HEK293T and normal human skin keratinocytes (HaCaT) cells were obtained from the China Center for Type Culture Collection (Wuhan, China). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (Invitrogen, Karlsruhe, Germany) and incubated in a humidified 37°C, 5% CO₂ incubator. The primary antibody of P12^CDK2AP1 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). GAPDH antibody was purchased from Millipore (Billerica, MA, USA). SYBR-Green PCR Master mix for the quantification of CDK2AP1 mRNA was obtained from Takara (Kyoto, Japan) and oligonucleotides from Sangon (Shanghai, China).

The sequence of short hairpin RNA (shRNA) targeting the human CDK2AP1 gene was 5′-GATCTCAGTTCTGCGT TTA-3′, corresponding to the coding region positions of CDK2AP1 mRNA. The control plasmid was obtained from GenePharma Co., Ltd. (Shanghai, China). The human U6 promoter was amplified from genomic DNA and cloned in the AgeI and EcoRI sites of the pGCSIL-GFP plasmid (GeneChem, Shanghai, China). The oligonucleotides which were previously shown to induce RNAi against CDK2AP1 were cloned downstream of the U6 promoter. The sequence of the oligonucleotide (top strand) was: 5′-CcggtaGATCTCAGT TCTGCGTTTATTCAAGAGATAAACGCAGAACTGAGATCta-TTTTTg-3′. The underlined sequence corresponds to nucleotides of human open reading frame for CDK2AP1. The sequence of the construct was confirmed by automated fluorescent sequencing.

A self-inactivating lentivirus vector containing a cytomegalovirus (CMV)-driven enhanced green fluorescent protein (EGFP) reporter and a U6 promoter upstream of cloning restriction sites (AgeI and EcoRI) was purchased from GeneChem Co., Ltd. A control shRNA unrelated to human gene sequences was used as a negative control. We constructed 3 si-CDK2AP1 lentiviruses, KD-1, KD-2 and KD-3, targeting human CDK2AP1 mRNA. The following custom primers containing AgeI and EcoRI sites at their 5′ termini were used for PCR amplification: 5′-CCTATTTCCCATGATTCCT TCATA-3′ and 5′-GTAATACGGTTATCCACGCG-3′. The resulting PCR fragment was digested with AgeI and EcoRI and cloned into AgeI- and EcoRI-digested pLenti vector. Oligonucleotides for constructing the negative control and 3 si-CDK2AP1 lentiviruses were synthesized from Sangon. Correct insertions of shRNA cassettes were confirmed by restriction mapping and direct DNA sequencing. KD-3 was identified as the most efficient and was selected for this study.

Cells were replated at 5×10³ cells/well in 96-well plates along with recombinant lentivirus encoding for shRNA against CDK2AP1 at different multiplicity of infections (MOIs) in serum-free growth medium containing 5 μg/ml polybrene at 37°C and 5% CO₂. After 4 h, serum-containing growth medium was added to the cells, and there was complete replacement of growth medium after 48 h. After 3 days of post-infection, reporter gene expression was examined using fluorescent microscopy.

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The expression of CDK2AP1 mRNA was detected using the SYBR-Green miRNA assay (Takara) (13), and normalized using the 2^−ΔΔCt method (14) relative to human β-actin (Sangon). Primer sequences and the size of the products are shown in Table I. To ensure specificity of the PCR product amplification, the melting curves for standard and sample products were analyzed. All the real-time RT-PCR assays were performed three times.

Cells were seeded in 6-well plates and infected after 12 h. The cells were harvested 3 days following infection, washed once in phosphate-buffered saline (PBS) and lysed. Protein concentration was determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). Aliquots (60 μg) were separated on a 15% SDS-PAGE and transferred to PVDF membrane. The membrane was incubated with the specific antibody followed by peroxidase-conjugated secondary antibodies. An enhanced chemiluminescence detection solution was then applied (Pierce). The relative protein level in different groups was normalized to the signal intensity of GAPDH. Quantitative analysis of the blots was performed using the NIH-ImageJ program.

HaCaT cells were seeded at a density of 2×10³ cells/well in 96-well plates containing 0.2 ml DMEM (with 7% FBS) and cultured for 9 days. During this period, the medium was replaced with fresh complete medium every 3 days. Six wells from each group were selected randomly for the MTT assay each day. After 4 h of incubation, the reaction was stopped by adding 150 μl of dimethyl sulfoxide (DMSO; Sigma) to each well, followed by a 10-min incubation. The percentage of viable cells was determined by measuring the absorbance at 490 nm on a multiscanner reader (TECAN-spectra mini; Tecan Austria GmbH, Grödig, Austria). Cell growth curves were drawn by using average absorbance at 490 nm from 3 independent experiments.

The cells (72 h) were harvested, washed twice with ice-cold PBS, fixed with 70% ethanol overnight at 4°C, washed and resuspended in 100 μl of PBS containing a final concentration of 50 μg/ml RNase A for 30 min at room temperature. The cells were then stained with 20 μg/ml propidium iodide in a final volume of 300 μl for 20 min. DNA content and cell cycle were analyzed using a flow cytometer (FACSCalibur; Becton-Dickinson, Franklin Lakes, NJ, USA), and ModFit and CellQuest software. The experiments were conducted three times.

The experiments were performed in triplicate and standard deviations were calculated. The statistical analysis was performed by a two-tailed paired Student’s t-test or one-way ANOVA using SPSS 11.5 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To determine the lentiviral infection efficiency in HaCaT cells, EGFP expression was examined by microscopy at different MOIs 3 days after infection. The efficiency of lentiviral vector infection in HaCaT cells was >90% at an MOI of 40 (Fig. 1). The percentage of EGFP-expressing cells remained relatively unchanged when MOI was >40. Based on these results, an MOI of 40 was chosen. Our data suggested that lentivirus shRNA vector pGCSIL-GFP had high efficiency for infecting HaCaT cells.

The subconfluent cells were infected with either negative control or shCDK2AP1 lentivirus. Two days post-infection, the cells were collected and lysed for analysis. Real-time RT-PCR results showed the mRNA level of CDK2AP1 in HaCaT cells infected with lenti-shCDK2AP1 to be ~80% lower compared to that of HaCaT control cells (Fig. 2A). As shown in Fig. 2B and C, the level of P12^CDK2AP1 expression was markedly reduced in HaCaT cells infected by lenti-shCDK2AP1 compared to that in the control cells, a fact which was consistent with the downregulation of CDK2AP1. This study indicated that lentiviral siRNA was able to provide highly efficient and specific CDK2AP1 knockdown in HaCaT cells.

To determine whether silencing P12^CDK2AP1 by RNAi had a stimulative effect on HaCaT cell growth, determination of cell proliferation was performed using the MTT assay. After 3 days of infection, lenti-shCDK2AP1 did not positively affect HaCaT cell proliferation. However, lenti-shCDK2AP1 significantly increased HaCaT cell proliferation 4–9 days following infection (Fig. 3A). Notably, the suppression of P12^CDK2AP1 led to HaCaT cell growth promotion in a time-dependent manner and this stimulative effect was not observed before day 3.

The different proliferation rates of HaCaT cells infected with lenti-shCDK2AP1 vs. control HaCaT cells may partly be due to differences in cell cycle regulation. Therefore, cell cycle distribution was detected using flow cytometry. On day 7, a lower number of HaCaT cells infected by lenti-shCDK2AP1 were arrested in the G0/G1 compared to the S and G2/M phases of the cell cycle (Fig. 3B).