Inhibition Of N-glycosylation Regulates The Malignancy Of Nasopharyngeal Carcinoma Cells Through TGFβ/Smad Pathway And Its Possible Mechanisms

BackgroundNasopharyngeal carcinoma(NPC)is one of the most common malignant tumors in Southern China,and its cause is not yet clear.Genetic factors,Epstein-Barr virus(EBV)infection as well as environmental factors have been reported to be associated with the etiology of NPC.Radiotherapy alone is routinely the primary and the only curative treatment to early-stage disease,the five-year suvival rate is up to 90%.but for the majority undifferentiated later period disease,local recurrences or distant metastases are the crucial elements that restrict the effect.Therefore,elucidating the molecular mechanisms of NPC malignancy and finding some new molecular targets will help to develop effective therapeutic clues,resulting in better clinical outcomes.Transforming growth factor(TGF-β)is a multifunctional cytokines released by blood platelets and various stromal components.It takes part in a variety of cellular processes,such as cell invasion,immune regulation,and microenvironment modification.It has been confirmed that there is an autocrine of TGFβ1 in nasopharyngeal carcinoma,and the increase of TGFβ1 may be related to poor radiotherapy,recurrence and metastasis of tumor.TGFP first plays a biological effect with the membrane surface TβRⅠ,Ⅱ and other receptors,by activating the downstream of the classic Smad pathway or non-Smad pathway,and then regulate the transcription of the target gene.The abnormalities of TGFβ signaling pathway are thought to be closely related to the occurrence,development and metastasis of tumor.At present,there is little research focused on the mechanism of TGFβ1 affecting the malignant biological behavior of nasopharyngeal carcinoma cell lines,and the activation of its downstream signaling pathway is not clear.Protein glycosylation is an important post-translational modification that affects cell growth,differentiation,and tumor metastasis.N-acetylglucosaminyltransferase V(GnT-V)is an important glycosyltransferase located in the Golgi apparatus and can catalyze the formation of N-glucoside β-1,6 branches of various glycoproteins such as EGFR,TβR,NGFR,while the latter proved to be one of the key factors in the invasion and metastasis of multiple malignancies.Studies have shown that N-glycosylation modification of TβR protein by GnT-V facilites it to anchor in the cell membrane and not easy to endocytosis,thus lead cells to be sensitive of the stimulation of TGFβ1.The excessive expression of GnT-V can be found in the liver cancer,breast cancer and other malignant tumors,which is associated with poor prognosis.In our previous study,down-regulation of GnT-V expression can significantly inhibit tumor proliferation and metastasis in nasopharyngeal carcinoma and small cell lung cancer.Swainsonine(SW)is an a-mannosidase inhibitor that specifically inhibits the expression of N-glycoprotein beta-1,6 branches on the surface of tumor cell membrane,thereby inhibiting the growth and metastasis of tumor cells.Therefore,it is the focus of this paper to explore the molecular mechanism and the activation of downstream signaling pathway in nasopharyngeal carcinoma cell line.Through this study,we found that the continuous activation of TGFβ1 and downstream TGFβ/Smad pathway is an important factor affecting the malignant biological behavior of nasopharyngeal carcinoma.N-glycosylation inhibition can be achieved by reducing the expression of TβR and limiting the activation of TGFp/Smad pathway,reversing the malignant biological behavior of nasopharyngeal carcinoma cell lines,enriching the nasopharyngeal carcinoma comprehensive treatment strategy and providing a certain experimental basis.ObjectiveIn this study,we used nasopharyngeal carcinoma cell lines CNE-1 and CNE-2 to investigate the effect of N-glycosylation on the malignant biological behavior ofTGFβ1-mediated nasopharyngeal carcinoma cell line proliferation,invasion and migration and its downstream TGFβ/Smad pathway activation.Materials and methods1、lentivirus transfection constructs stable down-regulated Gnt-V nasopharyngeal carcinoma cell lines2、RN A extraction and qRT-PCR detection of the corresponding cell lines mRNA levels3、protein extraction and Western blot were used to detect the expression of protein in the corresponding cell lines4、Lectin-Blot test to detect the expression of total β-1,6 branches5、CCK-8 proliferation assay to detect cell proliferation6、clone formation test to detect cell cloning ability7、flow cytometry detection of apoptosis8、scratch healing test to detect cell migration ability9、Transwell test to detect the ability of cells to attack10、statistical analysisResults1、Explore the effect of TGFβ1 on the malignant biological behavior of nasopharyngeal carcinoma cell linesThe results showed thatthe stimulation of TGFβ1 could significantly promote the proliferation and clone formation of nasopharyngeal carcinoma cell lines(CNE-1 and CNE-2),promote its invasion and metastasis,but had no obvious effect on cell apoptosis.2、Explore the reversal effect of N-glycosylation inhibitor SW on malignant biological behavior of TGFβ1-mediated nasopharyngeal carcinoma cell lineThe effect of N-glycosylation inhibitor SW on nasopharyngeal carcinoma cell line was pretreated for 24 hours and then stimulated with TGFβ1.The results showed that the pretreatment of SW could significantly inhibit nasopharyngeal carcinoma cell line proliferation and the clone formation,reverse its invasion and metastasis abilities,but had no obvious effect on cell apoptosis.3、The effect of SW on TβR and TGFβ1 downstream signaling pathwayRT-PCR and Western blotting showed that SW could significantly reduced the expression of TβR1,TβR2 mRNA and protein,reduced the expression of Smad2 and 3 protein and inhibit the phosphorylation of Smad2,and inhibited the activation of TGFβ/Smad pathway.The results of lectin showed that TGFβ1 increased the expression of totalβ-1 and 6 branches,while SW decreased the expression of total β-1 and 6 branches.4、Lentivirus construction of stable low expression of GnT-V nasopharyngeal carcinoma cell linesRT-PCR and Western blotting were used to verify the downregulation of GnT-V.5、Lentivirus construction of stable low expression of GnT-V nasopharyngeal carcinoma cell linesRT-PCR and Western blotting showed that the expression of TβR1,T(3R2 mRNA and protein was significantly decreased in thelow expression of GnT-V nasopharyngeal carcinoma cell lines,as well as Smad2,3 and the phosphorylation Inhibited,suggesting that inactivation of TGFβ/Smad pathway.Conclusion1、TGFβ1 promotes the proliferation,invasion and migration of nasopharyngeal carcinoma cell lines,but has no significant effect on cell apoptosis.2、SW can inhibit the activation of TGFβ/Smad pathway by down-regulating the expression of TβR receptor protein and inhibit the biological behavior of TGFβ1-mediated nasopharyngeal carcinoma cell line.3、Targeting GnT-V shRNA lentivirus transfection can significantly decreased the GnT-V mRNA and protein expression.4、Down-regulation of GnT-V can significantly reduce the expression of TβR and inhibit the activation of TGFβ/Smad pathway.