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Introduction

Leukemia is the most common malignancy in children (0–14 years) and young adults (15–39 years). According to GLOBOCAN 2012 data, 351,965 new cases of leukemia were diagnosed in 2012 (2.5% of total cancer cases), and leukemia accounts for ~265,471 cases of cancer-associated mortality worldwide every year. Among these newly diagnosed cases, 30% (105,436/351,965 cases) occurred in the USA and China (1). In addition, leukemia has the highest incidence and mortality rate of any cancer in children in China. The majority of cases of leukemia are acute myeloid leukemia (AML). AML represents a heterogeneous hematological malignancy characterized by uncontrollable proliferation and survival, and impaired differentiation of neoplastic cells or blasts (2). In the USA, 19,950 cases of AML and 10,430 cases of AML-associated mortality are projected in 2016 (3). AML is often fatal, due to the following reasons: Firstly, the majority of cases of AML initially respond to traditional chemotherapy (4); however, relapse is common indicating resistance of malignant cells to chemotherapy (4–7); and secondly, the acuity and severity of AML is often poor at diagnosis. Recently, studies have focused on identifying novel genes, which target tumor cell growth and survival signaling pathways (8–11). Identification of these genes may provide a novel strategy for the optimization of AML therapy or diagnosis (12).

The ribosome is comprised of two ribonucleoprotein subunits: 40S and 60S, which are known as the 'small' and 'large' subunits, respectively. It has previously been reported that ribosomal proteins exhibit extra-ribosomal functions, which include, but are not limited to, DNA repair, cell death, inflammation, tumorigenesis and transcriptional regulation (13). Ribosomal protein S15a (RPS15A), which is a component of the 40S ribosomal subunit, is able to promote mRNA/ribosome interaction during the early stage of translation. RPS15A has been shown to suppress the cdc33 mutation growth arrest phenotype by binding and stabilizing eukaryotic initiation factor 4E (14), thus indicating that RPS15A has a function in cell growth regulation. In addition, RPS15A has been identified as a novel internal control gene for use in quantitative polymerase chain reaction (qPCR) analysis of prepubertal bovine mammary tissue (15). Akiyama et al (16) demonstrated that RPS15A is upregulated in response to transforming growth factor-β in the A549 human lung carcinoma cell line, which has a central role in the regulation of proliferation, differentiation, apoptosis and carcinogenesis. Furthermore, Zeller et al (17) identified RPS15A as a responsive gene of the Myc oncogenic transcription factor. Krüppel-like factor 4 is a tumor suppressor (18), which is able to inhibit the GC content of the RPS15A promoter to downregulate its expression (19). In addition, RPS15A was shown to be overexpressed in hepatitis B virus-encoded X antigen-positive cells, and overexpression of RPS15A stimulated cell growth, colony formation and tumor formation in SCID mice in vivo (20), thus indicating that RPS15A may have a key role in hepatocellular carcinogenesis. A meta-analysis of cancer gene expression signatures revealed that RPS15A is highly expressed in astrocytoma, colorectal cancer and prostate cancer (21).

Despite reports indicating that RPS15A may stimulate growth in yeast, plants and human cancer (22), its functional role in AML remains unknown. The present study demonstrated that knockdown of RPS15A by lentivirus-mediated short hairpin RNA (shRNA) was able to inhibit AML cell proliferation and induce apoptosis in vitro. Furthermore, RPS15A knockdown suppressed cell cycle progression via G0/G1 phase arrest. Collectively, RPS15A may modulate AML cell growth and have a prominent role in AML; therefore, RPS15A may be considered a potential therapeutic target for the treatment of AML.

Materials and methods

Cell lines and culture conditions

Human embryonic kidney 293T (HEK 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; Biowest SAS, Nuaillé, France). Human acute myeloid leukemia (AML) U937 cells were cultured in RPMI-1640 (Hyclone; GE Healthcare Life Sciences) supplemented with 10% FBS. The cell lines were purchased from the Cell Bank of Chinese Academy of Science (Shanghai, China), and were maintained at 37°C in a humidified incubator containing 5% CO2.

Preparation of shRNA-expressing lentiviruses and cell infection

Two targeted sequences (S1, S2) designed to be homologous to RPS15A were cloned into the lentiviral expression vector pFH-L (Shanghai Hollybio, Shanghai, China), which was digested by NheI and PacI restriction enzymes (Takara Bio, Inc., Otsu, Japan). The sequences of the two shRNA targeting RPS15A were as follows: S1, 5′-GTGCAACTCAAAGACCTGGAACTCGAGTTCCAGGTCTTTGAGTTGCACTTTTT-3′ and S2, 5′-GCATGGTTACATTGGCGAATTCTCGAGAATTCGCCAATGTAACCATGCTTTTT-3′ (Shanghai Hollybio). The lentiviruses containing S1 and S2 sequences were classified as Lv-shRPS15A (S1) and Lv-shRPS15A (S2), respectively. As a negative control, a scrambled shRNA was used (Shanghai Hollybio), which had the following sequence: 5′-GCGGAGGGTTGAAAGAATATCTCGAGATATTCTTTCAAACCCTCCGCTTTTTT-3′. This sequence was classified as Lv-shCon. The uninfected control cells were classified as Con. Each single-stranded oligonucleotide became a double-stranded oligonucleotide using an annealing system. Briefly, the annealing reaction mixture, containing 5 µl each of forward and reverse primers (Shanghai Hollybio), 10 µl 5X annealing buffer (Beyotime Institute of Biotechnology, Shanghai, China) and 30 µ1 double-distilled H2O (ddH2O), was run on a Bioer TC-96/G/H(b)A (BIOER, Hangzhou, China) thermal cycler with the following program: Initial denaturation at 94°C for 1 min, followed by 50 cycles of denaturation at 80°C for 30 sec and extension at 30°C for 30 sec, followed by cooling to 4°C at the end of the PCR. Subsequently, the double-stranded oligonucleotide was cloned into the linearized pFH-L vector.

The ligation product was used to transform the Escherichia coli DH5α strain, and was extracted using a plasmid purification kit [Tiangen Biotech (Beijing) Co., Ltd., Beijing, China]. The plasmid was confirmed by PCR and sequencing. The shRNA-expressing lentiviruses were produced by co-transfection of 10 µg recombinant expression shRNA vectors and packaging pHelper plasmids (7.5 µg pVSVG-I and 5 µg pCMVΔR8.92; Shanghai Hollybio) into the HEK 293T cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. Supernatants containing either the lentivirus expressing the RPS15A shRNA or the control shRNA were harvested 24, 48 and 72 h post-transfection. The lentiviruses were purified using ultracentrifugation, and the titer of the lentiviruses was determined as previously described (23). U937 cells were infected with the concentrated virus at a multiplicity of infection of 80 and mock-infected cells were used as negative controls. After 96 h of infection, the expression of green fluorescent protein was observed using a fluorescent microscope (CKX41; Olympus Corporation, Tokyo, Japan) in order to assess the infection efficiency. The efficiency of RPS15A knockdown was evaluated by reverse transcription (RT)-qPCR and western blot analysis.

RNA isolation and RT-qPCR

U937 cells were harvested 5 days post-lentiviral infection. Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The purity and integrity of the RNA was assessed by spectrophotometry (Epoch Microplate Spectrophotometer; Biotek Instruments, Inc., Winooski, VT, USA) and 3% agarose gel electrophoresis, respectively. First-strand cDNA was synthesized from 2 µg total RNA using RT reagents containing 1 µl Oligo dT (0.5 µg/µl), 4 µl M-MLV Buffer, 1.25 µl dNTPs, 0.5 µl RNasin, 0.75 µl M-MLV-RTase and Nuclease-free water, to a final volume of 20 µl (Promega Corporation, Madison, WI, USA). PCR primers (Shanghai Hollybio) were designed to amplify fragments that span intron/exon boundaries and the sequences were as follow: RPS15A, forward 5′-TGACGTGCAACTCAAAGACC-3′, reverse 5′-CCAGAGTCCATGAGGCATT-3′; β-actin, forward 5′-GTGGACATCCGCAAAGAC-3′, and reverse 5′-AAAGGGTGTAACGCAACTA-3′. RT-qPCR was performed in the linear range using the SYBR Green Core Reagents kit (Takara Bio, Inc.) on a Bio-Rad CFX96 Touch™ Real-Time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR reaction mixture consisted of 5 µl cDNA, 10 µl 2X SYBR Premix Ex Taq (Takara Bio, Inc.), 0.8 µl PCR primers (2.5 µM) and 4.2 µl ddH2O. The PCR cycling conditions were as follows: Initial denaturation at 95°C for 60 sec, 45 cycles of denaturation at 95°C for 5 sec, annealing and extension at 60°C for 20 sec, followed by cooling to 4°C. The absorbance values were read at the extension stage. β-actin was used as the internal control for all normalizations and the relative expression levels were calculated using the 2−ΔΔCq method (24).

Western blot analysis

U937 cells were harvested 5 days post-lentiviral infection, were washed twice with ice-cold phosphate-buffered saline (PBS), and were lysed in ice-cold 2× sodium dodecyl sulfate (SDS) Lysis Buffer [100 mM Tris-HCl (pH 6.8), 10 mM EDTA, 4% SDS, 10% Glycine]. The protein concentration of the cell lysates was determined using the bicinchoninic acid protein assay kit (Thermo Scientific Pierce, Rockford, IL, USA). Total protein samples (30 µg) were separated by 10% SDS-polyacrylamide gel electrophoresis and were transferred onto nitrocellulose membranes. The membranes were blocked with Tris-buffered saline and Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) containing 5% non-fat milk at room temperature for 2 h, after which they were incubated with the following antibodies: Anti-RPS15A (1:1,000; cat. no. AP4804a; Abgent, San Diego, CA, USA) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:40,000; cat. no. 10494-1-AP; Proteintech Group, Inc., Chicago, IL, USA) at 4°C overnight. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000; cat. no. sc-2054; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Signals were detected using an enhanced chemiluminescence test kit (Amersham; GE Healthcare Life Sciences, Chalfont, UK). GAPDH served as the internal standard. Density analysis was performed using Quantity One software, version 4.62 (Bio-Rad Laboratories, Inc.).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay-growth curve

In vitro cell viability was analyzed using the MTT assay. U937 cells were seeded at a density of 3,500 cells/well in 96-well plates 96 h post-lentiviral infection. After 24 h, 20 µl 5 mg/ml MTT solution (Sigma-Aldrich) was added to each well daily between days 1 and 5, and the plates were incubated for 4 h at 37°C. Subsequently, 100 µl stop buffer (0.012 M HCl, 10% SDS, 5% isopropanol) was added to each well and gently agitated for 10 min. Absorbance values were measured at a wavelength of 595 nm using the Epoch Microplate Spectrophotometer.

Cell cycle analysis

To determine cell cycle distribution, 8×104 U937 cells were seeded in 6 cm dishes a total of 6 days after lentiviral infection. Following a 40 h culture, the cells were washed twice with ice-cold PBS and were resuspended in PBS containing 50 µg/ml RNase A (Sigma-Aldrich) and 50 µg/ml propidium iodide (Sigma-Aldrich). Cells were incubated at 37°C in the dark for 1 h. The percentage of cells in each phase of the cell cycle was measured using FACScan (BD Biosciences, San Diego, CA, USA) and results were analyzed using ModFit software, version 3.2 (Verity Software House, Topsham, ME, USA).

Apoptosis analysis

To assess the apoptotic rate, 1×105 U937 cells were seeded in 6 cm dishes. Apoptosis was detected 6 days following lentiviral infection. Cells were harvested and the experiment was conducted according to the Annexin V-allophycocyanin (APC)/7-aminoactinomycin D (7-AAD) Apoptosis Assay kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). A total of 1×106 cells were resuspended in 100 µl 1X Annexin V binding buffer with 5 µl Annexin V-APC and 5 µl 7-AAD and were incubated for 15 min at room temperature in the dark. The cells were analyzed on a FACSCalibur (BD Biosciences) using CellQuest Pro software (BD Biosciences). The percentage of each quadrant was calculated.

Statistical analysis

Results are presented as the mean ± standard deviation. Differences between the groups were assessed using the Student's t-test. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using SPSS 13.0 statistical software (SPSS, Inc., Chicago, IL, USA).

Results

Lentivirus-mediated shRNA inhibits the expression of RPS15A in U937 cells

The U937 cell line was infected with shCon, shRPS15A (S1) and shRPS15A (S2) lentiviral particles. Infection efficiency was >70%, as determined by detecting the expression of green fluorescent protein 96 h post-infection (Fig. 1A). qPCR analysis demonstrated that the RPS15A mRNA expression levels were significantly reduced in the shRPS15A (S1; P<0.001) and shRPS15A (S2; P<0.001) groups compared with the shCon and control groups (Fig. 1B). The protein expression levels of RPS15A were also markedly decreased in the shRPS15A (S1) and shRPS15A (S2) groups compared with the shCon and control groups (Fig. 1C). The RPS15A knockdown efficacy of shRPS15A (S1) and shRPS15A (S2) was 96.0 and 93.2%, respectively. These results suggest that lentivirus-mediated shRNA knockdown of RPS15A expression was specific, and the off-target effects were eliminated. These results indicate that lentivirus-mediated RPS15A shRNA was able to significantly downregulate RPS15A expression in U937 cells.

Figure 1 Knockdown of ribosomal protein S15a (RPS15A) in U937 cells by lentivirus-delivered short hairpin RNA (sh)RPS15A (S1)/(S2). (A) Detection of lentiviral infection efficiency. The U937 cells were infected with shCon, shRPS15A (S1) or shRPS15A (S2), or remained uninfected (control; Con). Bright (upper panel) or green fluorescent protein (GFP) (lower panel) images were obtained 96 h post-infection. Scale bar, 100 µm. (B) Analysis of RPS15A mRNA expression in U937 cells transduced with shCon, shRPS15A (S1) or shRPS15A (S2) by quantitative polymerase chain reaction. (C) Western blot analysis of RPS15A protein expression in U937 cells transduced with shCon, shRPS15A (S1) or shRPS15A (S2). Data are presented as the mean ± standard deviation of three independent experiments. ***P<0.001, compared with the shCon group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Knockdown of RPS15A inhibits proliferation of U937 cells

To examine the effects of RPS15A knockdown on U937 cell growth, shRPS15A (S1)-, shRPS15A (S2)- and shCon-infected U937 cells were subjected to the MTT assay. As shown in Fig. 2, cell proliferation in the shRPS15A (S1) and shRPS15A (S2) groups was significantly reduced; proliferation decreased by >80% in the shRPS15A (S1) group (P<0.001) and by >84.5% in the shRPS15A (S2) group (P<0.001; Fig. 2) at days 4 or 5. Since proliferation inhibition occurred to a greater extent in the shRPS15A (S2) group, these cells were selected for further experimentation. Notably, the proliferative index [proliferative index = (S + G2/M)/(G0/G1 + S + G2/M)] of shRPS15A (S2)-transduced U937 cells was slightly lower than that of the shCon and control cells (35.45±3.2 vs. 50±3.8 and 51.3±4.2%; data from Fig. 3). These results indicate that the proliferation of U937 cells was significantly inhibited following RPS15A knockdown compared with in the shCon and control groups.

Figure 2 Knockdown of ribosomal protein S15a (RPS15A) inhibits the growth of U937 cells in vitro. Growth curves of control (Con), short hairpin RNA (sh)Con, shRPS15A (S1) and shRPS15A (S2) groups, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Data are presented as the mean ± standard deviation of three independent experiments. ***P<0.001, compared with the shCon group. OD, optical density.

Figure 3 Knockdown of ribosomal protein S15a (RPS15A) blocks cell cycle progression of U937 cells. Cell cycle distribution was analyzed by flow cytometry. (A) Representative images of control (Con), short hairpin RNA (sh)Con and shRPS15A (S2) cell cycle progression, as determined by flow cytometric analysis. (B) Proportion of cells in different cell cycle phases. (C) Proportion of cells in sub-G1. Data are presented as the mean ± standard deviation of three independent experiments. ***P<0.001, compared with the shCon group.

Knockdown of RPS15A arrests cell cycle progression of U937 cells

To investigate whether cell cycle arrest contributed to growth inhibition, flow cytometric analysis was conducted. The proportion of cells in G0/G1 phase was significantly increased (P<0.001), and the percentage of cells in S phase were significantly decreased (P<0.001), in the shRPS15A (S2) group compared with the shCon and control groups (Fig. 3A and B). These results indicate that shRPS15A (S2)-induced growth suppression may be partly mediated by cell cycle arrest at G0/G1 phase. In addition, the proportion of cells in sub-G1 was significantly increased in the shRPS15A (S2) group compared with the shCon and control groups (P<0.001; Fig. 3C).

Knockdown of RPS15A enhances apoptosis of U937 cells

Results of the flow cytometric analysis indicated that the percentage of early apoptotic (Annexin V-positive/7-AAD-negative) and late apoptotic (Annexin V-positive/7-AAD-positive) cells was significantly higher in the shRPS15A (S2)-transduced cells compared with in the shCon and control cells (P<0.001; Fig. 4A and B). Following transduction, the apoptotic rate (early and late apoptotic cells) of U937 cells in the shRPS15A (S2) group was 18.35±0.28%, which was significantly higher than in the shCon and control groups (7.97±0.12 and 8.2±0.06%). The apoptotic rate of U937 cells was significantly increased following RPS15A knockdown, as compared with that in the shCon and control groups.

Figure 4 Effects of ribosomal protein S15a (RPS15A) knockdown on U937 cell apoptosis. Cell apoptosis was determined using flow cytometry. (A) Representative images of control (Con), short hairpin RNA (sh)Con and shRPS15A (S2) apoptosis, as determined by flow cytometry. (B) Proportion of each quadrant [Annexin V+/7-aminoactinomycin D (7-AAD)-, Annexin V+/7-AAD+, Annexin V−/7-AAD−, Annexin V−/7-AAD+]. Data are presented as the mean ± standard deviation of three independent experiments. ***P<0.001, compared with the shCon group. Lv, lentivirus.

Discussion

AML has the lowest survival rate among all types of leukemia (25), and knowledge regarding its basic biology remains to be completely elucidated. Numerous molecules have been identified as potential targets; however, only a few have pivotal roles in AML cell proliferation and survival (12,26–33). Therefore, identification of novel therapeutic targets, and the development of novel therapeutic regimens that more effectively regulate cellular function are of central importance. RPS15A has been reported to be overexpressed and have an important role in regulating carcinogenesis in several types of human cancer. Elevation of RPS15A expression in tumor cells leads to phenotype changes that are characteristic of more aggressive malignancy (20,22).

It has been reported that RPS15A gene expression was upregulated in leukemia tissues at the mRNA level (34–36). Furthermore, in order to ensure the specificity of RPS15A silencing, two RPS15A shRNA expression vectors were used, which resulted in a marked decrease in RPS15A expression in U937 cells. Knockdown of RPS15A inhibited proliferation of U937 cells, and led to cell cycle arrest at G0/G1 phase, as determined by flow cytometry. Notably, downregulation of RPS15A in hepatocellular carcinoma cells has previously been shown to potently suppress cell growth via cell cycle arrest at G0/G1 phase (22). The results of the present study also indicated that RPS15A may have a crucial role in regulating AML cell growth. In addition, the present study demonstrated that the majority of shRPS15A-transduced cells underwent apoptosis. These results strongly suggested that RPS15A may have a central role in AML carcinogenesis and the maintenance of malignant phenotypes. It may be hypothesized that RPS15A dysregulation may affect the translation of proteins that specifically govern the cell cycle. Therefore, the identification of RPS15A downstream target proteins through high-throughput proteomics is the focal point of our future research, which will facilitate the elucidation of the mechanisms underlying the effects of RPS15A on AML development.

In conclusion, to the best of our knowledge, this is the first study to examine the function of RPS15A in AML cells. The results demonstrated that inhibition of RPS15A significantly reduced U937 cell proliferation, and induced G0/G1 phase arrest and apoptosis, thus providing a future target for AML therapy. Further investigations regarding the regulatory mechanisms underlying the effects of RPS15A on AML may help to better understand AML carcinogenesis.

Acknowledgments

The authors of the present study are grateful for the financial support received from the National Natural Science Foundation of China (grant no. 81573772) and the Shandong Provincial Science and Technology Development Projects (grant no. 2014GSF118141).

References