The Gluconeogenic Enzyme FBP1 And PCK1 Inhibit HCC Tumorigenesis

Objective: Primary liver cancer is one of the ten most common malignant tumors in the world,ranking sixth in cancer incidence rate and fourth in mortality.Primary liver cancer mainly includes three different pathological types: hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma(ICC)and HCC-ICC mixed type.The three types have great differences in pathogenesis,biological behavior,histological morphology,treatment and prognosis,among which the proportion of HCC is more than 85%-90%.The research of tumor is mainly based on the instability of genome.With the development of tumor molecular biology,there are some new universal characteristics.Traditional tumor research mainly includes six aspects: 1.Signal of continuous proliferation 2.Escape from growth inhibitory factor 3.Resistance to cell death 4.Unlimited replication 5.Induction of angiogenesis 6.Activation of tumor invasion and metastasis.The instability of genome is hidden behind these six markers.Otto Warburg,a famous German biochemist and Nobel Prize winner in physiology and medicine,found that when oxygen is sufficient,about 80% of glucose in tumor cells mainly produces ATP through aerobic glycolysis,accompanied by the production of a large number of lactic acid,rather than through oxidative phosphorylation.It is the famous Warburg effect.The emergence of metabolism is a new angle and is a new sign of tumor.The study of metabolizing enzymes is a hot pot in tumor metabolism.In recent years,the study of tumor aerobic glycolysis has been the focus of the study of glycometabolism,but the opposite side of glycolysis is rarely studied.Gluconeogenesis is a process in which glucose and glycogen are synthesized from simple non sugar precursors(lactic acid,glycerin,amino acid,etc.).Fructose-1,6-bisphosphotase(FBPase)is the key enzyme of gluconeogenesis,which catalyzes the transformation of fructose-1,6-diphosphate into fructose-6-phosphate.It is the second rate limiting reaction of gluconeogenesis.There are two kinds of fructose-1,6-diphosphate,FBP1 and FBP2.FBP1 is the rate limiting enzyme of gluconeogenesis in liver and kidney.FBP2 only exists in muscle tissue.Phosphoenolpyruvate carboxykinase(PCK)is the first key enzyme in gluconeogenesis,which catalyzes the formation of phosphoenolpyruvate from oxaloacetic acid.There are two kinds of phosphoenolpyruvate lysosomes to form autophagic lysosomes and degrade the contents(proteins and organelles).Autophagy plays an important role in resisting tumor cell death,regulating cell life and death.It has been proved that glucose-6-phosphatase(G6PC),a key enzyme in gluconeogenesis,can regulate autophagy,but it has not been reported that FBP1 regulates autophagy in liver cancer.In this study,we first observed the expression of glycoallogeneic key enzyme FBP1 in clinical liver cancer tissue samples and liver cancer cell lines,and verified that overexpression of FBP1 inhibited autophagy and proliferation of liver cancer cells in vitro,and found that the overexpression of FBP1 in liver cancer cells after low glucose treatment resulted in significant apoptosis.PCK1 was found to promote the development of liver cancer by knocking out mice with liver specific PCK1 and inducing mice with diethylnitrosamine / carbon tetrachloride.The above experiments will help us to understand the role of key gluconeogenesis enzymes FBP1 and PCK1 in the occurrence and development of liver cancer,which is expected to provide new ideas and strategies for clinical prevention and treatment of liver cancer.Method: 1.Through TCGA(the Cancer Genome Atlas)database,we analyzed the expression of FBP1 in clinical liver cancer and adjacent tissues,and analyzed the relationship between the expression of FBP1 and the prognosis of patients.The expression of FBP1 in clinical liver cancer and adjacent tissues was detected by western blot.The expression of FBP1 in normal hepatocytes and different hepatoma cell lines was detected by western blot.2.Obtaining a hepatoma cell model with over expression of FBP1.The recombinant plasmid of FBP1 was constructed,and the over expression vector of FBP1 was constructed by AdEasy adenovirus vector system.The over expression efficiency of FBP1 recombinant adenovirus was identified by Western blot.3.Analyzing the effect of FBP1 on autophagy level in hepatoma cell lines and clinical hepatoma tissues: To observe the effect of FBP1 on autophagy level of hepatoma cells by Western blot and laser confocal technology.Western blot was used to detect the expression of autophagy LC3 in clinical liver cancer and adjacent tissues.4.The effect of FBP1 on the proliferation and apoptosis of hepatoma cell line was detected by cell observation in vitro.The effect of FBP1 on the proliferation of hepatocarcinoma cells was observed by clonogenesis and MTS experiments.Finally,the effect of FBP1 on the apoptosis of hepatocarcinoma cells was detected by cell apoptosis and Western blot experiments.5.The construction of PCK1 gene liver specific knockout mouse liver cancer model with diethylnitrosamine / carbon tetrachloride.Result: 1.TCGA database analysis showed that the expression of FBP1 in HCC was significantly lower than that in adjacent tissues(P < 0.01),and the survival time of HCC patients with low expression of FBP1 was significantly lower than that of patients with high expression of FBP1.Western blot showed that the expression of FBP1 protein in 9 out of 12 pairs of tumor tissues decreased,accounting for 66.6%.Western blot showed that the expression of FBP1 in HCC cells was lower than that in normal hepatocytes.2.The recombinant plasmid of FBP1 adenovirus was successfully constructed and packaged into high titer adenovirus.Western blot was used to identify the recombinant adenovirus.3.Western blot showed that the level of LC3,an autophagy index of hepatoma cells overexpressing FBP1,decreased significantly.The results of laser confocal detection showed that the number of autophagy decreased significantly.The level of autophagy LC3 was higher in 8 out of 9 liver cancer tissues with low expression of FBP1.4.The results of clonogenesis and MTS showed that overexpression of FBP1 significantly inhibited the proliferation of hepatoma cells.The results of apoptosis and Western blot showed that the hepatoma cells of overexpression of FBP1 group had significant apoptosis after low glucose treatment.5.PCK1 gene liver specific knockout mouse liver cancer model was successfully constructed.Conclusion:In this study,we first confirmed that the expression of FBP1 in liver cancer tissue was significantly lower than that in adjacent tissues,and then proved that FBP1 can inhibit autophagy and proliferation of liver cancer cells in vitro,and that over expression of FBP1 can promote apoptosis of liver cancer cells.The liver cancer model of PCK1 liver specific knockout mice was successfully constructed,and it was confirmed that PCK1 knockout could promote the development of liver cancer in vivo.