The Role And Mechanism Of O-GlcNAcylation In Hepatocellular Cancer Proliferation And Metastasis

Backgrounds:Enormous studies have linked metabolism disorder with the malignancies.As a major metabolic organ,liver plays a key role in remaining nutrients including glucose,amino acid,fatty acid and nucleotide as a complicated dynamic equilibrium.Recently,it is wildly accepted that the abnormal change of specific protein function is the initiating factor of tumor development and the acquisition of malignant biological phenotype.The function of protein is closely related to its expression,and it is more dependent on the change of its own activity.However,post-translational modification(PTM)is the main way to regulate the functional activity of proteins.O-GlcNAcylation,as a type of wildly studied post-translational modification,also has been reported to involve in various cancers.O-GlcNAcylation is catalysed by a single enzyme,termed O-linked N-acetylglucosamine transferase(OGT),which transfers N-acetylglucosamine from uridine diphosphate Nacetylglucosamine(UDP-GlcNAc)to protein substrates.Furthermore,a single enzyme known as N-acetyl-?-glucosaminidase(OGA;also known as O-GlcNAcase),which is encoded by the MGEA5 gene,removes the O-GlcNAc.Currently,studies have shown that O-GlcNAcylation plays an important role in the NASH-HCC and the progression of high glucose stimulation HCC.The YAP protein can be modified by O-GlcNAcylation,and can play an important role in the development of hepatocellular carcinoma with high glucose stimulation.However,because of the universality of O-GlcNAcylation,we believe that more complicated regulatory networks may have significant influence on the malignant phenotype of HCC.Therefore,it is of great significance to continue to study the role of O-GlcNAcylation in liver cancer.Aims:The aim of this study is to investigate the potential role of O-GlcNAcylation in HCC progression and find out the possible underlying mechanisms.1.To determine the expression level of O-GlcNAcylation in HCC tissues and cell lines.2.To explore the regulation effect of O-GlcNAcylation of hepatocellular carcinoma in vitro and in vivo.3.To explore the potential molecular mechanism of O-GlcNAcylation in hepatocellular cancer.Methods:1.The expression of O-GlcNAcylation in HCC and normal tissues was detected by IHC.The correlation between O-GlcNAcylation expression and clinical parameters of HCC and the association of O-GlcNAcylation and the overall survival was analyzed by Kaplan–Meier analysis.2.The expression of O-GlcNAcylation and OGT level in HCC cell lines and established immortalized liver cell line were detected by western blot experiment.3.The effect of O-GlcNAcylation in HCC proliferation was detected by in vitro CCK8,BrdU and colony forming assay as well as in vivo subcutaneous xenograft tumor model in nude mice.4.The effect of O-GlcNAcylation in HCC metastasis was detected by automated high-content image analysis system,Transwell,scratch assay and 3D spheroid invasion assay.5.Using mass spectrometry to analyze the related proteins that may be modified by O-GlcNAcylation in HCC.The results of mass spectrometry are further analyze by GO annotation and KEGG pathway analysis.6.The O-GlcNAcylation sites of target protein was predicted by the online database(http://www.cbs.dtu.dk/services/YinOYang/)The regulation of O-GlcNAcylation on target protein in HCC was verified by western blot,co-IP assay and the detecting of ubiquitination level.7.To observe whether the down-regulation of target protein could reverse the effect of the increased O-GlcNAcylation level,the method of siRNA was used to interfere with the level of target protein in the HCC cell lines overexpressed OGT.Results:1.Compared with normal tissues,the level of O-GlcNAcylation was significantly raised in hepatocellular cancer tissues.And statistics results showed that high level of O-GlcNAcylation was associated with pathology grade of tumor.Higher O-GlcNAcylation expression had worse outcomes than those with lower O-GlcNAcylation expression.2.O-GlcNAcylation and OGT expression levels were significantly higher in HCC cell lines,especially,highest expressing in BEL-7404 cell line,than in immortalized liver cell line,THLE-3.3.Drug and lentivirus infection can effectively change the level of O-GlcNAcylation level in cells.Elevated levels of O-GlcNAcylation promoted HCC cell growth,colony formation efficiency,migration,and invasion ability.At the same time,the nude mice tumorigenesis test results showed that the increase of O-GlcNAcylation could promote the growth of cancer cells in vivo.On the contrary,the reduction of O-GlcNAcylation level decreased the proliferation of hepatocellular carcinoma cell lines,colony formation efficiency and the ability of invasion and metastasis.5.We analysis the results of OGT-IP samples using mass spectrum and found 214 eligible candidates for O-GlcNAcylation.Among the potential candidates,we found that there are two factors involved in the KEGG map05202,which represents for the transcription misregulation in cancer,SIX1 and P53.SIX1,which is closely related to the malignant phenotype of liver cancer and glycolysis,was chosen to be the research objective for the next step.6.The result of online database showed that SIX1 was predicted to have 10 O-GlcNAcylation sites and the results of co-IP showed that SIX1 could directly bind with OGT,which further illustrated that SIX1 could be O-GlcNAcylation.Then we found O-GlcNAcylation could inhibit the ubiquitination level of SIX1 and sustain it and its downstream factors in high expression levels in HCC.7.The results of loss of function assays of SIX1 showed that the oncogenic effect of O-GlcNAcylation in HCC cell is partially dependent on SIX1 and its downstream elements.Conclusion:The level of O-GlcNAcylation was significantly raised in hepatocellular cancer tissues.Higher O-GlcNAcylation expression had worse outcomes than those with lower O-GlcNAcylation expression and hyper O-GlcNAcylation plays an oncogenic role in HCC cell lines.The specific nutrient uptake pattern of tumor cells increased the concentration of O-GlcNAc in the cells,thus increasing the level of O-GlcNAcylation of a series of proteins including SIX1.Hyper O-GlcNAcylation could inhibit the ubiquitination SIX1 level and sustain it and its downstream factors in high levels in HCC,through which the malignant phenotype of liver cancer is promoted.