conditioned medium (vs. all other samples, P < 0.01). The MMP9 activity was very low in the conditioned medium, as compared with that in the conditioned medium from HT1080 cells, and no significant variations were found between HTR-8/SVneo cells treated with HGF or transfected with siHLX1.