The Molecular Mechanism Of HMGB1 In The Invasion And Metastasis Of Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in the world,and ranking the third major factor of tumor associated deaths.Surgery is the most effective method for the treatment of HCC.The prognosis of patients diagnosed with HCC is very poor and the 5-year survival rate is less than 20%due to the high rate of invasiveness and recurrence after surgery.HMGB1 is a member of damage pattern recognition molecules(DAMPs),which mainly exists in the cellular nucleus,and plays a critical role in maintaining the stability of the genome and the regulation of the related genes.In the condition of stress,HMGB1 can be actively released to the outside of the cell.Accumulating evidences show that whether endogenous HMGB1 located in cells or exogenous HMGB1 are both closely related to the prognosis of patients with hepatocellular carcinoma.Many studies have indicated that HMGB1 can promote the proliferation,invasion and metastasis of hepatocellular carcinoma.However,the underlying molecular mechanisms of HMGB1 in the progression of HCC remain unclear.Therefore,we will study the mechanism of endogenous HMGB1 and exogenous HMGB1 in the invasion and metastasis of hepatocellular carcinoma.We will further clarify the role of HMGB1 in the development of HCC,and provide a new theoretical basis and drug target for the treatment of HCC.Purpose:1.The relationship between the level of endogenous HMGB1 expression and the EMT phenotype of HCC cells.2.Explore how autophagy inhibits the EMT phenotype in HMGB1 knockout HCC cells.3.Study the effect of the formation of SIAH2-ZEB1-p62 complex on the EMT phenotype of HMGB1 knockout cells4.Explore the changes on glucose metabolism in HMGB1 knockout HCC cells5.The relationship between exogenous HMGB1 and the formation of HCC stem cells.6.Explore the role of Rage/JNK/miR-21 signal axis in the formation of HCC stem cells induced by exogenous HMGB1.7.Evaluate of the role of HMGB1 inhibitor on the effect of chemotherapy on HCC.Methods:The relationship between the expression level of HMGB1 and the phenotype of EMT was evaluated by immunohistochemistry,Q-PCR and Western.Lentivirus transfection was used to knock out the expression of HMGB1 in various HCC cell lines,and the phenotypic changes of EMT were detected by Q-PCR,Western and immunofluorescence.The invasiveness of HCC cells in HMGB1 knockout cells was detected by scratch test,Transwell invasion experiment and tail vein injection experiments.The changes of autophagy were detected by electron microscopy,immunofluorescence,Q-PCR and Western after HMGB1 knockout.The effects of autophagy on the EMT phenotype in HMGB1 knockout cells were investigated by autophagy related inhibitors.The formation of SIAH2-ZEB1-p62 complex was detected by immunofluorescence co-localization and immunoprecipitation.The role of AMPK/mTOR pathway in HMGB1 knockout cells was studied by Western experiment and AMPK siRNA.The glucose and lactate detection kits were used to determine the detect glucose metabolism changes in HMGB1 knockout cells.The changes of HMGB1 and autophagy were detected in cells treated by different concentrations of glucose medium.The role of exogenous HMGB1 in the proliferation,invasion and tumor sphere forming ability of HCC cells were evaluated.The role of Rage/JNK/miR-21 signal axis under HMGB1 stimulation was detected by Q-PCR,Western and immunofluorescence.The causes of HMGB1 release caused by chemotherapy were investigated by flowcyte,Western,cytotoxicity test and so on.HMGB1 inhibitors,ethyl pyruvate,were used to assess the effect of HMGB1 inhibitors on the efficacy of chemotherapy.Results:1.The expression of HMGB1 is positively correlated with the phenotype of EMT in HCC tissues.2.HMGB1 knockout could significantly inhibit the EMT phenotype of HCC cells.3.The increase of autophagy level is responsible for the inhibition of the EMT phenotype of HMGB1 knockout cells.4.Autophagy related degradation mediated by SIAH2-ZEB1-p62 complex is the direct cause of EMT phenotypic inhibition in HMGB1 knockout cells5.HMGB1 knockout results in glucose uptake disorder6.Low glucose culture can reduce the expression of HMGB17.Exogenous HMGB1 can promote the proliferation,invasion and formation of HCC cells8.Rage/JNK/miR-21 is the underlying molecular mechanism for the HCC stem cells formation by HMGB1 stimulation9.Chemotherapy promotes the release of HMGB1 by up-regulation of ROS10.HMGB1 inhibitors can increase the killing effects of chemotherapeutic drugs and reduce drug resistanceConclusion1.HMGB1 knockout mediates the autophagy-related degradation of ZEB1 by promoting the formation of the SIAH2-ZEB1-p62 complex,and then inhibits the EMT phenotype of the HCC cells.2.HMGB1 knockout can activate the AMPK/mTOR signaling pathway by inhibiting glucose uptake,and then up regulation of autophagy.3.Exogenous HMGB1 promotes the formation of HCC stem cells by activating the Rage/JNK/miR-21 signal axis.4.HMGB1 inhibitors can reduce the release of HMGB1 by removing ROS and reduce the chemotherapeutic resistance of HCC.