Oxaloacetic Acid Influences The Hepatoma Cells Malignant Phenotype By Regulating Hyaluronan

Background:Primary liver cancer has always been a disease threatening human health.The highest incidence of primary liver cancer is hepatocellular carcinoma(HCC).HCC ranked fifth in the incidence and third in the fatality rate of all cancers worldwide in the world epidemiological statistics.In the process of tumor cell growth,in addition to changes in the internal components of the relevant cells,there are also changes in a kind of non-cellular components,which is the extracellular matrix(ECM).ECM can assist tumor progression by virtue of its dynamic structure and regulate the malignant phenotype of tumor cells through resynthesis and metabolism of its own structure.Hyaluronic acid(HA)is a part of ECM.Due to its highly hydrophilic physicochemical properties,HA can regulate tissue homeostasis and resistance to compressive forces.HA is usually present in cartilage and connective tissue,and can serve as tissue repair signal.HA is a long chain of glycosaminoglycans composed of repeated disaccharides,each of which is a condensation of D-glucuronic acid(Glc UA)and N-acetylglucosamine(Gle NAc).The de novo synthesis of HA is derived from glucose,and the two precursors of HA are derived from 6-phospho-glucose(G-6-P)and6-phospho-fructose(F-6-P),respectively.Therefore,the synthesis of HA is related to common competitive substrates in glycolysis.A large number of studies have shown that excessive accumulation of HA content in malignant tumors can promote tumor proliferation,migration and angiogenesis,and is associated with poor prognosis.Oxaloacetic acid(OAA)is an important intermediate product in the tricarboxylic acid cycle.In addition to participating in oxidative phosphorylation(OXPHOS)as a substrate,OAA has many other biological functions.The activity of human hepatoma cell line Hep G2 increased firstly and then decreased with the increase of OAA concentration.It has been found that low OAA concentration can up-regulate VEGF m RNA expression and promote angiogenesis,while high OAA inhibits glycolysis and enhances OXPHOS in human hepatoma cell line Hep G2.Apoptosis was induced by reversing Warburg effect.Since different concentrations of OAA have different or even opposite effects on Hep G2,and both HA and OAA are closely related to glycolysis,we used this as a starting point to explore the reasons for different effects of OAA at different concentrations on Hep G2 and the relationship between HA and OAA.Research materials and methods:The optimal concentration of HA and HA inhibitor 4-methylumbelone(4-MU)was detected by cell counting.The effect of HA on the viability of hepatocellular carcinoma Hep G2 and normal liver LO2 cells was verified by CCK-8 cell proliferation assay.The effect of HA on proliferation of hepatocellular carcinoma Hep G2 was verified by clonal formation assay.The effect of HA on migration of Hep G2 cell line was verified by scratch test.The effect of HA on invasion of hepatocellular carcinoma Hep G2 was verified by Transwell assay.The effects of different concentrations of OAA on the proliferation,migration and invasion of hepatoma Hep G2 cells were detected by the same method.The effects of different concentrations of OAA on HA synthesis were detected by ELISA.QRT-PCR assay was used to detect the expressions of HAS1,HAS2 and HAS3 m RNA in hepatocellular carcinoma Hep G2 and normal liver cells LO2,as well as in different concentrations of OAA,to verify that the effects of different concentrations of OAA on HA synthesis were independent of HAS transcription.The effects of different concentrations of OAA on the activity of hexokinase,a key enzyme of glycolysis,and lactate production of glycolysis product were detected by the kit.Finally,the protein expression of AKT/ p-AKT under different concentrations of OAA was detected by Western blot assay.Research results:Our study found that 40-100 k Da HA promote the cell number and proliferation of HCC cells Hep G2 and normal liver cells LO2.The optimal concentration of exogenous HA was determined to be 100 μg/ml and the optimal concentration of4-MU was 0.3m M.CCK-8 showed that HA could increase the viability of Hep G2 and LO2,but had a greater effect on Hep G2.Cloning and formation experiments showed that HA promoted the proliferation of Hep G2 cells.Scratch test showed that HA promoted the migration of hepatoma Hep G2 cells.Transwell assay showed that HA promoted the invasion of hepatocellular carcinoma cells Hep G2.Enzyme-linked immunosorbent assay(ELISA)showed that 20 m M OAA promoted the synthesis of HA in Hep G2 cells,and 50 m M OAA inhibited the synthesis of HA in Hep G2 cells.CCK-8 assay,clone formation assay,scratch assay and transwell assay showed that20 m M OAA promoted the proliferation,migration and invasion of Hep G2,and50 m M OAA inhibited the proliferation,migration and invasion of Hep G2.QRT-PCR showed that m RNA expression of HAS1 and HAS3 was low in Hep G2 cell lines.Two concentrations of OAA had no effect on HAS in LO2 cell lines,and 20 m M OAA had no effect on three HAS in Hep G2 cell lines.50 m M OAA increased the m RNA levels of HAS1 and HAS3 in Hep G2 cell lines,demonstrating that the effect of OAA on HA synthesis was independent of HAS transcription.HK assay showed that 20 m M OAA increased HK activity and 50 m M OAA inhibited HK activity.Both OAA and OAA inhibited the production of lactic acid.Western Blot analysis showed that20 m M OAA activated AKT phosphorylation and 50 m M OAA inhibited AKT phosphorylation.Analysis conclusions:1.Different concentrations of OAA have different effects on the malignant phenotype of hepatocellular carcinoma cells,low concentrations of OAA promote the malignant phenotype of hepatoma cells,high concentrations of OAA inhibit the malignant phenotype of hepatocellular carcinoma cells.2.Low concentration of OAA promotes malignant phenotype of hepatocellular carcinoma cells by increasing HA synthesis,and high concentration of OAA inhibits the malignant phenotype of hepatocellular carcinoma cells by inhibiting HA synthesis and glycolysis,and the effect of OAA on HA is not related to HAS.3.This study suggests that higher concentrations of OAA have potential pharmacological significance for the treatment of liver cancer.