Studies On The Molecular Mechanisms Of Fucosyltransferase ? Promoting The Proliferation And Migration Of Thyroid Follicular Cancer Cells

BackgroudsThyroid cancer is the most common malignant tumor of endocrine system,and its incidence is increasing year by year all over the world.Follicular thyroid carcinoma(FTC)is a common type of thyroid cancer with characteristics of high invasion and metastasis,often infiltrate capsule and blood vessels,accompanied by distant organ metastasis such as lung,bone,brain and liver.Currently,there is no definite molecules that can predict the risk of metastasis and evaluate the prognosis of thyroid follicular carcinoma,and its pathological morphology does not accurately indicate its biological behavior.Therefore,investigating the molecular mechanisms that promote the proliferation and metastasis of thyroid follicular carcinoma is of great significance for searching for biomarkers that reflect the invasiveness of the tumor,exploring effective therapeutic targets and improving patients' prognosis.Glycosylation of proteins is an important and common type of post-translational modification of proteins.Glycosyltransferases covalently bind carbohydrates to protein polypeptide chains to form glycosidic bonds,which play a key regulatory role in the processes of cell recognition,receptor activation and signal transmission.Protein glycosylation can be divided into N-glycosylation,O-glycosylation,C-glycosylation and glycosyl phosphatidyl inositol anchoring connections according to the different glycosylation sites.According to different types of catalytic substrates,glycosyltransferase can be divided into various types,such as fucosyltransferase(FUT),glucosetransferase,N-acetyl-glucosetransferase and N-acetyl galactosyl transferase.FUTs,as a member of the glycosyltransferase family,catalyze the transfer of L-focose from the nucleoside diphosphate fucose to the sugar chain terminal of the glycan,forming connections with the ?-glycosidic bond.So far,13 species of fucosyltransferase have been identified in the human genome.According to different types of glucosidase catalyzed,fucosyltransferase can be divided into three categories:FUT1 and 2,which catalyze ?1,2-glucoside bonds formation;FUT3,4,5,6,7,9,10 and 11,which catalyze ?1,3-/1,4 glucoside bonds formation,and FUT8 with catalyzes the formation of ?l,6-glycoside bonds.In addition,there are two types of fucosidase that catalyze the synthesis of O-fucosidase bonds,including protein O-fucosidase 1 and 2(POFUTl and 2).Fucosyltransferase and fucosylated proteins have been shown to be involved in a variety of physiological and pathological processes,including lymphocyte homing,sperm and egg binding,and tumor progression.FUT7 is one of the members of the?l,3-Fucosyltransferase family,which catalyzes the synthesis of al,3-fucose and sialyl Lewis X(sLeX)oligosaccharides.Accumulating evidence shows that FUT7 is highly expressed in tumors,such as hepatocellular carcinoma,lung cancer,breast cancer and so on,closely relates to tumor cell proliferation,migration,invasion,transformation of epithelial mesenchymal(EMT)and poor prognosis.However,the role of FUT7 in the proliferation,migration and invasion of thyroid follicular cancer cells and its related molecular mechanisms are still unclear.Abnormal cell cycle progression is the main mechanism leading to uncontrolled tumor growth.Abnormal expression of CyclinD1,CyclinEl and cell cycle protein-dependent kinase inhibitors P21CIP1,is closely related to abnormal cell proliferation,and tumor formation and development.Matrix metalloproteinase(MMP)is a group of highly homologous zinc-dependent peptides that can degrade the extracellular matrix and basement membrane,thus relating to tumor invasion and metastasis,which is an important indicator of tumor invasion.EMT is an important step leading to the invasion and metastasis of malignant tumors.During the process of EMT,epithelial phenotype markers such as E-cadherin are gradually eliminated,while the expression of mesenchymal markers such as N-cadherin and Vimentin are up-regulated.At present,few studies have been reported on the effects of FUT7 on the proliferation,EMT,migration and invasion of thyroid follicular cancer cells.Epidermal growth factor receptor(EGFR)is a tyrosine kinase receptor.When it binds to EGF,conformational changes occur,forming homodimers and self-phosphorylation to initiate the downstream signaling cascades,including mitogen-activated protein kinase(MAPK)and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(PKB/Akt)pathways.They play important roles on the biological behavior of tumor cells,including proliferation,migration,and apoptosis.Glycosylation regulates the structure and function of EGFR receptor,and can affect the receptor's localization on cell surface,ligand binding,conformational stability,dimerization and cell signal transduction,so as to regulate the proliferation,migration and invasion of tumor cells.Whether FUT7 and the ?1,3-fucose catalyzed by FUT7 regulate the EGF/EGFR signaling pathways in thyroid follicular cancer cells is worthy of in-deepth exploration.This study mainly includes:1.Clinical studies on 49 thyroid follicular carcinoma cases(including 5 metastasis cases)to analyze the expression characteristics of FUT7 in thyroid follicular carcinoma and its correlation with tumor biological behavior.2.The expression levels of FUT families in different invasive capacities of thyroid follicular cancer cells were examined using low invasive FTC-133 and high invasive FTC-238 cell lines.The effects of FUT7 on the proliferation,migration and invasion of human thyroid follicular cancer cells were investigated.3.Studies on the effects of FUT7 on EGFR and its downstream MAPK pathway and PI3K-Akt pathways to explore the molecular mechanisms of FUT7 regulating the proliferation,EMT and invasion and migration of thyroid follicular cancer cells.With the main focus on FUT7,this study innovatively explores the molecular mechanisms underlying the proliferation,invasion and metastasis of thyroid follicular carcinoma,in order to find potential biomarkers for the prognosis assesment of thyroid follicular carcinoma,explore novel thereupetic targets of thyroid follicular carcinoma,and build a foundation for improving the prognosis of thyroid follicular carcinoma.In this study,the regulatin on tumorigenesis and progression were studied from the perspective of posttranslational protein modification--glycosylation.We deeply explored the regulatory effects of FUT7 on the proliferation and migration of thyroid follicular cancer cells that have higher levels of invasiveness and metastasis.This is the first study investigating the molecular mechanism of FUT7 on regulation of the proliferation and migration of thyroid follicular cancer cells.Our innovative study aims to find potential biomarkers for the prognosis of thyroid follicular carcinoma,explore new effective targets for the treatment of thyroid follicular carcinoma,and lay the foundation for improving the prognosis of thyroid follicular carcinoma This study is divided into the following three parts:1.Studies on the expression of FUT7 in human thyroid follicular carcinomaMethods:(1).Immunohistochemistry was performed to detect the expression of FUT7 in 49 cases of thyroid follicular carcinoma and their paracancerous thyroid tissues.The relationship between the expression levels of FUT7 and the clinical and pathological features of thyroid follicular carcinoma was analyzed.(2).The amount of sleX oligosaccharides and ?1,3-fucose in thyroid follicular carcinoma tissues were examined using immunofluorescence and lectin fluorescent staining methods.(3).MMP2 expression levels were analyzed in 11 age-and sex-matched high and low FUT7 expression thyroid follicular carcinoma cases by immunohistochemistry.(4).The expression levels of FUT7,E-cadherin and Vimentin in the primary and metastatic tissues of thyroid follicular carcinomas were analyzed by immunohistochemical staining.Results:(1).FUT7 was expressed in the cytoplasm of thyroid follicular cancer cells.The expression prevalance and expression levels of FUT7 in thyroid follicular carcinoma were higher than those in paracancerous thyroid tissues.The expression levels of FUT7 in thyroid follicular carcinoma were positively associated with tumor size.(2).The amount of sleX oligosaccharides and al,3-fucose in thyroid follicular carcinoma tissues were increased compared to the paracancerous tissues.(3).MMP-2 expression was increased in thyroid follicular carcinoma tissues in the FUT7 high-expression group.FUT7 expression was positively associated with MMP-2 expression.(4).The expression levels of FUT7 and Vimentin were increased,while E-cadherin were decreased in thyroid follicular carcinoma metastases tisses compared to the primary tumor issues.Conclusions:(1).The expression levels of FUT7 were increased in thyroid follicular carcinoma and was positively associated with tumor size.(2)The amount of sleX oligosaccharides and ?-1,3 fucose were increased in thyroid follicular carcinoma.(3).FUT7 might play a role in promoting the migration and invasion potential of thyroid follicular cancer cells.(4).FUT7 might play a role in promoting the process of epithelial mesenchymal transformation of thyroid follicular cancer cells.2.Studies on the effects of FUT7 on the proliferation,migration and invasion of thyroid follicular cancer cellsMethods:(1).The gene expression levels of the members of the FUT families in low invasive thyroid follicular cancer cells FTC-133 and high invasive follicular cancer cells FTC-238 were examined by real-time PCR.(2).The expression levels of FUT7 in FTC-133 cells and FTC-238 cells were compared by Western blot and immunocytochemistry.(3).The expression levels of FUT7 in FUT7-overexpressing FTC-133 cells and FUT7-RNA interferenced FTC-238 cells were detected by Real-time PCR and Western blot.(4).The amount of ?1,3-fucose levels were compared between FUT7-overexpressing FTC-133 cells and FUT7-RNA interferenced FTC-238 cells by Lectin blot and Lectin fluorescent staining.(5).The expression levels of CyclinD1,CyclinE1 and P21CIP1 in FUT7-overexpressing FTC-133 cells and FUT7-RNA interferenced FTC-238 cells were examined by Western blot and Real-time PCR.(6).The proliferation capacity of FUT7-overexpressing FTC-133 cells and FUT7-RNA interferenced FTC-238 cells was examined by CCK8 assay and EdU assay,respectively.(7).The expression levels of epithelial mesenchymal transition markers(E-cadherin,N-cadherin and Vimentin)in FUT7-overexpressing FTC-133 cells and FUT7-RNA interferenced FTC-238 cells were examined by Real-time PCR and western-blot.(8).The expression levels of MMP-2 and MMP-9 in FUT7-overexpressing FTC-133 cells and FUT7-RNA interferenced FTC-238 cells were examined by Real-time PCR,Western-blot and Gelatin zymography assay.(9).Migration and invasion capacity of FUT7-overexpressing FTC-133 cells and FUT7-RNA interferenced FTC-238 cells were examined by Migration and Invasion Transwell experiments.Results:(1).The expression levels of FUT7 gene in highly invasive FTC-238 thyroid follicular carcinoma cells were higher than those of other FUTs.(2).The expression levels of FUT7 mRNA and protein in highly invasive FTC-238 thyroid follicular carcinoma cells were higher than that of weekly invasive FTC-133 cells.(3).FUT7 expression levels and the amount of ?1,3-fucose were increased in FUT7-overexpressing FTC-133 cells but decreased in FUT7-RNA interferenced FTC-238 cells compared to control groups,.(4).Overexpression of FUT7 upregulated the mRNA and protein levels of PCNA,CyclinD1 and Cyclin El in FTC-133 cells,while the expression levels of P21CIP1 were decreased.Knocking-down of FUT7 expression decreased the mRNA and protein levels of PCNA,CyclinDl and Cyclin El in FTC 238 cells,while the expressions of P21CIP1 were increased.(5).Overexpression of FUT7 promoted the proliferation of FTC-133 cells while knocking-down of FUT7 inhibited the proliferation of FTC-238 cells.(6).Overexpression of FUT7 decreased the expression of E-cadherin in FTC 133 cells,while the expression of N-cadherin and Vimentin were increased.Knocking-down of FUT7 increased the expression of E-cadherin in FTC-238 cells,while the expression of N-cadherin and Vimentin were decreased.(7).Overexpression of FUT7 increased the expression of MMP-2 and MMP-9 in FTC-133 cells.Knocking-down of FUT7 decreased the expressions of MMP-2 and MMP-9 in FTC-238 cells.(8).Overexpression of FUT7 enhanced the migration capacity of FTC-133 cells.Knocking-down of FUT7 inhibited the migration capacity of FTC-238 cells.Conclusions:(1).FUT7 expression is higher in highly invasive thyroid follicular carcinoma than that in weekly invasive thyroid follicular carcinoma.(2).FUT7 promotes the proliferation,EMT and migration of thyroid follicular cancer cells.3.Studies on the effects of FUT7 on EGFR and its downstream signaling pathways in thyroid follicular cancer cellsMethods:(1).The ?1,3-fucosylation levels of EGFR in FUT7-overexpressing FTC-133 cells and FUT7-RNA interferenced FTC-238 cells were examined by lectin fluorescent staining.(2).The expression and phosphorylation of EGFR in FUT7-overexpressing FTC-133 cells and FUT7-RNA interferenced FTC-238 cells were examined by immunofluorescence.(3).The expression levels of EGFR,phosphorylated EGFR,Erk1/2,phosphorylated Erkl/2,Akt and phosphorylated Akt in FUT7-overexpressing FTC-133 cells and FUT7-RNA interferenced FTC-238 cells were examined by Westen blot.(4).The expression levels of EGFR,phosphorylated EGFR,Erk1/2,phosphorylated Erkl/2,Akt and phosphorylated Akt in FUT7-overexpressing FTC-133 cells and FUT7-RNA interferenced FTC-238 cells were examined by Westen blot in culture in the absence of serum or with EGF stimulation.(5).The expression levels of EGFR,phosphorylated EGFR,Erk1/2,phosphorylated Erkl/2,Akt,phosphorylated Akt,PCNA,CyclinDl,Vimentin,N-cadherin and MMP-2 were examined by Westen blot after FTC-133 and FTC-238 cells were treated with different concentrations of EGFR inhibitors(Erlotinib).(6).The effects of overexpression of FUT7,RNA interference of FUT7 and Erlotinib on the levels of EGFR,phosphorylated EGFR,Erk1/2,phosphorylated Erk1/2,Akt and phosphorylated Akt,as well as the expression levels of PCNA,CyclinD1,Vimentin,N-cadherin and MMP-2 were compared in thyroid follicular cancer cells FTC-133 and FTC-238 after EGF stimulation by Westen blot.Results:(1).Overexpression of FUT7 increased the amounts of ? 1,3-fucose in FTC-133 cells.Knocking-down of FUT7 decreased the amounts of ? 1,3-fucose in FTC-238 cells.(2).Overexpression of FUT7 increased EGFR phosphorylation levels in FTC-133 cells.Knocking-down of FUT7 decreased the phosphorylation levels of EGFR in FTC-238 cells.(3).Overexpression of FUT7 upregulated the phosphorylation levels of EGFR,erk1/2 and Akt in FTC-133 cells.Knocking-down of FUT7 decreased decreased the phosphorylation levels of EGFR,Erk1/2 and Akt in FTC-238 cells.(4).After EGF stimulation,the phosphorylation levels of EGFR,Erk1/2 and Akt were further increased in FUT7 overexpressing FTC-133 cells;while the phosphorylation levels of EGFR,Erk1/2 and Akt were further decreased in FUT7 knocking down FTC-238 cells.(5).EGFR inhibitor,Erlotinib,reduced the phosphorylation levels of EGFR,Erk1/2 and Akt in FTC-133 cells and FTC-238 cells,as well as the expression levels of PCNA,CyclinD1,Vimentin,N-cadherin and MMP-2 proteins.(6).Overexpression of FUT7 further promoted the EGF-dependent phosphorylation and activation of EGFR,Erk1/2 and Akt,increased the expression of downstream molecules such as PCNA,CyclinD1,Vimetnin,N-cadherin and MMP-2 in FTC-133 cells.Knocking down of FUT7 inhibited the EGF-dependent phosphorylation and activation of EGFR,Erk1/2 and Akt,reduced the expression of downstream molecules such as PCNA,CyclinD1,Vimetnin,N-cadherin and MMP-2 in FTC-238 cells.Conclusions:(1).FUT7 promotes the ?1,3-fucosylation and phosphorylation of EGFR in thyroid follicular cancer cells.(2).FUT7 enhances the phosphorylation of ERK1/2 and Akt in follicular thyroid cancer cells,thus promotig the activation of MAPK and PI3K/Akt signaling pathways.(3).FUT7 increases the expression levels of PCNA,CyclinD1,Vimentin,N-cadherin and MMP-2 proteins in thyroid follicular cancer cells,suggesting that FUT7 regulates the intracellular signal transduction and its downstream regulators on tumor cell proliferation and metastasis through EGFR-dependent pathways.