The Suppression Role Of SGK196 N-glycosylation On Metastasis Of Basal-like Breast Cancer

Objectives To explore the role and mechanism of SGK196 protein and its N-glycosylated modification in the invasion and metastasis of basal-like breast cancer.Methods1.Online database information was used to analyze the relationship between m RNA expression level of SGK196 with breast cancer patients' prognosis.Western blot and Real-time PCR were used to analyze protein and m RNA levels of SGK196 in different breast cancer cell lines.Differences in protein band patterns were also detected by Western blot.The above results were statistically analyzed using SPSS 13.0 statistical software.2.Protein lysates of breast cancer cell lines were digested with glycosidase(PNGase F and Endo H)and analyzed by Western blot.The N-glycosylation modification site of SGK196 was predicted by software and verified by site-directed mutagenesis of SGK196 in HEK293 T cells.Analysis of the results of mass spectrometry indicated that the RPN1 protein(ribophorin I)may interact with SGK196,which was further verified by CO-IP.3.Using lentiviral and retroviral vectors,stable cell lines that interfering with SGK196 or overexpressing SGK196 were constructed in basal-like breast cancer cell lines(MDA-MB-231,BT-549),and the efficiency of transfection was verified by Western blot;Cell proliferation ability was examined by CCK-8 kit;Cell cloning ability was detected by plate cloning assay,and cell invasion ability was detected by Transwell chamber invasion assay.In order to further clarify the biological function of Nglycosylated SGK196 and to exclude the influence of endogenous SGK196 expression,we constructed a plasmid re-expressing WT-SGK196 and 4NQ-SGK196 after knocking down SGK196.Then,we also used CCK8 to detect cell proliferative ability,plate cloning assay to detect cell cloning ability,Transwell chamber invasion assay to detect cell invasion ability among different cell groups.Moreover,we established a nude mouse model of abdominal metastasis to study the inhibition effect of SGK196 on the invasion and metastasis of basal-like breast cancer cells in vivo.Afterwards,we examined the effect of SGK196 on tumor-associated signaling pathways by Western blot.The above results were calculated using SPSS 13.0 statistical software.Results1.Survival analysis of the Kaplan-Meier Plotter database showed that the higher the expression level of SGK196 m RNA in breast cancer patients,the better the patient's recurrence-free survival rate(RFS),especially in patients of basal-like breast cancer type.Moreover,Western blot assay showed that the SGK196 protein band shifted in different breast cancer cell lines.1.SGK196 protein is ubiquitously N-glycosylated in different subtypes of breast cancer cell lines,and other forms of glycosylation(O-glycosylation?)are also present in some breast cancer cell lines;N-glycosylation site of SGK196 are as follows: Asn67,Asn 165,Asn 220 and Asn 235.There is a mutual binding between SGK196 protein and RPN1 protein,and the mutation of N-glycosylation site of SGK196 does not affect the binding of the two prtoeins.RPN1 protein can partially regulate the N-glycosylation of SGK196 protein and affect m RNA and protein expression of SGK196.2.Western blot analysis showed that the stable cell line that interfered with SGK196 and overexpressed SGK196 was successfully established in MDA-MB-231 and BT-549.The migration and invasion abilities of SGK196-interferenced cell line were significantly stronger than those of the control group;There were no significant differences of the proliferation and cloning abilities between experimental groups and the control group;the migration and invasion abilities of the stably-transfected cell line overexpressing SGK196 were significantly weaker than those of the control group.Similarly,the proliferation and cloning abilities of the SGK196-overexpressing group were not significantly different from the control group.After re-expressing WTSGK196 and 4NQ-SGK196 on the basis of interference with SGK196,we found that N-glycosylated SGK196 protein could significantly inhibit the invasive ability of tumor cells,while non-glycosylated SGK196 protein almost lost such function;however,regardless of overexpression of N-glycosylated SGK196 protein or non-glycosylated SGK196 protein,both of them have no significant effect on cell proliferation and cloning abilities.In animal experiments,we also get the same results of in vitro experiments: the metastasis rates of liver and lung in the nude mice that interfered with SGK196 group were significantly higher than those of the control group,while Nglycosylated SGK196 protein could inhibit the liver and lung metastasis and nonglycosylated SGK196 protein group could not.Detecting tumor-associated signaling pathways,we found that N-glycosylated SGK196 protein could inhibit activity of the PI3K-AKT-GSK3?-Snail pathway,whereas non-glycosylated SGK196 protein lost its inhibitory effect on this signaling pathway.ConclusionsThe human SGK196 protein exists glycosylated modification.Asn67,Asn165,Asn220,and Asn235 are N-glycosylation modification sites of human SGK196.RPN1 and SGK196 could interact with each other and RPN1 could partially regulate the glycosylation modification of SGK196 protein.Both in vitro and in vivo experiments confirmed that N-glycosylated SGK196 protein can significantly inhibit the invasion and metastasis of basal-like breast cancer cells,and play the above-mentioned inhibition by PI3K-AKT-GSK3?-Snail signal transduction pathway.