| Glycosylation is considered to play a key role in the malignant transformation of tumors and is closely related to the pathways of cancer progression.Sialylation changes are common in cancer,and sialylation products mediate pathophysiological reactions at different stages of tumor progression.Intracellular sialylation is a biochemical process catalyzed by intracellular sialyltransferase.Sialyltransferase is a series of enzymes that catalyze the synthesis of sugar chains in disaccharides,polysaccharides and sugar complexes.In malignant tumors,ST6GALNAC1 adds sialic acid to the ?-2,6 chain of serine or threonine residues,which plays an important role in various types of tumors.Wnt/?-catenin signal pathway is a part of Wnt signal pathway,which plays an important role in human development and the maintenance of adult homeostasis.Its abnormal activation is closely related to a variety of human cancers.When Wnt protein binds to the Frizzled receptor family on the cell surface,it will cause a series of intracellular reactions,including the activation of Dishevelled receptor family proteins and the final change of ?-catenin level in the nucleus.Dishevelled is a key component of cell membrane-related Wnt receptor complex.It binds to Wnt and activates,which inhibits the degradation of downstream protein ?-catenin.When ?-catenin receptor degradation complex is inhibited,?-catenin can stably exist in the cytoplasm.Part of ?-catenin enters the nucleus to interact with TCF/LEF transcription factor family and promote the expression of specific genes,such as cell cycle and apoptosis-related genes c-myc and Cyclin D1,growth factor VEGF,gastrin,HGF,Cmurmer,and genes involved in tumor progression,such as MMP7,CD44,ITF-2,COX2 and so on.Therefore,the abnormal activation of Wnt signal is one of the important causes of cancer.The change of glycosylation level will affect many physiological responses,including Wnt/?-catenin signaling pathway.Wnt protein itself is N-glycoprotein,for example,N-glycan modification in Wnt3 a is necessary for its function.But whether Wnt3 a can be modified by O-glycan is still unknown.According to the study,the proportion of CD44 in exosomes of abnormal O-glycosylated cells in colorectal cancer is significantly higher than that of normal cells,so we know that glycosylation can affect the secretion of exosomes.Wnt3 a is a secretory protein.In order to perform its function,it will depend on exosomes to secrete into the cell and play a role.We speculate that the glycosylation modification of Wnt3 a may affect its exosomes secretion.Many studies have shown that drug resistance is related to glycosylation.For example,in multidrug resistant tumor cells,the ability of ceramide glycosylation becomes stronger,which blocks the glycosylation of ceramide,and then increases the expression level of ceramide in cells,and induces apoptosis of multidrug resistant cell(Multidrug Resistance,MDR).However,the role of ST6GALNAC1 in drug resistance is not clear.Based on the above research background,we found that ST6GALNAC1 mediated sialidation plays an important role in the process of tumor,but its mechanism is not clear.In addition,Wnt3 a,as an important ligand of Wnt signaling pathway,its glycosylation modification is related to its function and secretion.However,whether it can be sialylated and whether it can affect its secretion has not been reported.The results of our on-line tool analysis suggest that Wnt3 a has a site that can be sialylated.Therefore,we hypothesized that the abnormal expression of ST6GALNAC1 in tumor cells may affect its abnormal secretion by sialyzing Wnt3 a,and then affect the classical Wnt/?-catenin signaling pathway,and finally affect the tumor progression and drug sensitivity.In order to test the hypothesis,we will start from the following experimental purposes.Purpose of the study: To explore the effect of ST6GALNAC1 on sialylation modification and function of Wnt3 a.To explore the effect of ST6GALNAC1 on the invasion and migration ability of cancer.To explore the role of ST6GALNAC1 in cisplatin-resistant ovarian cancer cells.Experimental methods:(1)Lentiviral vector system was used to construct A375,SK-MEL-28 and A2780 s cell lines with stable knockdown of ST6GALNAC1 gene.(2)Online prediction and detection of sialylation of Wnt3 a protein by lectin precipitation reaction.(3)Western Blot and real-time fluorescence quantitative PCR were used to verify whether the ST6GALNAC1 gene was knocked down.(4)Western Blot was used to detect whether ST6GALNAC1 affected the expression of proteins related to Wnt/ ?-catenin signal pathway.(5)CCK-8 test,scratch test and Transwell test were used to detect the changes of proliferation,migration and invasion of cells after knockdown of ST6GALNAC1 gene.(6)The expression of Wnt3 a protein in exosome was detected by Western Blot.(7)Western Blot and real-time fluorescence quantitative PCR were used to detect the expression of ST6GALNAC1 gene in drug-resistant cells.(8)A2780cp cell line stably knocking down ST6GALNAC1 gene was constructed by lentiviral vector system.(9)The change of drug resistance of cells was detected by CCK-8 test.(10)Scratch test and Transwell test were used to detect the changes of cell migration and invasion.Experimental results:(1)Western Blot and real-time fluorescence quantitative PCR showed that the expression of ST6GALNAC1 was decreased after the stable knock-down ST6GALNAC1 gene was constructed by lentivirus vector system.(2)Online prediction and lectin precipitation showed that Wnt3 a protein could be sialylated by ST6GALNAC1.(3)After knocking down ST6GALNAC1 gene,the results of CCK-8 assay showed that the ability of cell proliferation decreased,the results of scratch test showed that the ability of cell migration decreased,and the results of Transwell test showed that the ability of cell invasion decreased.(4)The results of Western Blot experiment showed that the expression of proteins related to Wnt/?-catenin signal pathway decreased after ST6GALNAC1 gene knockdown.(5)Western Blot showed that the amount of Wnt3 a transported in the exocrine body decreased,and the amount of exocrine body secreted by cells decreased.(6)Western Blot and real-time fluorescence quantitative PCR were used to detect the high expression of ST6GALNAC1 gene in drug-resistant cells.(7)CCK-8 assay showed that ST6GALNAC1 increased the resistance of A2780cp-resistant cells to cisplatin.(8)Scratch test and Transwell test showed that ST6GALNAC1 inhibited the migration and invasion of A2780cp-resistant cells.Experimental conclusion: ST6GALNAC1 can glycosylate Wnt3 a protein and change the expression of ST6GALNAC1 leads to the change of protein related to Wnt/?-catenin signal pathway,while the change of protein related to Wnt/?-catenin signal pathway leads to phenotypic changes such as proliferation,migration and invasion of cancer.The change of ST6GALNAC1 expression leads to the change of glycosylation level,and the change of glycosylation level leads to the change of exosome secretion.ST6GALNAC1 is highly expressed in drug-resistant A2780 cp cells,which can enhance the resistance of cells to cisplatin and increase the ability of invasion and migration of cells. |
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