The human NPC cell lines (CNE-1, CNE-2, HNE-1, HONE-1 and SUNE-1) and the human immortalized nasopharyngeal epithelium cell line NP69 were preserved by our laboratory. CNE1 is a nasopharyngeal, well-differentiated squamous cell carcinoma cell line; CNE2, HNE1, HONE1 and SUNE1 are nasopharyngeal, poorly differentiated squamous cell carcinoma cell lines; NP69 is an immortalized nasopharyngeal epithelial cell line, showing squamous epithelioid and adherent growth, with normal characteristics of the nasopharyngeal epithelium. The five NPC cell lines were cultured in an RPMI-1640 medium (Gibco, Life Technologies Inc., Carlsbad, CA, USA) containing 10% fetal bovine serum (Tianhang Biotechnology Co., Ltd., Zhejiang, China). NP69 cells were cultured in a keratinocyte/serum-free medium (K-SFM; Gibco) supplemented with bovine pituitary extract (Gibco) and epidermal growth factor (human recombinant; Gibco).

The cells were all cultured at 37°C with 5% CO2. The SGK3 shRNA plasmids were all produced by GeneCopoeia Inc., Rockville, MD, USA, and included sh1025, sh1132, sh1206 and sh1284; the sequences are 5′-GCTAGTGCATTGGGTTACTTA-3′, 5′-GCTTTGTAAAGAAGGAATTGC-3′, 5′-GCCGAGATGTTGCTGAAATGT-3′ and 5′-GGTCCATTCTGGAAGAACTCC-3′, respectively. The negative control (NC) plasmid, with a sequence 5′-GCTTCGCGCCGTAGTCTTA-3′, was also purchased from GeneCopoeia Inc. To obtain the best sequence of the plasmid on gene silencing, the cells were seeded before they were transfected and allowed to grow to a density of 60% on the second day. The cationic polymer PEI (obtained from Aladdin Inc.) was used to transfect the plasmids with different sequences of ShRNA (silent different locus of SGK3) into cells. After 72h, cell proteins were extracted, and western blotting was used to observe the silencing effects of ShRNA on SGK3, and select the plasmid with the best sequence on silencing. The best sequence is 5′-GCTAGTGCATTGGGTTACTTA-3′.

A carbon dioxide incubator was produced by Thermo Fisher Scientific Inc., USA. An HM340E-rotary slicer and a hematoxylin–eosin (HE) staining fully automatic dyeing machine were produced by MICROM Inc., Germany. A vertical electrophoresis apparatus and transmembrane system were produced by Bio-Rad Inc., Hercules, California, USA. The ChemiDoc™ XRS+chemiluminescence imaging system was also produced by Bio-Rad Inc. The BD FACSCalibur™ flow cytometer was produced by BD biosciences Inc., California, USA. An inverted fluorescence microscope was produced by Carl Zeiss Co. Ltd., Germany.

Forty-two NPC paraffin-embedded tissue samples and nine chronic nasopharyngitis tissue samples were obtained from the Department of Otolaryngology of Zhujiang Hospital. The clinicopathological data is presented in Tables 1 and 2. The SGK3 expression in sections was assayed via immunohistochemistry with the Elivision two-step detection kit (Kit-0015; Fuzhou Maixin Biotech. Co., Ltd., China). These sections were deparaffnized and pretreated by boiling the slides in citrate buffer (pH 6) for 10min. The sections were then immersed in 3% hydrogen peroxide for 10min at room temperature to block endogenous tissue peroxidase activity. Following washing with phosphate-buffered saline (PBS), sections were incubated with Rabbit SGK3 polyclonal antibody (catalog no. ab153981; 1:200; Abcam Company, USA) at 4°C overnight, and then sections were incubated with 50μL amplifer polymer (reagent A), and 50μL horse radish peroxidase-conjugated anti-mouse/rabbit IgG (reagent B) for 30min at room temperature. Following washing with PBS, the sections were stained with diaminobenzidine chromogenic solution (DAB) and counterstained with hematoxylin, differentiated using hydrochloric acid in ethanol, blued by washing with water and sealed with conventional resin.17 The results were evaluated by the HSCORE method which included both the intensity and the distribution score of the specific staining, evaluated by two pathologists independently.18 The distribution scores of SGK3 expression were recorded as percentages of positively stained cells in each of the four intensity categories, which were scored as 0 (no staining), 1 (weak staining), 2 (distinct staining) and 3 (strong staining). The HSCORE of each tissue was derived by summing the four distribution scores (D) of each category multiplied by the intensity scores (i) as follows: HSCORE=ΣD(i), where D varied from 0 to 100% and i=0, 1, 2 or 3. The maximum of HSCORE was 300, and the minimum was 0. An HSCORE=75 was used as the cut-off point to distinguish positive and negative expression.

This study was approved by the Ethics Committee (no. 2016-EBYHK-002).

Total proteins were extracted from cells using RIPA lysis buffer (Kaiji Biotechnology Co., Ltd., Jiangsu, China). Protein concentrations were determined using protein assays (Beyotime Biotechnology Co., Ltd., Shanghai, China). Purified proteins (50μL) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked for 2h at room temperature in 5% non-fat milk and incubated overnight at 4°C with an anti-SGK3 antibody (Abcam Company, 1:1000). Following incubation with a horseradish peroxidase (HRP), linked secondary antibody (Biosharp Biotechnology Co., Ltd., Hefei, China) (1:5000), the membranes were washed, and proteins were visualized using an enhanced chemiluminescence kit (PerkinElmer Inc., Waltham, MA, USA). Western blotting results were semi-quantitatively analyzed by Image-Pro Plus software.19

Logarithmic phase CNE-2 cells were seeded in 6-well plates for 24h before transfection to achieve 80–90% confluence. Cells in each well were transfected with 4μg of SGK3 shRNA or NC plasmid and 10μL of Lipofectamine 2000 (Invitrogen, Life Technologies Inc., Carlsbad, California, USA). Non-treated cells were used as the control. All cells were cultured at 37°C for 24h. After determining the success of transfection according to the degree of green fluorescent protein (GFP) by fluorescence microscopy, the cells were cultured for another 48h and harvested for western blot analysis to identify the plasmid with the greatest effect on SGK3 knockdown.20 The treated cells were harvested for subsequent experiments. Cells successfully transfected with the SGK3 shRNA or NC plasmid were referred to as the shSGK3 or NC group, respectively. CNE-2 cells served as a blank control (control group). All in vitro experiments were performed for each group.

CNE-2 cells in the three groups were cultured in 96-well plates (5000cells/well in five wells). Cell growth was examined using a 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay with one plate conducted at the indicated time-points for three consecutive days. Each well contained 20μL of MTT reagent and was then cultured at 37°C for 4h. The medium was discarded, and crystals that had formed were dissolved in 150μL of dimethyl sulfoxide (DMSO). The viable cells in each well were determined each day by measurement at an absorbance wavelength of 490nm. Each experiment was repeated independently three times. Cell growth curves were subsequently generated.20

CNE-2 cells in the three groups were seeded in 6-well plates (50000cells/well) and cultured for 48h. At the end of incubation, all cells were trypsinized, washed twice with pre-cooled PBS (HyClone, Logan, UT, USA), collected and resuspended in 100μL of binding buffer. Then, 5μL of annexin V-APC and 5μL of PI were added, and the plates were incubated for 30min in the dark at room temperature. Cells were immediately examined by the flow cytometer. Data were analyzed by ModFit LT 2.0 software.21

CNE-2 cells were added to 6-well plates (50000cells/well). Once the cells had grown to approximately 100% confluence, scratch tests were performed using a 200μL sterile pipette tip to scratch the confluent monolayer. The scratched area was washed with PBS until the detached cells were removed. Serum-free medium was added, and the cells were cultured at 37°C. The distance between the cells in the scratched area at 0, 24 and 48h was measured by microscopy.22 Horizontal cell migration was analyzed by Image J software and calculated using the following formula: the relative mobility=1 (immediate scratch width/original scratch width).

Statistical evaluation was performed using SPSS 22.0 or Graph Pad Prism 6 software. All data were acquired from at least three independent experiments, and the results are presented as the mean±standard deviation. One-way analysis of variance and Student's t-tests were used to compare the differences in means among groups.

The association of SGK3 expression with the clinical features in NPC was assayed by logistic regression analysis. A value of p<0.05 indicated a significant difference.

Fig. 1A shows SGK3 protein expression in tissues. The positive expression rates of SGK3 in NPC tissues and chronic nasopharyngitis tissues were 90.50% (38/42) and 11.10% (1/9), respectively. The SGK3 expression rate at different NPC stages and in chronic nasopharyngitis tissues was assessed according to the HSCORE.

Figure 1.

Representative immunohistochemical staining of SGK3 in NPC tissues. (A) Immunohistochemical results showed that SGK3 protein expression was mainly located in the cytoplasm, with strongly positive SGK3 expression in NPC cancer nests and no expression or weak expression in chronic nasopharyngitis tissues. (B) SGK3 expression rate in different stages of NPC and chronic nasopharyngitis tissues according to the HSCORE. The SGK3 expression in stage I, II, III and IV NPC tissues was significantly higher than that in chronic nasopharyngitis tissues (p<0.01). However, no significant difference was observed among the different NPC stages (p>0.05).

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The SGK3 expression level was significantly higher in NPC than in chronic nasopharyngitis tissue (p<0.01) (Fig. 1B), indicating that SGK3 expression is related to NPC carcinogenesis. However, SGK3 expression was not correlated with gender, age, TNM stage or clinical stage (p>0.05) (Table 3).

Expression of the SGK3 protein in different NPC cell lines and NP69 cells was as shown in Fig. 2. SGK3 was more highly expressed in most NPC cells (CNE-2, HNE-1, SUNE-1) than in NP69 cells (p<0.01). Moreover, CNE-2 and HNE-1 cells exhibited higher levels of SGK3 expression than other NPC cell lines (p<0.01). The results of the western blot analysis were consistent with those of the immunohistochemistry analysis. These results also indicated that SGK3 might be closely related to NPC development.

Figure 2.

Detection of SGK3 protein expression in NPC cell lines. (A) Representative SGK3 protein expression in different NPC cell lines and NP69 cells detected by western blot analysis. (B) Analysis of the gray value of SGK3 protein expression as the ratio of SGK3 to β-actin in the western blot results; SGK3 was more highly expressed in most NPC cell lines (CNE-2, HNE-1, SUNE-1) than in NP69 cells (p<0.01).

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SGK3 shRNA and NC plasmids were successfully transfected into CNE-2 cells (Fig. 3A). The screening results of the most effective shRNA plasmid targeting SGK3 (shSGK3) are presented in Fig. 3B, which shows that the SGK3 protein was notably reduced in the sh1025 group compared with the other plasmid and control groups (p<0.01) (Fig. 3B and C). The sh1025 plasmid had the most significant effect on SGK3 knockdown; therefore, sh1025 group cells were referred to as the hSGK3 group and used in subsequent experiments. The western blot results showed that the SGK3 protein level was markedly reduced in the shSGK3 group compared with that in the NC group and control group (p<0.01) (Fig. 3D and E), and no significant difference in SGK3 expression was observed between the NC group and the control group.

Figure 3.

The best SGK3 shRNA screening and efficiency of SGK3 silencing by the specific shRNA in CNE-2 cells. (A) GFP expression in CNE-2 cells observed by fluorescence microscopy after transfection with the SGK3 shRNA or NC plasmid. (B) Representative SGK3 protein expression in CNE-2 cells treated with different SGK3 shRNA plasmids determined by western blot analysis. (C) Analysis of the gray value of SGK3 protein expression as the ratio of SGK3 to β-actin in western blot results. The sh1025 plasmid had the most significant effect on SGK3 knockdown (p<0.01). (D) Representative SGK3 protein expression in shSGK3, NC and control groups determined by western blot analysis. (E) Analysis of the gray value of SGK3 protein expression as the ratio of SGK3 to β-actin in western blot results; expression of SGK3 in the shSGK3 group was obviously reduced compared with that in the NC and control groups (*#p<0.01).

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To confirm the cell inhibition effect of SGK3 shRNA, we evaluated its cytotoxic effects. Fig. 4A shows the lack of toxicity in the NC group and obvious cytotoxicity in the shSGK3 group at 24, 48 and 72h (p<0.01).

Figure 4.

The effect of SGK3 downregulation on the proliferative ability and apoptosis of CNE-2 cells. MTT assay (A) and apoptosis analysis (B, C) by flow cytometry in the different groups. The cell viability rate in the shSGK3 group was obviously reduced compared with that in the NC and control groups, and the cell apoptosis rate in the shSGK3 group was obviously increased compared with that in the NC and control groups (*#p<0.01).

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To determine whether SGK3 shRNA could effectively induce cell apoptosis, we determined the percentage of cell apoptosis after the CNE-2 cells were transfected with SGK3 shRNA. As shown in Fig. 4B, CNE-2 cells displayed limited apoptosis in the NC group compared with that in the control group, which was consistent with the results of the cytotoxicity analysis. The percentages of cell apoptosis (including early and late apoptosis) in the shSGK3 group, the NC group and the control group after 48h were 17.85, 7.49 and 6.70%, respectively. The cells treated with SGK3 shRNA exhibited a more obvious increase in apoptosis (p<0.01) (Fig. 4C).

To detect the invasion and metastasis ability of tumor cells, the cell scratch test was conducted in the control group, NC group and shSGK3 group. As shown in Fig. 5A, the horizontal cell migration rate in the shSGK3 group after 24h was 20.50%, whereas these rates were 51.60 and 56.60% in the NC group and control group, respectively. After 48h, cells in the shSGK3 group had not yet recovered from the damage. The horizontal cell migration rate was 46.70%, but the scratches in the NC group and control group were hardly visible. These results suggested that the migratory ability of the shSGK3-treated cells was obviously decreased compared with that in the NC group and the control group (p<0.01) (Fig. 5B).

Figure 5.

The effect of SGK3 downregulation on the motility of CNE-2 cells. (A) Distance at different times after the scratch test (original magnification: ×5). Scratch distances in the shSGK3 group did not obviously change after 24h, whereas cells in the NC and control groups exhibited higher migration rates (p<0.01). After 48h, cells in the shSGK3 group had not yet recovered from the damage (p<0.01), but cell scratches in the NC and control groups were hardly visible. (B) Comparison of the cell migration rates at different times by the scratch test; the cell migration rates in the shSGK3 group were higher than those the NC and control groups after 24 and 48h (△◊ p<0.01).

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