Molecular Mechanism Of Glycosytransferases PST And FUT8 In Breast Cancer

Glycosylation is one of the most essential post-translational modifications in proteins and plays important roles in regulation of protein function by affecting the folding,degradation and the trafficking of protein in cels.While,aberrant glycosylation is always involved in the signal pathway of cancer cells,proliferation and invasion of tumor cels,the interaction between the cell and matrix,angiogenesis and immune regulation,and plays critical roles in the occurrence and development of tumors.At present,the research of protein glycosylation is mainly focused on the new tumor targets and their biological function,but the regulation mechanism of glycosyltransferase is less attended.Sialylation and core-fucosylation are the most important modifications in the development of tumor,therefore,the study on the molecular regulation mechanism of related glycosyltransferases will contribute to understand the role of sialylation and core-fucosylation in the development of cancer and provide theoretical basis for the treatment of cancer.In previous study,we found that PSA enhanced NCAM-mediated cell proliferation and migration and attenuated NCAM-mediated cell adhesion.PSA is catalyzed by two polysialyltransferases,ST8 Sia II(STX)and ST8 Sia IV(PST).However,their expression at mRNA levels in EMT-undergoing NMuMG cels showed significant difference compared to that in control cels.Therefore,elucidation of regulatory mechanism of STX and PST will provide foundational basis for understanding the role of polysialic acid and polysialyltransferase in tumor metastasis.miRNAs(MicroRNAs)are single-stranded noncoding RNAs and growing evidence showed that miRNAs play pivotal roles in many biological processes,ranging from development,organogenesis,proliferation to apoptosis.Aberrant miRNA expression is associated with diverse pathophysiological diseases,such as cancer.However,the mechanism how miRNAs exert their biological function and induce tumor malignant transformation via glycosylation further needs to be illustrated.In this study,expression of glycosyltransferases was analyzed with a glycan-related gene chip in miR-10b-overexpressed MCF10 A cells.Results showed that overexpression of miR-10 b resulted in the significant changes of glycosyltransferases,especially,FUT8.FUT8 specifically catalyzes the transfer of a fucose reside to the innermost GlcNAc residue of N-linked type complex glycopeptide.Upregulation of core fucosylation is associated with the process of carcinogenesis.Therefore,elucidating the mechanism of miR-10 b on FUT8 expression will contribute to better understand the biological function of miRNAs and provide theoretical basis for breast cancer therapy.In this study,we used the mammary gland cels and breast cancer cells of human and mouse as cell models.We clarified the mechanism that different expression of STX and PST in EMT model,analyzed the effect of miR-10 b on glycosyltransferase and glycan,and revealed the mechanism of miR-10 b on regulation of FUT8.The main results are as follows:(1)Differential effects of Pax3 on expression of polysialyltransferases STX and PST.Firstly,we confirmed the change of STX and PST,and detected the expression of STX,PST and transcriptional factor Pax3 between NMuMG and 4T1 cells using RTPCR.Secondly,we analyzed the changes of STX and PST in Pax3-overexpressed NMuMG cels or Pax3-knockdown 4T1 cels.Results showed that Pax3 not only promoted the expression of STX but also down-regulated the expression of PST.Further,luciferase assay and electrophoretic mobility shift assays found that Pax3 can directly bind to the proximal promoter of PST and attenuates its expression.Finally,we found that Pax3 enhanced the expression of NCAM and PSA-NCAM,and promoted cell migration,but showed no effect on cell proliferation.(2)Expression of N-glycan and O-glycan in miR-10b-overexpressed MCF10 A cels.Firstly,the expression of glycosyltransferases was analyzed using a glycan-related gene chip in miR-10b-overexpressed MCF10 A cells.Next,the alteration of N-glycan and O-glycan was analyzed.Finally,expression of glycosyltransferase gene FUT8,MGAT3,OGT and corresponding glycans were evaluated by RT-PCR,western blotting and lectin blotting,respectively.We found that miR-10 b can induce a global influence on glucose metabolism by changing the expression of glycosyltransferases and significantly enhanced the expression of FUT8,MGAT3,OGT.(3)Enhanced cell motility and proliferation by miR-10b/FUT8/p-AKT axis in breast cancer cels.Firstly,we detected the effect of FUT8 on cell motility and proliferation in FUT8-overexpressed MCF10 A cells and FUT8-knockdown MDA-MB-231 cells.Secondly,we detected the expression of FUT8 and p-AKT,and analyzed the effect of miR-10 b inhibitor on cell motility and proliferation in Twist-overexpressed MCF10 A cells and TGF-β-treated MCF10 A cells where miR-10 b was significantly upregulated.In summary,miR-10 b promoted expression of FUT8 and AKT,and enhanced cell motility and proliferation.(4)Modulation of FUT8 expression by miR-10b/TFAP2C/STAT3 pathway in breast cancer cells.Firstly,expression of FUT8 in breast cancer cells and breast cancer tissues was analyzed,and overexpression of FUT8 was validated in breast cancer by immunohistochemistry on breast cancer tissue chip.Bioinformatics analysis showed that FUT8 was modulated by TFAP2 C via the regulation of p-STAT3.Analysis with 1205 breast tissues(including 112 normal breast tissues and 1903 breast cancer tissues)showed a negative correlation between TFAP2 C and FUT8,and the result was demonstrated with immunohistochemistry.Finally,we found that miR-10 b can directly bind to 3’UTR of TFAP2 C and repress its expression confirmed by the luciferase report assay.