O-GlcNacylation Promotes Crc Metastasis Via The MiR-101/O-GlcNAc/EZH2 Regulatory Feedback Circuit

Background:Tumor metastasis represents a multistep cellular biological event termed the invasion-metastasis cascade,whereby epithelial cells in primary tumors disseminate as cancer cells to anatomically distant organs and subsequently adapt to the foreign microenvironments.Increased risk of cancer-related death is a serious consequence of metastasis.Despite significant advances in the detection and treatment of colorectal cancer(CRC),many patients still die from local or distant metastasis.Although a substantial number of molecules have been identified that reveal important aspects of CRC metastasis,the critical molecular basis behind CRC metastasis is still largely unknown.In addition,most of the current investigations are focused on genetic mechanisms and cellular signaling pathways,and little is known about the metabolic and epigenetic mechanisms involved in this process.The abnormal change of specific protein function is the initiating factor of tumor development and obtaining malignant biological phenotypes.The function of protein is not only related to its expression level,but also closely depends on its own activity.Posttranslational modification(PTM)of proteins is one of the main ways to regulate their activities.With the development of oncology research,it has been found that abnormal PTM of proteins play an important role in tumor development.O-GlcNAcylation has been identified as a dynamic and reversible posttranslational modification that regulates diverse cellular processes,such as cell signal transduction,protein translation and proteasomal degradation.Recently,O-GlcNAcylation was found to be increased in CRC cells to enable the proliferative and migratory properties of these cells.However,the potential effect of O-GlcNAcylation and the mechanism of its action in CRC metastasis are still not well understood.Aims:To explore the mechanism of O-GlcNAc glycosylation and its key enzyme OGT increasing in colorectal cancer and its molecular mechanism of promoting CRC metastasis.1.To determine the expression of O-GlcNAc in CRC and its correlation with invasion and metastasis of CRC;2.To explore the mechanism of O-GlcNAc promoting the invasion and metastasis of CRC;3.To clarify the effect of O-GlcNAc on the stability and function of EZH2;4.To reveal the reason for the increase of O-GlcNAc and OGT in CRC;5.To explore the mechanism of feedback regulation of miR-101 expression by O-GlcNAc and EZH2;6.To verify the correlation between O-GlcNAc,EZH2,OGT and miR-101 in CRC.Methods:1.Immunohistochemistry and CRC tissue chip were used to detect O-GlcNAc,OGT and OGA expression levels in cancer tissue and its matched adjacent tissues and lymph node metastasis foci,and the correlation between O-GlcNAc and clinicopathological parameters was analyzed;Oncomine database was used to analyze OGT and OGA m RNA expression levels in CRC and its adjacent tissues;protein and m RNA of CRC cell line and immortalized normal colorectal epithelial cell line were extracted for detection of OGlcNAc,OGT and OGA expression levels;O-GlcNAc level of SW-480 and SW-620 were manipulated by lentivirus or small interfering RNA transfection or OGA inhibitor treatment and the function of O-GlcNAc in CRC invasion and metastasis was determined by Transwell migration and invasion assay,3D spheroid basal membrane extract(BME)cell invasion assay and tumor metastasis model in nude mice;2.Western blot,q RT-PCR and immunofluorescence were used to detect the changes of EMT related markers;Co-IP and mass spectrometry were used to detect the potential O-GlcNAc modified proteins;small interfering RNA or EZH2 inhibitors were used to detect the necessity of EZH2 for O-GlcNAc to promote the invasion and metastasis of colorectal cancer;3.Co-IP was used to verify the binding of OGT and EZH2 in CRC cells;Yin OYang1.2 database was used to predict the potential O-GlcNAc sites of EZH2 protein;the halflife and ubiquitination level of EZH2 protein were detected by Western Blot to evaluate the effect of O-GlcNAc on EZH2 stability and function;4.Several web-based target prediction algorithms(Target Scan S,miRanda,Pictar,and PITA)were used to identify miRNAs that could potentially target OGT and a luciferase reporter assay was performed to further verify this regulation;Mi R-101 mimics or inhibitor was transfected into SW-620 or SW-480 cells,and the protein and m RNA were extracted to verify the regulation of miR-101 on OGT,O-GlcNAc and EZH2levels;Transwell migration and invasion assay and 3D spheroid BME cell invasion assay were used to explore the role of miR-101 in invasion and metastasis of CRC;5.Ch IP-q PCR,agarose gel electrophoresis and miR-101 precursor detection were implemented to explore the feedback regulation of O-GlcNAc on miR-101 transcription;6.EZH2 and OGT protein expression levels were analyzed by western blotting and the miR-101 level was analyzed by real-time PCR in 30 freshly collected CRC tissues and adjacent normal tissues;Immunohistochemistry and three consecutive sections of CRC tissue chips were used to detect O-GlcNAc,OGT and EZH2 expression levels in cancer tissue and its matched adjacent tissues,and the correlation between O-GlcNAc,OGT and EZH2 were analyzed by Pearson correlation analysis.Results:1.The IHC staining of the CRC tissue microarrays showed that O-GlcNAcylation was significantly elevated in cancer tissues compared with that in normal tissues,and highest in lymph node metastases foci.The patients with higher O-GlcNAcylation had shorter overall survival than those with lower O-GlcNAcylation,indicating that the OGlcNAcylation level was an independent risk factor for a poor prognosis in CRC patients.Importantly,the cell lines with a high metastatic potential,i.e.Lo Vo and SW620,which were established from distant metastatic foci,exhibited higher expression levels of OGlcNAcylation than the other CRC cell lines.Transwell migration and invasion assay,3D spheroid BME cell invasion assays and xenograft model confirmed that O-GlcNAcylation promotes the migratory and invasive capacities of colon cancer cells in vitro and in vivo.2.Western Blot and Immunofluorescent assay showed the expression of fibronectin,vimentin and Snail 1,which are the major markers of mesenchymal cells,decreased when OGT was downregulated in the SW620 cells,whereas the localization of Claudin 7 and Ecadherin on the cell membrane was increased.Consistent with these results,these markers showed the opposite change when OGlcNAcylation was upregulated by PUGNAc or TMG treatment in the SW480 cells.EZH2 was identified as a potential O-GlcNAc modifying protein by Co-IP and LC-MS/MS.O-GlcNAc promotes the invasion and metastasis of CRC by up regulating the expression of EZH2 and H3K27me3.3.The Co-IP assays showed that exogenous OGT coprecipitated with endogenous EZH2 and nine amino acids with potential for direct O-GlcNAcylation were predicted by using the Yin OYang 1.2 Server(www.cbs.dtu.dk/services/Yin OYang).Co-IP experiments showed that the binding capacity of EZH2 and SUZ12,another indispensable component of PRC2,was increased when the O-GlcNAcylation of EZH2 was upregulated.OGlcNAcylation enhanced EZH2 protein stability by prolonging the half-life of degradation and suppressing its ubiquitination.4.miR-101 was simultaneously identified by several web-based target prediction algorithms(Target Scan S,miRanda,pictar,and PITA)as potential regulators of OGT expression and then verified by a luciferase reporter assay and miR-101 mimic or inhibitor transfection assays.miR-101 was significantly decreased in all CRC cell lines compared with that in the HCo Epi C cell line and was negatively linearly correlated with the expression of OGT and O-GlcNAcylation.Western blot analyses showed that the protein level of fibronectin and the O-GlcNAcylation level were decreased following the transfection with the miR-101 mimics in the SW620 cells,whereas E-cadherin was increased.Consistent with these results,these markers showed the opposite change following the transfection with the miR-101 inhibitors in the SW480 cells.In addition,the overexpression of OGT or EZH2 in the SW480 cells partially blocked the inhibitory effects on the EMT that were induced by the miR-101 mimic transfection.5.The levels of mature miR-101,precursor miR-101-1 and precursor miR-101-2were increased when OGT or EZH2 were downregulated.Ch IP-q PCR analysis of the transcriptional start regions(TSSs)of miR-101-1 and miR-101-2 precursors showed that the miR-101 promoter regions are highly enriched in EZH2,H3K27me3,and OGlcNAcylation.The EZH2 knockdown in the SW480 cells almost completely eliminated the enrichment of O-GlcNAcylation and H3K27me3 in these regions,and when OGlcNAcylation was upregulated,the H3K27me3 enrichment in these regions was significantly increased.6.The expression of miR-101 in fresh tissues of colorectal cancer was significantly lower than that in adjacent normal tissues and OGT and EZH2 protein expression had a negative relationship with the miR-101 level.The results of immunohistochemistry staining in three consecutive sections of CRC tissue chips and Pearson correlation analysis showed that there was a positive correlation among the levels of OGT,EZH2,and OGlcNAcylation in vivo.Conclusion:In conclusion,in colorectal cancer cells,miR-101/O-GlcNAcylation/EZH2 signaling forms a double-negative feedback loop that promotes metastasis.The down-regulation of miR-101 in CRC promotes the elevation of O-GlcNAcylation and,thus,enhances EZH2 protein stability and function,which,in turn,further reduces the expression of miR-101.Our findings provide a new mechanistic insight into the basic theory of cancer metastasis and suggest that blocking the miR-101/O-GlcNAcylation/EZH2 axis may represent a potential therapeutic strategy for metastatic colorectal cancer.From the perspective of metabolic and epigenetic mechanisms,the current study elucidates a crucial role of OGlcNAc modification in metastatic CRC and provides a new potentially potent strategy of metastatic CRC treatment and intervention.Meanwhile,it further improves the theoretical system of O-GlcNAc modification regulating tumor malignant phenotypes and enriches the understanding of PTM in cancer.