The Role Of GBA In The Progression Of Hepatocellular Carcinoma And The Mechanism Of CBE Enhancing The Efficacy Of Sorafenib

Background Hepatocellular carcinoma（HCC）is the the main type of liver cancer,and ranks third in the cause of tumor death.Sorafenib,a multi-target and multi kinase inhibitor,has become the standard drug for the treatment of advanced HCC,but the tumors treated by sorafenib can not completely subside and have high drug resistance.Therefore,it is urgent to describe the driving factors of drug resistance and develop new treatment strategies to improve the efficacy of sorafenib.Glucosaminidase（GBA）,a member of glycoside hydrolase family 30,plays an important role in complex lipid degradation,cell membrane turnover and cholesterol metabolism.It is reported that inhibition of GBA enzyme activity can enhance the sensitivity of gastric cancer and breast cancer to chemotherapy.However,there are no reports on the effect of GBA on HCC progression and its regulatory mechanism.Therefore,exploring the regulation and mechanism of GBA on HCC may provide new ideas for clinical HCC treatment.Cholesterol biosynthesis is considered to be a hallmark of cancer.Evidence has emerged to indicate that the biosynthesis of fatty acids and cholesterol is essential for the development and progression of a wide variety of tumors,owing to their critical nature as building blocks for membrane components.In addition,increased intracellular cholesterol levels were closely associated with the subsequent alterations of oncogenic growth signaling and motility in cancer cells.Although the cholesterogenic pathway is considered to be a promising pharmaceutical target for cancer treatment,the ability to sensitize HCC cells to the effect of cholesterol-lowering drugs and improve the anti-cancer effect has been poorly studied.In this study,we evaluated the carcinogenic effect of GBA in HCC cell lines in vitro,and constructed hepatocyte specific GBA knockout(GBA^LKO)mice to assess its effect on the development of HCC in vivo.We also clarified the mechanism of GBA promoting HCC,and evaluated the anticancer effect of GBA inhibitor CBE in the treatment of HCC and the combination of CBE and sorafenib in xenograft HCC and orthotopic HCC mouse models.Objective The purpose of this study was to explore the expression level of GBA in HCC cancer tissues;To explore the role and molecular mechanism of GBA in the progression of HCC;To explore the potential value and mechanism of GBA inhibitor CBE in the treatment of HCC;To explore the effect and mechanism of GBA inhibitor CBE and sorafenib in the treatment of HCC.Methods The cancer samples and paired adjacent samples of 12 pairs of clinical HCC patients were collected,and analyze the differentially expressed protein molecules in the tissue samples by LC-MS/MS mass spectrometry.The expression level of GBA in tissue samples was detected by real time PCR,Western blot and immunohistochemistry.The expression levels of GBA in different clinicopathological features of HCC and the correlation between GBA and HCC survival were retrieved and analyzed in TCGA（Cancer Genome Atlas）database.In order to explore the role and regulatory mechanism of GBA in the progression of HCC,overexpression vector and specific small interfering RNA（si RNA）were used to overexpress and silence GBA gene in human hepatoma cell lines in vitro,and cell lines with stable and high expression of GBA（Hep G2-GBA）and stable knockout of GBA（SKHEP-sg GBA）were constructed.After GBA overexpression or knockdown treatment,hepatoma cells were treated,CCK-8 experiment and cell clone formation experiment were used to detect the effect of GBA on the proliferation of HCC cells,Transwell experiment was used to detect the effect of GBA on the migration and invasion of HCC cells,flow cytometry and TUNEL experiment were used to detect the effect of GBA on HCC cell apoptosis,ELISA was used to detect the content of intracellular cholesterol and cholesterol ester,and Western blot was used to detect the protein expression level of key molecules of intracellular cholesterol synthesis.In vivo,GBA stable knockout cell line（SKHEP-sg GBA）and GBA stable high expression cell line（Hep G2-GBA）（2×10⁶/100μL PBS）was injected subcutaneously into the left back of 6-week-old male BALB/c nude mice.The effect of GBA on xenograft HCC mouse model was detected by observing the tumor volume,the size of tumor nodules and tumor histopathological analysis.Hepatocyte specific GBA knockout mice(GBA^LKO mice)were constructed.The orthotopic HCC model of mice was constructed by hydrodynamic injection technology.The growth of liver tumor,the content of cholesterol and cholesterol ester in liver tumor,the pathological changes of liver tumor,the protein level of key molecules of cholesterol synthesis in hepatocytes and the effect of GBA on orthotopic transplanted HCC mouse model were detected by lipomics analysis.In order to evaluate the potential of GBA inhibitor conduritol B epoxide（CBE）in the treatment of HCC,different concentrations of CBE（0μM-800μM）were given in different liver cancer cells in vitro.After a certain time of drug action,the levels of proliferation and apoptosis of hepatoma cells after intervention with different concentrations of CBE were analyzed.Give CBE（600μM）or Sorafeni（5μm）drugs to interfere with liver cancer cells,analyze the effect of combined treatment of GBA and Sorafeni in vitro.In vivo experiments,HCC mouse models of xenotransplantation and orthotopic transplantation were constructed respectively,and the mice were randomly divided into four groups:control group were given PBS（intraperitoneal injection,once every three days）,GBA inhibitor treatment group（CBE）received CBE with a dose of 100 mg/kg（intraperitoneal injection,once every three days）,the Sorafenib treatment group（SF）received Sorafenib with a dose of 60 mg/kg（oral,daily）,and the CBE+SF treatment group received 100 mg/kg CBE+60mg/kg SF.The mice were euthanized after treatment for three weeks,and the tumor masses of mice were collected for molecular biological and histopathological analysis.Results Proteomic analysis of liver tumor tissues and normal tissues of clinical HCC patients showed that GBA was significantly overexpressed in HCC liver tumor tissues,and there was significant statistical difference;The results of real time PCR,Western blot and immunohistochemical staining showed that the m RNA level and protein level of GBA in HCC liver tumor were significantly increased.TCGA（Cancer Genome Atlas）database analysis showed that GBA expression was elevated in HCC and was associated with poor prognosis in patients with liver cancer.The results in vitro showed that GBA could promote the proliferation,migration and invasion of hepatoma cells.When GBA was inhibited,the proliferation,migration and invasion of hepatoma cells were also inhibited;The high expression of GBA can inhibit the apoptosis of hepatoma cells,and the low expression of GBA can induce the apoptosis of hepatoma cells;The contents of cholesterol,total cholesterol,free cholesterol and cholesterol esters in hepatoma cells increased when the expression of GBA was high,and their contents also decreased when the expression of GBA was low;When GBA is highly expressed,the key proteins of intracellular cholesterol biosynthesis（SREBP2,LDLR）and multiple receptors on the plasma membrane（itgav,ITGB4 and TGF）which are important for tumor growth and metastasisβThe protein expression level of R1)increased significantly,the PI3K/AKT/m TOR carcinogenic signal pathway was activated,the protein expression level of cell proliferation molecules increased significantly,and the protein level of Pro apoptotic molecules decreased.The growth rate and volume of xenograft tumor in nude mice inoculated with GBA stable and high expression cells were significantly higher than those in the control group;PIK3CA/c-Met plasmid was transfected into hepatocyte specific GBA knockout mice(GBA^LKO)and control mice(GBA^fl/fl)by hydrodynamic injection.The results showed that the incidence of liver tumors in GBA^LKO mice was significantly lower than that in GBA^fl/fl mice,the contents of cholesterol and cholesterol esters in GBA^LKO mice,and the protein level of cholesterol synthesis signal pathway in GBA^LKOmice.The proliferation level of hepatoma cells was significantly inhibited,while the level of apoptosis was significantly increased,the content of intracellular cholesterol and cholesterol ester was significantly reduced,and the protein level of key genes of intracellular cholesterol synthesis was also inhibited;After the combined action of CBE and sorafenib（SF）,the proliferation level of hepatoma cells decreased significantly,and the inhibition efficiency was higher than that of CBE or SF alone,and the combined action of CBE and SF significantly induced apoptosis of hepatoma cells,and the proportion of apoptosis was higher than that of CBE or SF alone;The therapeutic effect of CBE or CBE combined with SF was further verified in xenotransplantation and orthotopic transplantation of HCC mouse models.The results showed that CBE alone or combined with SF could inhibit the growth of tumors in vivo,and the inhibitory effect of CBE combined with SF was more obvious.Conclusion GBA drives the occurrence of HCC by promoting the accumulation of intracellular cholesterol or cholesterol esters.Inhibiting GBA can reduce intracellular cholesterol synthesis,inhibit tumor growth in vivo and enhance the sensitivity of sorafenib to HCC treatment.