The Role And Mechanism Of O-Glycosylation-regulated Endoplasmic Reticulum Stress In Hepatocellular Carcinoma

[Background/Aims]The number of patients with liver cancer continues to expand,and has become the main malignant tumor of tumor-related death.As one of the new ten characteristics of malignant tumors,metabolic changes during overnutrition provide ’driving energy’ for unlimited proliferation of tumor cells,and also participate in complex intracellular signal transduction,which has become an important central link in the development of malignant tumors.However,the molecular mechanism between excessive obesity caused by overnutrition and metastasis and invasion of HCC has not been clarified.O-linked β-N-acetylglucosamine(O-GlcNAc)modification is a reversible post-translational modification of proteins.Studies have shown that O-glycosylation is relatively high in cancer tissues.At present,the understanding of Oglycosylation modification in HCC is limited.The only research shows that the O-glycosylation level in HCC tissue is significantly higher than that in normal liver tissue.At the same time,OGT plays a vital role in maintaining the survival of islet cells.As a key nutritional sensor,OGT integrates the signaling pathways necessary for glucagon secretion and regulation of α-cell quality.However,the specific mechanism of O-glycosylation modification regulating the occurrence and development of HCC under overnutrition has not been reported.ER stress often occurs in cells with nutritional disorders,and ER stress plays an important role in the progression of HCC.In addition,FoxO1 as a nutritional sensor plays an important role in regulating obesity and related diseases.At the same time,our previous studies have confirmed that FoxO1 can up-regulate ER stress.Therefore,we speculated that O-glycosylation modification could affect the ER stress level through FoxO1 and thus affect the progress of HCC.[Methods]The clinical samples receiving liver surgery were collected to detect the expression of OGT and FoxO1,and to determine the correlation between OGT and FoxO1.The correlation between OGT/FoxO1 and prognosis of patients was analyzed in clinical samples.A high-fat diet mouse model was established to analyze the growth of subcutaneous tumors in nude mice in the high-fat group and the control group.The effect of FoxO1 overexpression or interference on the expression of endoplasmic reticulum stress-related proteins,and to determine the correlation between FoxO1 and endoplasmic reticulum stress.Double luciferase reporter gene assay and chromatin immunoprecipitation were used to confirm the regulatory mode of FoxO1 on endoplasmic reticulum stress.Effect of FoxO1 knockdown by lentivirus on migration and invasion of hepatocellular carcinoma cells.In vitro knockout of OGT or ST045849 inhibited the effect of OGT expression on FoxO1 expression level and migration/invasion of HCC cells.The expression level of FoxO1 was detected after mutation of specific O-GlcN acylation sites on SRF by pcDNA3.1.Lentivirus knock-down OGT expression was used for nude mouse subcutaneous tumor growth.[Results]Compared with adjacent tissues,the expression levels of OGT and FoxO1 in cancer tissues were higher and correlated with poor prognosis,and they were positively correlated.In vivo experiments confirmed that high-fat feeding promoted subcutaneous tumor growth in nude mice.Knockdown of FoxO1 can down-regulate endoplasmic reticulum stress level,thereby inhibiting EMT and ultimately affecting the migration/invasion of liver cancer cells.FoxO1 can bind to the promoter region of PERK to promote its transcription and activate endoplasmic reticulum stress.SRF O-glycosylation site mutation can downregulate FoxO1 expression.Knockdown of OGT can inhibit the growth of subcutaneous tumors.Inhibition of OGT expression in vitro can inhibit the migration/invasion of hepatocellular carcinoma cells by down-regulating FoxO1 expression.[Conclusion]Overnutrition increased the O-glycosylation level of SRF,induced FoxO1 expression,and up-regulated endoplasmic reticulum stress to promote epithelial-mesenchymal transition in hepatocellular carcinoma.The O-glycosylation modification of SRF is associated with the invasion and metastasis of hepatocellular carcinoma through the SRF/FoxO1/PERK axis.The O-glycosylation modification targeting SRF is expected to become a new strategy for the treatment of hepatocellular carcinoma.