The Expression Of SGK3 And Its Effect On Cell Biologic Characteristics In Nasopharyngeal Carcinoma

BackgroundNasopharyngeal Carcinoma(NPC)is one of the most common head and neck tumors that originating from nasopharyngeal mucosa.NPC frequently occurs in Southern China and Southeast Asia,due to its features of regional distribution and familial aggregation.Most of the pathological types of NPC are low differentiated squamous cell carcinoma,which have been high degree of malignancy and cervical lymph node metastasized at early stage.NPC is almost highly sensitive to radiotherapy.Although the local control rate has been greatly improved thanked to concurrent chemo-radiotherapy,the distant metastasis rate was no significantly decreased.Local recurrence and distant metastasis are still the main reasons leading to the failure of NPC treatment.Various pathogenic factors,including family history,genetic dysregulation,environmental alteration,Epstein-Barr virus(EBV)infection and trace elements,correspond to NPC development.However,the molecular processes involved in the NPC pathogenesis have not yet been fully explained.Therefore,it is of great significance to further study in the occurrence and development of NPC,in order to seek new therapeutic approaches and improve the survival of NPC patients.Serum and glucocorticoid-kinase kinases(SGKs)are serine/threonine kinases functioning downstream of phosphoinositide 3-kinase(PI3-K)signaling pathway.SGKs have 3 isoforms in mammals(SGK1,SGK2,and SGK3)and belong to the AGC kinase family(protein kinase A,protein kinase G,and protein kinase C)as like protein kinase B/AKT.SGKs share great homology with AKT in the kinase domain and also involve in PI3-K signaling pathway,regulating cell processes similar to AKT.SGK3 has been suggested to play a pivotal role in tumor-genesis and development,such as breast,liver and prostate cancers.Its activation and phosphorylation at Thr320 by phosphoinositide-dependent kinase-1(PDK1)is considered to be an AKT-independent manner of signal:ing in cancer.However,the functions of SGK3 in human NPC still remain unknown.Our study was designed to investigate the expression of SGK3 in NPC tissues and cells,and the effects of SGK3 on biological behaviors in NPC cells.Exploring the association between SGK3 expression and NPC development.And to provide evidences for individual therapy.Materials and methods1.Inmunohistochemical analyses were performed to assess the SGK3 protein expression in formalin-fixed,paraffin-embedded specimens from various nasopharyngeal tissues,including 42 NPC samples and 9 normal nasopharyngeal epithelial samples.The difference between two groups was compared by the chi-square test.Logistic regression analysis was used to analyze the association between the expression level of SGK3 protein and the clinicopathological features of NPC,which were grouped by the age,gender,TNM stage and clinical stage.SPSS 22.0 software was used for statistical analysis.P<0.05 was considered statistically significant.2.Western blot was used to detect the expression of SGK3 protein in five NPC cell lines CNE-1,CNE-2,HNE-1,SUNE-1,HONE-1 and human immortalized nasopharyngeal epithelium cell line NP69.The relative expression intensity of SGK3 protein was semi-quantitative analyzed by Image Pro Plus software.The difference of the gray values of each group was compared by one-way ANOVA test.3.Lipofectamine 2000 was used to transient transfect CNE-2 cell lines.Fluorescence microscope determined the success of transfection according to the degree of green fluorescent protein(GFP)after 24 hours and western blot detected the expression of SGK3 protein after 48 hours.The most obvious plasmid for the knockdown of SGK3 gene was selected as the best plasmid,establishing NPC cells capable of silently expressing SGK3 for the following experiments.4.Cell biological function experiments.The proliferation capacity of NPC cells transfected by the best plasmid was detected using MTT assays.The apoptotic rates were detected by flow cytometry.The scavenging ability was measured using scratch test by microscopy.The survival and apoptotic rates were analyzed by univariate analysis of variance(ANOVA).The scratch tests were analyzed by variance analysis of factorial design.Results1.Immunohistochemical results showed that the expression of SGK3 protein was mainly found in the cytoplasm.The HSCORE values were calculated according to the immunohistochemical semi-quantitative scoring method.The positive expression frequency of SGK3 protein significantly increased in NPC samples(90.5%)in comparison to normal nasopharyngeal epithelial samples(11.1%).However,the expression level of SGK3 protein was not correlated with gender,age,TNM stage and clinical stage(P>0.05).2.Western blot results showed that SGK3 protein was highly expressed in the NPC cell lines and low level in the NP69 cell line.Moreover,some NPC cell lines,especially CNE-2 and HNE-1 cell lines,had higher levels of SGK3 protein than that of other NPC cell lines(P<0.01).3.Successful construction of CNE-2/shSGK3 silently expressing SGK3.Experimental data showed that reducing the expression of SGK3 suppressed both proliferation and migration,and promoted the apoptosis of NPC cells in vitro.Conclusion1.SGK3 was highly expressed in NPC tissues and cells.But there was no significant correlation between the expression level of SGK3 protein and the clinicopathological features of NPC patients.Indicating that SGK3 expression is involved in human NPC occurrence.2.SGK3 expression disorder can affect the biological characteristics of NPC cells.Indicating that SGK3 may be an oncogene that promotes the progression of NPC.