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. 2019 Nov-Dec;85(6):705-715.
doi: 10.1016/j.bjorl.2018.05.012. Epub 2018 Jul 18.

Serum- and glucocorticoid-inducible kinase 3 is a potential oncogene in nasopharyngeal carcinoma

Jing Chen  1 , Hai Liang Li  2 , Bo Bo Li  3 , Wei Li  1 , Dong Ma  4 , Yong He Li  5 , Tao Liu  6
Affiliations

Serum- and glucocorticoid-inducible kinase 3 is a potential oncogene in nasopharyngeal carcinoma

Jing Chen et al. Braz J Otorhinolaryngol. 2019 Nov-Dec.

Abstract
in English, Portuguese

Introduction: Serum- and glucocorticoid-inducible kinase 3, a serine/threonine kinase that functions downstream of the PI3K signaling pathway, plays a critical role in neoplastic processes. It is expressed by various tumors and contributes to carcinogenesis.

Objective: The objective was to investigate serum- and glucocorticoid-inducible kinase 3 expression in nasopharyngeal carcinoma, to study the anti-tumor effects of serum- and glucocorticoid-inducible kinase 3 shRNA by inhibiting its expression in nasopharyngeal carcinoma cells and to discuss the potential implications of our findings.

Methods: Serum- and glucocorticoid-inducible kinase 3 protein expression in nasopharyngeal carcinoma cell lines (CNE-1, CNE-2, HNE-1, HONE-1, and SUNE-1) and the human immortalized nasopharyngeal epithelium cell line NP69 were assayed by western blotting. Serum- and glucocorticoid-inducible kinase 3 expression in 42 paraffin-embedded nasopharyngeal carcinoma tissues were performed by immunohistochemistry. MTT assay, flow cytometry, and scratch tests were performed after CNE-2 cells were transfected with the best serum- and glucocorticoid-inducible kinase 3 shRNA plasmid selected by western blotting using lipofectamine to study its effect on cell proliferation, apoptosis, and migration.

Results: Serum- and glucocorticoid-inducible kinase 3 was overexpressed in human nasopharyngeal carcinoma tissues and cells. Serum- and glucocorticoid-inducible kinase 3 expression decreased markedly after CNE-2 cells were transfected with the serum- and glucocorticoid-inducible kinase 3 shRNA, leading to strong inhibition of cell proliferation and migration. In addition, the apoptosis rate increased in CNE-2 cells after serum- and glucocorticoid-inducible kinase 3 knockdown.

Conclusion: Serum- and glucocorticoid-inducible kinase 3 expression was more frequently observed as the nasopharyngeal epithelium progresses from normal tissue to carcinoma. This suggests that serum- and glucocorticoid-inducible kinase 3 contributes to the multistep process of NPC carcinogenesis. Serum- and glucocorticoid-inducible kinase 3 represents a target for nasopharyngeal carcinoma therapy, and a basis exists for the further investigation of this adjuvant treatment modality for nasopharyngeal carcinoma.

Introdução: A quinase 3 sérica induzida por glicocorticoide, uma serina/treonina quinase que funciona downstream da via de sinalização PI3K, desempenha um papel crítico nos processos neoplásicos. É expressa por vários tumores e contribui para a carcinogênese.

Objetivo: O objetivo foi investigar a expressão de quinase 3 sérica induzida por glicocorticoide no carcinoma nasofaríngeo, estudar os efeitos antitumorais do shRNA da quinase 3 sérica induzida por glicocorticoide, inibindo sua expressão em células de carcinoma nasofaríngeo e discutir as implicações potenciais de nossos achados.

Método: A expressão de proteína quinase 3 sérica induzida por glicocorticoide em linhagens de células de carcinoma nasofaríngeo (CNE-1, CNE-2, HNE-1, HONE-1 e SUNE-1) e a linhagem de células humanas imortalizadas do epitélio nasofaríngeo NP69 foram avaliadas por Western blot. A expressão da quinase 3 sérica induzida por glicocorticoide em 42 tecidos de CNF embebidos em parafina foi realizada por imunohistoquímica. Testes com MTT, citometria de fluxo e testes de raspagem foram realizados após as células CNE-2 terem sido transfectadas com o melhor plasmídeo shRNA da quinase 3 sérica induzida por glicocorticoide selecionado por Western blot, utilizando lipofectamina para estudar seu efeito na proliferação, apoptose e migração celular.

Resultados: Foi observada uma sobre-expressão da quinase 3 sérica induzida por glicocorticoide em tecidos e células de carcinoma nasofaríngeo humanas. A expressão de quinase 3 sérica induzida por glicocorticoide diminuiu acentuadamente após as células CNE-2 terem sido transfectadas com o shRNA da quinase 3 sérica induzida por glicocorticoide, conduzindo à forte inibição de proliferação e migração celular. Além disso, a taxa de apoptose aumentou nas células CNE-2 após o knockdown da quinase 3 sérica induzida por glicocorticoide.

Conclusão: A expressão de quinase 3 sérica induzida por glicocorticoide foi observada com maior frequência à medida que o epitélio nasofaríngeo progride de tecido normal para carcinoma. Isto sugere que a quinase 3 sérica induzida por glicocorticoide contribui para o processo multi-etapas da carcinogênese do carcinoma nasofaríngeo. A quinase 3 sérica induzida por glicocorticoide representa um alvo para a terapia do carcinoma nasofaríngeoe há uma base para a investigação adicional desta modalidade de tratamento adjuvante para o carcinoma nasofaríngeo.

Keywords: Apoptose; Apoptosis; Carcinoma nasofaríngeo; Migration; Migração; Nasopharyngeal carcinoma; Proliferation; Proliferação; Quinase 3 sérica e induzida por glicocorticoide; Serum- and glucocorticoid-inducible kinase 3; shRNA.

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Figures

Figure 1
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).
Figure 2
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).
Figure 3
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).
Figure 4
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).
Figure 5
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 24 h, whereas cells in the NC and control groups exhibited higher migration rates (p < 0.01). After 48 h, 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 48 h (△◊ p < 0.01).
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