Downregulation of NIMA-related kinase-7 inhibits cell proliferation by inducing cell cycle arrest in human retinoblastoma cells

Cell lines and culture

A human retinoblastoma cell line Y79 (cat. no. HTB-18™) and a normal retinal pigment epithelium (RPE) cell line (cat. no. CRL-4000™) were purchased from American Type Culture Collection (Manassas, VA, USA). The human retinoblastoma cell line SO-Rb50 (cat. no. TCHu213; Cell Bank of Chinese Academy of Science, Shanghai, China) was obtained from the Department of Pathology of the Zhongshan Ophthalmic Center, Sun Yat-sen University (Guangzhou, China). The human retinoblastoma cell line WERI-RB1 (cat. no. TCHu213) and 293T cells (cat. no. GNHu17) were purchased from the Cell Bank of Chinese Academy of Science. 293T cells were cultured in Dulbecco's modified Eagle's medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 100 µg/ml penicillin/streptomycin. Human retinoblastoma and RPE cells were cultured in RPMI 1640 (Hyclone; GE Healthcare Life Sciences) medium supplemented with 10% FBS. All cells were maintained at 37°C in a humidified incubator with 5% CO₂.

RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from human retinoblastoma cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocols. All RNA was quantified by using the Nanodrop spectrophotometer ND-2000 (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Only those RNA samples with 260/280 ratios of 1.8–2.0 were used for further investigation. Then, cDNA was synthesized using M-MLV Reverse Transcriptase (cat. no. 28025013; Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocols. Next, a SYBR GreenER™ qPCR SuperMix Universal kit (cat. no. 11762-100; Invitrogen, Thermo Fisher Scientific, Inc.) was used to determine the mRNA level of Nek7 in human retinoblastoma and RPE cells using a CFX96 Touch™ Real-Time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Primers used for amplification were as follows: Nek7 (forward), 5′-CACCTGTTCCTCAGTTCCAAC-3′; Nek7 (reverse), 5′-CTCCATCCAAGAGACAGGCTG-3′; β-actin (forward), 5′-GTGGACATCCGCAAAGAC-3′; and β-actin (reverse), 5′-AAAGGGTGTAACGCAACTA-3′. The PCR protocol was as follows: Initial denaturation at 95°C for 60 sec, 40 cycles of denaturation at 95°C for 5 sec, annealing and extension at 60°C for 20 sec. The relative Nek7 expression levels were calculated using the 2^−ΔΔCq method (19). β-actin was used as an internal control.

Western blot analysis

Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed with radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40 and 0.1% SDS) containing 1 mM phenylmethane sulfonyl fluoride, a serine/cysteine protease inhibitor (cat. no. ST506; Beyotime Institute of Biotechnology, Haimen, China). The supernatant was obtained for protein concentration determination by the BCA Protein Assay kit (cat no. 23235; Pierce; Thermo Fisher Scientific, Inc.). Samples of equal amount of protein were separated by 10–12% SDS-PAGE, and the resulting bands were transferred to polyvinylidene difluoride microporous membranes (EMD Millipore, Billerica, MA, USA). The membrane were blocked in 5% non-fat milk in Tris-buffered saline with Tween-20 (TBS-T) buffer for 1 h and then incubated overnight at 4°C with the following primary antibodies: Rabbit anti-Nek7 (cat. no. 3057; 1:1,000 dilution), cyclin D1 (cat. no. 2978; 1:1,000 dilution), cyclin-dependent kinase 2 (CDK2; cat. no. 2546; 1:1,000 dilution), cyclin E (cat. no. 20808; 1:1,000 dilution) and GAPDH (cat. no. 2118; 1:4,000 dilution) (all Cell Signaling Technology, Inc., Danvers, MA, USA). Subsequently, samples were washed three times with TBS-T buffer and incubated with goat anti-rabbit secondary antibodies conjugated with horseradish peroxidase (cat. no. 7074; 1:5,000 dilution; Cell Signaling Technologies, Inc.) for 1 h at 37°C. Signal intensity was detected using an ECL detection system (Pierce; Thermo Fisher Scientific, Inc.). GAPDH was used as the reference control.

Construction of lentivirus vector and transduction of retinoblastoma cells

The stem-loop-stem oligos were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), including two Nek7 shRNAs (shNek7-1, 5′-GGAUGAGCAAUCACAAGGA-3′; and shNek7-2, 5′-GCUAAUUCCUGAAAGAACU-3′) and a negative control shRNA (shNC), 5′-CGUCAGAGUAUACUAAUAU-3′. These oligos were annealed and ligated into the pLKO.1 vector (Addgene, Inc., Cambridge, MA, USA) according to the instructions provided by the manufacturer. Lentiviruses were generated by triple transfection of 80% confluent 293T cells with 8 µg shNek7-1/pLKO.1, shNek7-2/pLKO.1 or shNC/pLKO.1, along with packaging pHelper plasmids (pVSVG-I and pCMVΔR8.92) using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturer's procedure. The supernatant was collected 48 h after transfection and lentiviral particles were harvested by ultracentrifugation (speed, 4,000 × g) at 4°C for 10 min followed by filtration through a 45 µm filter, and the titer of lentiviruses was measured as previously described (20).

For cell transfection, the human retinoblastoma cell lines Y79 and SO-RB50 were transfected using Lipofectamine 2000 with 5 µg constructed lentiviruses containing shNek7-1, shNek7-2 or shNC, respectively at 37°C in a humidified incubator with 5% CO₂. After 96 h of transfection, cells were collected and the transfection efficiency was determined by examining the green fluorescence protein (GFP) expression (Addgene, Inc.) through a fluorescence microscope at ×100 magnification, while the knockdown efficiency was determined by RT-qPCR and western blot analysis.

Cell viability assay

Cell growth curves were determined using the MTT method. Briefly, following transfection as described earlier, Y79 and SO-RB50 cells were reseeded in 96-well plates at a density of 3,000 cells/well. The medium containing 0.5 mg/ml MTT was added into each well and then incubated at 37°C in a humidified 5% CO₂ atmosphere for 4 h. This was followed by the addition of 150 µl solubilization solution (0.01 M HCl, 10% SDS and 5% isopropanol) to each well and incubation of cells for a further 10 min at 37°C with gentle shaking. The optical density of the plates was measured using an Epoch Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA) at an absorbance of 595 nm.

Colony formation assay

Y79 and SO-RB50 cells were reseeded in 6-well plates at a density of 500 cells/well after 4 days of lentivirus transfection. Media were replaced every 3 days. After 8 days of culture, the cells were washed with PBS and fixed with PBS containing 4% paraformaldehyde for 30 min at room temperature. Next, fixed cells were stained with 1% crystal violet (cat no. C0121; Beyotime Institute of Biotechnology) for 10 min, gently washed with ddH₂O and air dried. Images of the 6-well plates were captured with a camera, and visible colonies with >50 cells were counted under a microscope. Image analysis was conducted using Image-Pro Plus version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Cell cycle analysis

After 4 days of lentivirus transfection, Y79 and SO-RB50 cells (1×10⁶ cells per well) were harvested and washed twice with cold PBS, followed by fixation with ice-cold 70% ethanol overnight at 4°C. After washing twice with PBS containing 0.5% Triton X-100, the cells were incubated with 50 µg/ml propidium iodide (PI) and 50 µg/ml RNaseA (both BD Biosciences, San Jose, CA, USA) for 30 min at room temperature. The cells were then analyzed using a fluorescence-activated cell sorting (FACS) system with a FACSCalibur with CellQuest software (version 5.1) (both BD Biosciences). The data were analyzed using the ModFit software (version 4.0; Verity Software House, Topsham, ME, USA).

Statistical analysis

All observations were confirmed by at least three independent experiments. Quantitative data are expressed as the mean ± standard deviation. Two-tailed Student's t-test was performed for paired samples. P<0.05 was considered to indicate a statistically significant difference.

Nek7 is abnormally upregulated in human retinoblastoma cell lines

To investigate the biological function of Nek7 in human retinoblastoma, the expression of Nek7 was examined in several human retinoblastoma cell lines and normal RPE cells. The results identified that the mRNA expression level of Nek7 was commonly upregulated in human retinoblastoma cell lines (Y79, SO-RB50 and WERI-RB1), as compared with that in RPE cells (Fig. 1A; P<0.001). To further confirm this finding, the protein level of Nek7 was determined by western blot analysis. Consistently, the Nek7 protein level was higher in retinoblastoma cell lines compared with that in normal RPE cells (Fig. 1B). Thus, these results suggested that Nek7 may be involved in retinoblastoma development. Notably, Y79 and SO-RB50 cells that demonstrated higher Nek7 expression were selected for subsequent analysis.

Lentivirus-mediated shRNA transfection strongly suppresses Nek7 expression in Y79 and SO-RB50 cells

The Y79 and SO-RB50 cells were transfected with shNC, shNek7-1 or shNek-2 lentivirus particles. As shown in Fig. 2A, the majority of cells presented GFP-positive signals following shRNA transfection (with shNC, shNek7-1 and shNek7-2), indicating satisfying transfection efficiency. In order to verify that the Nek7 gene was silenced by the lentivirus, the mRNA and protein levels in Y79 and SO-RB50 cells were assessed using RT-qPCR and western blot assays, respectively. In Y79 cells, the mRNA and protein levels of Nek7 were significantly decreased in shNek7-1 and shNek-2 groups compared with the shNC group (Fig. 2B and C). Similarly, the expression of Nek7 was strongly reduced in shNek7-1 and shNek-2 groups compared with the shNC group in SO-RB50 cells (Fig. 2D and E). These results suggest that lentivirus-mediated Nek7 shRNA significantly downregulated Nek7 expression in Y79 and SO-RB50 cells. Notably, the knockdown efficacy of the shNek7-1 vector was more evident in comparison with that of shNek-2 in the Y79 and SO-RB50 cells.

Knockdown of Nek7 inhibits proliferation and colony formation of Y79 and SO-RB50 cells

To investigate the effect of Nek7 knockdown on cell growth, Y79 and SO-RB50 cells transfected with shNC, shNek7-1 and shNek-2 were subjected to MTT assay The results revealed that the viability of Y79 cells was markedly decreased, with the proliferation ability decreasing by 73.3% in the shNek7-1 group and 40.2% in the shNek7-2 group (P<0.001, Fig. 3A) at 5 days after transfection. Furthermore, it was observed that the proliferation ability reduced by 75.9% in shNek7-1-transfected and 52.6% in shNek7-2-transfected SO-RB50 cells (P<0.001; Fig. 3B) at 5 days. Given the aforementioned findings, it is suggested that shNek7-1 had a more powerful effect on inhibiting the cell proliferation ability as compared with shNek7-2.

The colony formation ability of Y79 and SO-RB50 cells was also determined in order to gain additional insight into the effect of shNek7-1 on cell proliferation. For Y79 cells, shNek7-1 resulted in smaller colony numbers compared with the shNC group (Fig. 3C and D; P<0.01). Similarly, smaller colony numbers were identified in SO-RB50 cells transfected with shNek7-1 (Fig. 3E and F; P<0.001). These results indicated that silencing Nek7 may inhibit the cell proliferation and colony formation abilities of Y79 and SO-RB50 cells, which correlates with the formation of human retinoblastoma.

Knockdown of Nek7 arrests the cell cycle progression of Y79 and SO-RB50 cells

To examine whether the growth suppression effect of Nek7 knockdown was associated with cell cycle regulation, flow cytometry with PI staining for cell cycle distribution profiles was performed in Y79 and SO-RB50 cells transfected with shNek7-1 (Fig. 4A). As demonstrated in the FACS analysis in Fig. 4B, the proportion of cells in the S phase was markedly decreased (from 27.16 to 18.24%; P<0.001), whereas the proportion of cells was increased in the G0/G1 (from 61.40 to 69.72%; P<0.001) and G2/M (from 11.43 to 12.04%) phases in Y79 cells transfected with shNek7-1 compared with the shNC group. A similar phenomenon was also observed in SO-RB50 cells transfected with shNek7-1, in which the proportion of cells in the S phase was significantly reduced (from 17.58 to 13.11%; P<0.01), while the proportion of cells was markedly enhanced in the G0/G1 (from 61.79 to 64.03%; P<0.01) and G2/M (from 20.63 to 22.87%; P<0.001) phases, compared with the shNC group (Fig. 4). These results suggest that the growth suppression mediated by shNek7-1 may be, in part, through arrest of the cell cycle in G0/G1 phase.

Knockdown of Nek7 modifies the expression of cell cycle-associated proteins

In order to further clarify the mechanism underlying the antiproliferative effect of Nek7 knockdown, several downstream cell cycle-associated regulators in Y79 and SO-RB50 cells were analyzed by western blot assay. The data revealed that the cell cycle checkpoint kinase CDK2 was clearly decreased in Y79 cells transfected with shNek7-1. In addition, cyclin D1 and cyclin E, which function as regulators of CDKs, were evidently decreased by silenced Nek7 (Fig. 5A). Similar results were also observed for SO-RB50 cells (Fig. 5B). These results clearly indicated that Nek7 silencing may inhibit the proliferation of Y79 and SO-RB50 cells by downregulating the expression of cell cycle-associated proteins.