The Study On The PpGalNAc-T4 Function And Specific Substrate In Human Breast Cancer Cells

Breast cancer,one of the most frequently diagnosed and aggressive malignancies,is the major cause of cancer-related death greatly threatening women health.Malignant proliferation and metastatic are main causes of poor prognosis and death in breast cancer patients.The study on the mechanism of breast cancer metastasis will provide an important experimental basis for the discovery of new target molecules for breast cancer diagnosis and treatment.The occurrence and development of breast cancer and other malignant tumors are closely related to the abnormal glycosylation of O-GalNAc protein.O-GalNAcylation is initiated by a family of up to 20 homologous genes encoding polypeptide N-acetylgalactosaminyltransferases(ppGalNAc-Ts),each displaying selective tissue and substrate protein specificity which is supposed to be one of the difficult points in glycobiology research.Studies have shown that ppGalNAc-T4,a member of the ppGalNAc-Ts family,plays a tumor suppressive role in a variety of epithelial tumors.However,the specific substrates and the glycosylation sites of ppGalNAc-T4 has rarely been reported.The function and mechanism of ppGalNAc-T4 in breast cancer remains unclear.This study aims to elucidate the role and mechanism of ppGalNAc-T4 in breast cancer metastasis,discover and confirm its specific substrates,and explore the potential application value of ppGalNAc-T4 in the diagnosis and treatment of breast cancer.The main word of this thesis is as follows:1.ppGalNAc-T4 inhibits human breast cancer cell proliferation and its molecular mechanism.The expression level of ppGalNAc-T4 in human breast cancer tissues is significantly higher than that in normal breast cancer tissues,and the high expression level of ppGalNAc-T4 is positively correlated with prognostic relapse-free survival rate of breast cancer patients from the analysis of gene expression based on TCGA and GEO database.Using MTT and colony formation assay,it was found that ppGalNAc-T4 inhibited cell proliferation when ppGalNAc-T4 was upregulated in high proliferative ability MDA-MB-231 cells and knocked down in MCF7 cells with low proliferative ability.Furthermore,cell cycle monitoring,transcriptome sequencing and differentially expressed gene analysis indicated that ppGalNAc-T4 inhibited the activity of Notch signaling pathway and regulated the expression of cell cycle-related proteins such as Cyclin D1 and p27,thereby slowing down the cell cycle process and inhibiting the proliferation of MDA-MB-231 and MCF7 in human breast cancer cells.2.ppGalNAc-T4 inhibits human breast cancer cell metastasis and its molecular mechanism.The human breast cancer cell lines MCF7 and MDA-MB-231 were used as experimental materials.Crystal violet staining showed that cells exhibited an epithelial morphology after ppGalNAc-T4 overexpression and a mesenchymal morphology after ppGalNAc-T4 knockout.Cell wound healing and Transwell chamber assay elucidated that ppGalNAc-T4 inhibited cell metastasis in breast cancer cells.Real-time PCR,Western blot and immunofluorescence indicated ppGalNAc-T4 suppressed breast cancer cells metastasis via TGF-β induced EMT process.3.Transforming growth factor-β type Ⅱ receptor(TβR Ⅱ)was found to be a new catalytic substrate for ppGalNAc-T4.Firstly,the interaction of ppGalNAc-T4 and TβR Ⅰ,TβR II was confirmed by co-immunoprecipitation experiment in human embryonic kidney HEK-293T cells,suggesting that TβR Ⅰ and TβR Ⅱ might be the substrates of ppGalNAc-T4.Secondly,MCF7 and HEK-293T cells were transfected with TβR Ⅰ and TβR Ⅱ vector,respectively.Lectin immunoprecipitation analysis using lectin VVL which can specifically recognize O-GalNAc modification showed that TβR Ⅰ and TβR Ⅱ were O-GalNAcylated and this modification was regulated by the expression of ppGalNAc-T4.Further,immunoprecipitation experiments showed that the O-GalNAc modification catalyzed by ppGalNAc-T4 inhibited the interaction between TβR Ⅰ and TβR Ⅱ.In addition,extracellular domain of TβR Ⅰ and TβR Ⅱ were proved O-GalNAcylated and this modification was regulated by the expression of ppGalNAc-T4 by immunoprecipitation assay in MCF7 and HEK-293T cells.Moreover,it was indicated that ppGalNAc-T4 directly catalyzed O-GalNAc modification of extracellular domain of TβR Ⅱ through high performance liquid chromatography(HPLC)and mass spectrometry(MS)in vitro.4.Ser31 was found to be the O-GalNAc modification site of TβR Ⅱ catalyzed by ppGalNAc-T4 which inhibited TGF-β signaling pathway.By means of site mutation,S31A was detected with no O-GalNAc modification.Intra-cellular mutation assay showed that ppGalNAc-T4 specifically catalyzed O-GalNAc modification of TβR Ⅱ at Ser31 which inhibited the dimerization of TβR Ⅰ and TβR Ⅱ,thereby suppressing the TGF-β signaling pathway in human breast cancer cells.The above results indicated that,ppGalNAc-T4 inhibiting breast cancer cells proliferation by which that ppGalNAc-T4 effected on Notch signaling pathway,regulated its downstream target genes Cyclin D1 and p27,slowed down cell cycle progression.TβR Ⅱ was new substrate of ppGalNAc-T4 and ppGalNAc-T4 catalyzed O-GalNAc modification of TβRⅡ inhibited TGF-β induced EMT,therefore suppressing metastasis potential of breast cancer cells.This paper revealed the function and molecular mechanism of ppGalNAc-T4 on breat cancer and provided an experimental basis for the study of ppGalNAc-Ts isoenzyme substrates and also a new target and idea for the diagnosis and treatment of breast cancer.