Elimination of Ras Suppressor-1 from Hepatocellular Carcinoma Cells Hinders their In Vitro Metastatic Properties

Materials and Methods

Cell lines. Two cell lines were used in this study; the hepatoma cell line PLC/PRF/5 (Alexander) and HCC cell line HEPG2. Both cell lines were purchased from ATCC (Manassas, VA, USA).

Antibodies and reagents. Anti-β-actin antibody (Sigma-Aldrich, St.Louis, MO, USA) was used as loading control. The Anti-RSU-1 antibody was purchased from Assay Biotechnology (Sunnyvale, CA, USA). The alamar Blue reagent was obtained from Invitrogen (Carlsbad, CA, USA).

Real time polymerase chain reaction PCR (RT-PCR). The RSU-1 mRNA expression level was assessed by RT-PCR, as described previously (6). Briefly, total RNA was extracted using TRIzol reagent (Gibco, Waltham, MA, USA). Preservation of 28S and 18S rRNA species was used to assess RNA integrity. Only samples with prominent 28S and 18S rRNA components were included in the study. Total RNA was reversed-transcribed to cDNA using SuperScript first-strand synthesis (Invitrogen). RT-PCR was performed using SYBR Green from Invitrogen in a Mini J Opticon Real Time PCR machine (Biorad, Hercules, CA, USA). Reactions were always performed in triplicate, while human porphobilinogen deaminase (PBGD) was used as housekeeping gene. The sequence of the primers used for RSU-1 was: Reverse 5’ CGT GCA ATC TCA AAA GCT CA 3’ and Forward: 5’-AGG CCA CAG AGC AAG GTC TA-3’.

Western blotting. Protein expression was assessed by Western blot analysis using the standard Western blotting protocol. Total protein was isolated from the two cell lines using 1% sodium dodecyl sulfate in RIPA buffer (20 mMTris/Cl pH7.5, 150 mM NaCl, 0.5% NP-40, 1% TX-100, 0.25% sodium deoxycholated, 0.6-2 μg/ml aprotinin, 10 μMleupeptin, 1 μM pepstatin). Protein concentration in the samples was determined by the BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amount of protein was loaded on each lane of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and detection of the antibodies was performed using super-signal ECL (Pierce).

Transfection with siRNAs. Both Alexander and HepG2 cells were treated for 48 h with 100 nM siRNA non-specific control (NSC) siRNA or RSU-1 siRNA using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the company's guidelines. The sequence of the NSC siRNA used was 5’AAA CUC UAU CUG CAC GCU GAC3’, while the RSU-1 siRNA sequence was 5’UCA ACG GCC UCU UUA CCU UdTdT 3’. Silencing efficiency prior to any experiment performed was tested by western blot and/or RT-PCR.

Cell adhesion assay. The cell adhesion assay was performed as described previously (7). Briefly, cells were transfected with a control NSC siRNA or RSU-1 siRNA. Forty eight hours post-transfection, 10⁴ cells/well were seeded in six (6) wells of a 96-well plate pre-coated with 0.1% gelatin. After a 60-min incubation at 37°C, three of the wells were washed three times with phosphate-buffered saline, while the remaining three were fixed with 4% paraformaldehyde (PFA). Washed wells were also fixed with PFA and then cells in all wells were quantified using crystal violet (7). Crystal violet was washed using ddH20 and cells were solubilized using acetic acid. Absorbance was measured at 570 nm using a PerkinElmer Enspire plate reader (PerkinElmer, Waltham, MA, USA). Adhesion was presented as the ratio of the absorbance at 570 nm of adhered cells (washed) divided by the absorbance at 570 nm of the total seeded cells (not washed). The data from two (2) independent experiments were analyzed using the Student's t-test. p-values <0.05 were considered statistically significant.

Cell proliferation assay. Twenty four hours following siRNA treatment, cells were subjected to alamarBlue assay (Invitrogen) (8) according to the company's guidelines. Briefly, cells were seeded in 96-well plates at a concentration of 10⁴ cells/ml and alamarBlue was added at a volume of 1/10 of the volume of medium in the well. Following incubation at 37°C for 2, 4 and 24 h post-plating, fluorescence was measured using a Perkin Elmer Enspire plate reader at 560/590 nm. Three independent experiments were performed.

Cell invasion. Cell invasion was assessed using the QCM™ Collagen Cell Invasion Assay in 24-well invasion chambers (8 μm) (Cat.#ECM551; Merck-Millipore, Billerica, MA, USA) following the manufacturer's protocol. Briefly, cells 24 h post-trasnfection with siRNA were trypsinized and a suspension of 0.5×10⁶ cells/ml was prepared in serum-free Dulbecco's modified Eagle's medium (DMEM) medium. DMEM supplemented with 10% Fetal Bovine Serum was used as chemoattractant at the bottom of the transwell. Cells were incubated at 37°C for another 24 h. At the end of the incubation period, the transwells were placed in staining solution and incubated for 20 min. Transwells were then washed several times with distilled water. The non-invaded cells that remained on the upside of the filter were removed, while the stained insert with invaded cells was transferred to a well containing 0.2 ml of extraction buffer. After 15min incubation at room temperature, the transwell was removed and the dye mixture was transferred to three wells of a 96-well plate. Optical density (OD) was measured at 560 nm using an automated Enspire Perkin Elmer spectrophotometer. Two independent experiments were performed.

Statistical analysis. Comparison of means using the Statgraphics software was used for the statistical analysis. The t-test was performed and a p-value <0.05 was considered statistically significant.