| Breast cancer is one of the most common malignancies in women.It is reported that the number of new cases and deaths of breast cancer in China accounts for 12.2% and 9.6% respectively in the world every year,and the number is increasing year by year.Breast cancer has a high degree of heterogeneity,potential invasiveness and complex biological behavior,which makes its diagnosis and treatment more difficult,and its drug resistance rate is increasing year by year.Therefore,the study of molecular markers of breast cancer have great significance for timely understanding of its malignancy,predicting metastasis and predicting prognosis.Glycosylation of proteins refers to the process in which glycosyltransferases transfer sugar chains to proteins and form glycosidic bonds with specific amino acid residues on proteins.Glycosylation plays an important role in protein modification and regulates protein function.According to the glycosidic chain types,mammalian protein glycosylation can be divided into three types,namely O-glycosidic bond type,N-glycosidic bond type,and GPI glycosylphosphatidylinositol anchor type.N-glycosylation occurs in the process of target protein translation.The glycosylated structure is Asn-X-Ser /Thr,Asn(asparagine),Ser(serine),Thr(threonine),and X is any amino acid except proline.Glycosylated proteins have been detected in human tumor cells for decades.In recent years,studies have shown that N-glycosylation is a key regulator of tumorigenesis.It has been reported that the N-glycosylation of the epithelial cadherin can increase the adhesion of gastric cancer cells when they invade.Therefore,the study of N-glycosylation will help us further explore the underlying mechanism of tumorigenesis.Epithelial cell adhesion molecule(EpCAM)is expressed in a variety of organizational type I transmembrane glycoprotein,EpCAM positioning of the human genetic code on chromosome 2 p21 the molecular weight of about 40 kd,aa is composed of 314 amino acids(amino acid)of peptides,the main structure is made up of 242 aa extracellular region(called EpEX),consisting of 23 aa area across the membrane,intracellular area of 26 aa(EpICD).Studies have shown that EpICD expression loss often occurs in human epithelial carcinoma.There are three glycosylation sites in the extracellular domain of EpCAM(N198,N111,N74),and mutation of N198 glycosylation site will lead to decreased expression of EpCAM.In addition,compared with the wild-type,three glycosylation sites knockout can reduce the half-life of the protein from 21 hours to 7 hours,so N-glycosylation sites play a crucial role in the biological activity of EpCAM.EpCAM is a marker of pluripotent stem cells,and its expression decreases as stem/group cells differentiate into different mature cell lines.Therefore,EpCAM variation may play an important role in human malignant tumors.The level of EpCAM is not only affected by gene expression change,but also by transcription and post-translation modification level.Therefore,glycosylation may play an important role in the regulation of EpCAM cell surface expression.Previous studies have found that EpCAM in breast cancer Epithelial mesenchymal transformation(EMT)played a role in the process,but cut the expression of EpCAM can inhibit the growth and metastasis of breast cancer cells,it is by inhibiting Ras/Raf extracellular signal regulating kinase pathway and matrix metalloproteinases MMP-9.These results suggest that EpCAM can not only serve as a regulatory molecule for breast cancer cells’ growth,but also as a potential marker for its prognosis.Autophagy refers to the process of degrading and circulating damaged organelles,proteins or lipids in vesicles.Degradation occurs after the fusion of autophagosomes and lysosomes,and the degradation products are recovered in the cytoplasm.Autophagy is very important for the clearance of damaged organelles such as mitochondria and unfolded proteins,and can maintain metabolic balance in vivo under stress conditions such as nutrient deficiency.Autophagy comes in three forms,macrophage,microautophagy,and chaperone mediated autophagy.Giant autophagy(hereinafter referred to as autophagy)is a kind of the processes of food,eating protein,complex or organelles cytoplasm into autophagy body(a cytoplasmic double membrane structure),autophagy body or be integrated into the late endosome,or be transported to the lysosomes,fusion with lysosomes to generate autolytic enzyme,then under the action of acid hydrolysis enzyme release and degradation.The resulting degradation products include nucleotides,amino acids,fatty acids and sugars,which are recycled for general cellular metabolism.Previous studies have found that MCF-7 and MDA-MB-231 cells of the glycosylated EpCAM can regulate apoptosis-related proteins through the ERK/MAPK signaling pathway and increase the cytotoxic effect of 5-fluorouracil(5-Fu).Studies have shown that it also playing an important role in apoptosis,the ERK pathway can also regulate different modes of cell death by regulating Akt and mTOR.Early reports indicated that the pathway involving ERK1/2 can cause autophagic cell death.Moreover,PI3K/Akt/mTOR is a recognized autophagy regulatory pathway.Based on the above literature,we speculate that EpCAM N-glycosylation can regulate autophagy of breast cancer cells through the PI3K/Akt/mTOR signaling pathway,and affect their proliferation and apoptosis.Experimental methods:1.Expressions of autophagy related proteins Beclin1,P62 and LC3 were detected by western-blot.2.pGFP-LC3 green fluorescence fusion protein assay and transmission electron microscopy were used to detect the number of autophagosomes in breast cancer cells modified by EpCAM N-glycosylation.3.Western-blot analysis of the expression of Caspase-3,Bcl2,Bax and proliferating protein PCNA in breast cancer cells with EpCAM N-glycosylated mutation after adding autophagy regulator.4.CCK8 assay was used to detect the proliferation of breast cancer cells with EpCAM N-glycosylated mutation after adding autophagy regulator.5.Western-blot detection of the expression of autophagy related proteins and the proteins of pathway PI3K/Akt/mTOR.6.pGFP-LC3 green fluorescent fusion protein assay and transmission electron microscopy were used to detect the number of autophagosomes in breast cancer cells with EpCAM N-glycosylated mutation after adding Akt inhibitor MK-2206.Experimental results:1.EpCAM N-glycosylation mutation in breast cancer cells,autophagy Beclin1 protein LC3 Ⅱ/LC3 Ⅰ increase,P62 decrease.2.The results of pGFP-LC3 green fluorescence fusion protein experiment and transmission electron microscopy showed that the autophagosome of breast cancer cells with EpCAM N-glycosylation mutation was significantly increased.3.Adding autophagy activator to breast cancer cells with EpCAM N-glycosylated mutation,the expression of Cleaved Caspase-3 and Bax were increased,while the expression of Bcl2 was decreased.PCNA expression of proliferation-related protein was decreased.4.CCK8 experiments showed that the proliferation ability of breast cancer cells increased when adding autophagy inhibitors.While adding of autophagy activator,the cell proliferation ability was weakened.However,the proliferation ability of breast cancer cells with EpCAM N-glycosylated mutation and the addition of autophagy regulator were significantly reduced.5.After adding of Akt inhibitor MK-2206,the expression of proteins p-Akt and p-mTOR in the PI3K/Akt/mTOR pathway of breast cancer cells with EpCAM N-glycosylated mutation was significantly reduced,while the expression of autophagy protein was increased.6.The results of pGFP-LC3 green fluorescence fusion protein experiment and transmission electron microscopy both showed that adding of Akt inhibitor MK-2206,the number of autophagosome in breast cancer cells with EpCAM N-glycosylation mutation was significantly increased.Experimental conclusion:1.Knockout of EpCAM N-glycosylation can enhance the autophagy ability of breast cancer cells.2.Breast cancer cells with EpCAM N-glycosylation knockout can inhibit their proliferation ability and enhance their apoptosis ability by upregulation of autophagy.3.EpCAM N-glycosylation knockout can enhance autophagy of breast cancer cells by inhibiting the PI3K/Akt/mTOR pathway... |
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