The effect of ROCK-1 activity change on the adhesive and invasive ability of Y79 retinoblastoma cells

Rb Y79 cell line was obtained from ATCC. RPMI 1640 media and fetal bovine serum (FBS) were purchased from Life Technologies Corporation. Y79 cells were cultured in RPMI 1640 medium supplemented with 15% heat-inactivated FBS, 0.1% ciprofloxacin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 4.5% dextrose. The cells were grown in 25 cm² culture flasks in the upright position in 10 mL aliquots of the culture medium. Incubation was performed at 37°C under a humidified atmosphere of 5% CO₂-95% air.

The short interfering RNAs (siRNAs) were synthesized (GenePharma, China) and used for transfection. The sense and antisense strands of the ROCK-1 siRNA were 5′-GGCAGAGGAAGAAUAUAAATT −3′ and 5′- UUUAUAUUCUUCCUCUGCCTT-3′; ROCK-2 siRNA were 5′-GCAGCUGGAAUCUAACAAUTT-3′ and 5′- AUUGUUAGAUUCCAGCUGCTT-3′; negative control were 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′. (designed and synthesized by GenePharma Co. Ltd, Shanghai). These siRNA were transfected into Y79 cells with Lipofectamine® 2000 (Invitrogen) following the manufacturer’s instructions. Briefly, a total of 5 × 10⁵ cells were plated in 6-well plates and transfected using 100 pmol siRNA and 5 μL of Lipofectamine® 2000 per well. After 24–48 hours of incubation, the cells were harvested for RT-qPCR or western blots analysis.

Y79 cells transfected with the siRNA were incubated for 24 hours. Cells were harvested for RT-qPCR analysis. Total RNA was extracted using TRIzol (Invitrogen), and single-stranded cDNA was synthesized with AMV Reverse Transcriptase System (Promega) according to the manufacturer’s instructions. Real-time quantitative polymerase chain (RT-qPCR) reactions were done with 10 ng cDNA in SYBR Green I mix (Takara Bio Inc.) and run on an ABI Prism 7300 HT 7300 Real-Time PCR System (Applied Biosystems). For all of cDNA, 40 cycles and annealing temperature of 60°C (31 seconds) were used. All PCR reactions were performed in triplicate. Primer sequences (designed by primer3 web (version 3.0.0) and synthesized by Sangon Biotech Co. Ltd, Shanghai) are:

ROCK-1: forward primer 5′-ACCTGTAACCCAAGGAGATGTG-3′ and reverse primer 5′- CACAATTGGCAGGAAAGTGG-3′;

ROCK-2: forward primer 5′-AAGTGGGTTAGTCGGTTG-3′ and reverse primer 5′-GGCAGTTAGCTAGGTTTG-3′;

β-actin: forward primer 5′-GGGACCTGACTGACTACCTCA-3′ and reverse primer 5′-GACTCGTCATACTCCTGCTTG-3′. Commercial software (SDS version 1.3; ABI) was used to calculate 2^-ΔΔCt relative expression values for ROCK-1 and ROCK-2 normalized to the β-actin endogenous control.

Y79 cells transfected with the siRNA were incubated for 48 hours. Cells were harvested for western blots analysis. Y79 cells were lysed for 5 minutes in cold lysis buffer. After centrifugation at 12,000 rpm for 5 minutes, the supernatant was collected as the total cellular protein extracts. The protein concentration was determined using Bradford method [15]. The total cellular protein extracts were separated on 8% SDS–PAGE. Proteins were electrotransferred to PVDF membranes (Millipore, USA) by a semi-dry transferor. The membranes were blocked in 5% skimmed milk in TBS-T containing 0.05% Tween 20 at room temperature (RT) for 2 hours, and then incubated at RT for 2 hours with antibodies to ROCK-1, ROCK-2, LIMK2, phospho-LIMK1 Thr508/LIMK2 Thr505, cofilin, phospho-cofilin Ser3 (1:1000, Cell Signaling Technology, Beverly, MA), and β-actin(1:5000, Cell Signaling Technology, Beverly, MA) diluted in 5% bovine serum albumin in TBS-T, respectively, followed by incubating with the appropriate HRP-linked secondary antibodies. Finally, the bands of specific proteins on the membranes were developed with Western Blotting Luminal Reagent (Millipore, USA) and quantified with Image J software (National Institutes of Health, USA).

Cell proliferation was detected by a Cell Counting Kit-8 (Beyotime, Jiangsu, China) assay. Y79 cells were suspended in RPMI 1640 medium supplemented with 15% heat-inactivated fetal bovine serum and subsequently seeded in 96-well plates and incubated for 24 h. After that, we added medium containing Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)- cyclohexanecarboxamide.2HCl, purchased from Enzo Life Sciences, Inc.) in 10, 50, 100 μM, respectively for 48 hours. Then the cultures were added 10 μl CCK-8 solution to each well and incubated at 37°C for another 2 hours. Optical density (OD) value of absorbance at 450 nm was measured by Thermo Scientific Fluoroskan Ascent FL (Thermo Fisher Scientific Inc.). The results were plotted as means ± SD of three independent experiments having three determinations per sample for each experiment.

The effects of Y-27632 on the adhesion ability of Y79 cells to ECM were examined using the adhesion assay. The binding of Y79 cells to matrigel were investigated. A 96-well plate was coated with matrigel (2 ug/50 μl). Cells (4 × 10⁵ cells, cultured in RPMI 1640 medium supplemented with 10% FBS) were seeded onto these components in 96-well Plate. Y-27632 was diluted at the concentration of 10 μM, 20 μM, 30 μM, 50 μM, 100 μM. The cells were treated for 1 h. They were allowed to adhere to each well for 30 min at 37°C and then gently washed twice in PBS. The adhesion Y79 cells were quantified by the Cell Counting Kit-8 assay according to the manufacturer’s instructions.

To study the effects of ROCK siRNA on cell adhesion, three groups of Y79 cells were transfected for 24 hr with control siRNA, ROCK-1 siRNA, or ROCK-2 siRNA, respectively. The cells in the forth group were treated with Y-27632 at 30 μM. Untransfected cells and cells transfected with control siRNA served as controls. Cells (4 × 10⁵ cells) were allowed to adhere to each well for 30 min at 37°C and then gently washed twice in PBS. The adhesion Y79 cells were quantified by the Cell Counting Kit-8 assay.

The 24-well plate Transwell system with a polycarbonate filter membrane of 8-mm pore size (Corning Inc.) was used to detect invasive capacity changes of Y79 cells. The Matrigel (BD Biosciences) layers were rehydrated for 2 hours at RT by adding 100 μL of serum-free media to the upper compartment. After rehydration, the media were removed from the upper compartment of the invasion chamber. Cell suspension containing 1 × 10⁶ cells/mL was prepared in serum-free media, 400 μl RPMI 1640 medium supplemented with 15% FBS were added to each of the lower compartments and 100 μL of the cell suspension were added to each of the upper compartments. After incubating for 48 hours at 37°C, invasive cells in lower compartments of the chamber were counted by Cell Counting Kit-8 assay.

The six groups of Y79 cells were treated with Y-27632 at 0, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, respectively. After incubating for 48 h at 37°C, invasive cells in lower compartments of the chamber were counted by Cell Counting Kit-8 assay. To study the effects of ROCK siRNA on cell invasion, three groups of Y79 cells were transfected for 24 hr with control siRNA, ROCK-1 siRNA, or ROCK-2 siRNA, respectively. The cells in the forth group were treated with Y-27632 at 30 μM. Untransfected cells and cells transfected with control siRNA served as controls. After incubating for 48 h, invasive cells in lower compartments of the chamber were counted by Cell Counting Kit-8 assay.

ROCK-1 and ROCK-2 mRNA expression in Y79 cell lines were examined by RT-qPCR.

The expression of mRNA of ROCK-1 was about twice as much as that of ROCK-2 ( 2^-ΔΔCt = 0.48 ± 0.03, P < 0.05) (Figure 1A). Treatment of Y79 cells with ROCK-1 siRNA for 24 hours reduced ROCK-1 mRNA levels against blank controls (2^-ΔΔCt = 0.38 ± 0.03, P < 0.05) and negative controls (2^-ΔΔCt = 0.40 ± 0.02, P < 0.05) (Figure 1B). Treatment of Y79 cells with ROCK-2 siRNA for 24 hours reduced ROCK-2 mRNA levels against blank controls (2^-ΔΔCt = 0.45 ± 0.05, P < 0.05) and negative controls (2^-ΔΔCt = 0.48 ± 0.05, P < 0.05) (Figure 1C).

Protein expression in Y79 cell line that received siRNA treatment for ROCK-1 and ROCK-2 were examined by using western blot analyses. Cells transfected with control siRNA were used as controls. The protein expression levels of ROCK-1 and ROCK-2 were reduced in the cells transfected with siRNA against ROCK-1 and ROCK-2, respectively, compared with the control cells (Figure 2).

To investigate the effects of ROCK inhibition on cellular proliferation of Rb cells, cell proliferation was evaluated by CCK-8 assay after exposure to various Y-27632 doses. Y-27632 treatment didn’t show any changes of Y79 cells proliferation (P > 0.05) (Figure 3). These results showed that ROCK inhibitor Y-27632 didn’t influence proliferation of Rb cell in vitro.

The Y-27632 dosage-dependent effect on Y79 cell adhesion capacity was studied. The adhesion capacity showed a positive correlation with increasing Y-27632 concentration (Figure 4A). The OD value of cells transfected with ROCK-1 siRNA were increased, comparing with blank and negative control group (P < 0.05) (Figure 4B), but it was similar to cells treated with Y-27632 at 30 μM (P > 0.05). There was no statistical difference between the cells transfected with ROCK-2 siRNA and negative control group (P > 0.05).

The Y-27632 dosage-dependent effect on Y79 cell invasion capacity was studied. After 48 hours incubation at 37°C, the invasion capacity showed an inverse relationship with increasing Y-27632 concentration (Figure 5A). The OD value of cells transfected with ROCK-1 siRNA were decreased, comparing with blank and negative control group (P < 0.05) (Figure 5B), but it was similar to cells treated with Y-27632 at 30 μM (P > 0.05). There was no statistical difference between the cells transfected with ROCK-2 siRNA and negative control group (P > 0.05).

We investigated the effects of Y-27632 and ROCK siRNA on the ROCK/LIMK2/cofilin signaling pathway by Western blot analysis. As we expected, Y-27632 or ROCK-1 siRNA could inhibit the levels of LIMK2 and cofilin phosphorylation in Y79 cells (Figure 6).