The Regulatory Mechanism Of ALDOA In The Development Of Hepatocellular Carcinoma

Hepatocellular carcinoma（HCC）,as a prevalent form of liver cancer,is the sixth most commonly diagnosed cancer and the third leading cause of cancer death globally.Most solid tumors,including hepatocellular carcinoma,have been found that metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis providing all the metabolites needed to meet the demands for rapid growth regardless of oxygen status.Metabolic reprogramming is considered to be an important factor affecting the occurrence and development of cancer.Fructose-bisphosphate aldolase A（ALDOA）is an important enzyme in the process of glycolysis metabolism,enhanced glycolysis can promote the expression of ALDOA gene in tumor cells.Our previous researches showed that ALDOA knockout significantly inhibit the proliferation of hepatocarcinoma cells,but the molecular mechanism is still unclear.Therefore,this study aims to explore the molecular mechanism of ALDOA regulating glycolysis metabolism of hepatocarcinoma cells and the occurrence and development of hepatocarcinogenesis.And it will provide a molecular theoretical basis for new clinical treatment strategies of hepatocarcinogenesis.Methods:（1）CCK8 assay,colony formation assays,flow cytometry,wound scratch assay and Transwell assay were performed to observe the effects of ALDOA on proliferation,cell cycle,migration and invasion of hepatoma cells in vitro,respectively.Subcutaneously implanted tumor models and orthotopic xenotransplant tumor models in nude mice were constructed to determine the effect of ALDOA on the proliferation and invasiveness of hepatoma cells in vivo,respectively.（2）Enzyme activity assays,glucose consumption kit,lactate assay kit and ATP detection kit were used to detect the aldolase activity,glucose consumption,lactate secretion and ATP production of ALDOA knockout cells,respectively.Seahorse XF technique detected the extracellular acidification rate（ECAR）of ALDOA knockout cells.¹³C-labeling glucose and glutamine were used as tracers to detect the intracellular metabolites in ALDOA knockout SMMC7721 cells.（3）Transcriptome sequencing was performed to detect the different expression genes of ALDOA knockout HCCLM3 cells.GO analysis and GESA analysis were used to enrichment the different expression genes.Co-Immunoprecipitation and immunofluorescence were performed to detect the binding and location of ALDOA and c-Jun.The levels of phosphorylated c-Jun were measured by Western Blot.The expression of PAK2 and c-Jun were silenced by siRNA transfection.ALDOA with c-Jun or their mutants were transiently co-transfected to HEK293T cells.The target genes were verified based on the ChIP peak annotation,comparison,and visualization of c-Jun by the“ChIPseeker”R package.The expressions of ALDOA,CXCL8,DUSP1,FGB and PPP1R15A were detected by qRT-PCR.（4）Diethylnitrosamine（DEN）was used to induce HCC mouse models.Hepatic Aldoa was knockdown by adeno-associated virus based on serotype 8（AAV8）.Western Blot was used to detect the expression of ALDOA、c-Jun、p-c-Jun（Thr93）and PAK2.qRT-PCR was used to detect the expression of Aldoa,Cxcl1,Dusp1,Fgb and Ppp1r15a.Results:（1）ALDOA knockout in SMMC-7721 and HCCLM3 cells markedly reduced cell proliferation by 25.07%and 47.82%compared to control at 96 h,respectively.ALDOA overexpression in HepG2 cells had greater cell proliferation.Knockout or overexpression of ALDOA had no significant effect on the invasion and migration of hepatoma cells.Cell cycle analysis demonstrated that ALDOA knockout increased the percentage of cells in the G1 phase in hepatoma cells.Overexpression of ALDOA decreased proportion of cells in the G1 phase in HepG2 cells.ALDOA knockout in SMMC7721 cells inhibited the growth of subcutaneous tumors in nude mice,mean tumor weights of SMMC7721-Ctrl and SMMC7721-sg ALDOA derived xenografts were 1.57±0.11 g and 0.78±0.13 g,respectively.On the contrary,ALDOA overexpression in HepG2 cells remarkably increased tumor weights,mean tumor weights of HepG2-NC and HepG2-ALDOA derived xenografts were 0.63±0.08 g and 1.17±0.10 g,respectively.（2）ALDOA deficiency markedly down-regulated the catalyze activity of aldolase enzyme in three cell lines,but did not affect the levels of glucose consumption,lactate secretion,and ATP production in these cells.ALDOA knockout had no undetectable decrease in glycolysis,glycolytic capacity and glycolytic reverse in three cell lines.We performed metabolic flow analysis（MFA）in SMMC-7721 cells using[U-¹³C]glucose as tracers,ALDOA knockout increased 1.28-fold M6-labeled fructose-6-phosphate（F6P）and2.38-fold M6-labeled fructose-1,6-diphosphate（FBP）derived from glucose carbons compared to the control group at 6 h.In addition,ALDOA knockout increased ¹³C M6-labeled F6P（1.10-fold）and FBP（1.32-fold）,and decreased ¹³C M3-labeled Serine（0.70-fold）and ¹³C M2-labeled Glycine（0.71-fold）at 20 h.Further intracellular content determination demonstrated ALDOA deficiency significantly downregulated the levels of Serine and Glycine in hepatoma cells,ALDOA knockout decreased 0.45-fold Serine and0.56-fold Glycine in SMMC7721-sg ALDOA,proving that the overproduction of ALDOA in HCC boosts Serine and Glycine synthesis.（3）GO analysis showed that ALDOA is closely related to the biological processes in the nucleus.GESA analysis suggested that ALDOA will affect the function of AP-1signaling pathway.Co-IP and Western Blot assays showed that ALDOA could directly interact with c-Jun and affect the phosphorylation of c-Jun at Thr91 and Thr93 in hepatoma cells.PAK2 silenced inhibited the p-c-Jun（Thr91 and Thr93）in hepatoma cells,but PAK2interact with c-Jun rather than ALDOA.The binding and co-localization of ALDOA and c-Jun were failed when ALDOA Y364 or c-Jun Y10 mutated,the phosphorylation of c-Jun（Thr91 and Thr93）was also inhibited,indicating that the Y364 of ALDOA and Y10 of c-Jun are the pivotal interaction site with each other.Ch IP-seq analysis and qRT-PCR assay showed that ALDOA regulated the transactivation of CXCL8,DUSP1,FGB and PPP1R15A by affecting c-Jun phosphorylation.（4）With the time extension of DEN-induction,the protein expression of ALDOA and p-c-Jun（Thr93）increased in livers.Hepatic Aldoa knockdown reduced the number of tumors and maximal tumor volume,and reduced the levels of AST and ALT in serum.Immunohistochemistry analysis showed that Aldoa knockdown inhibited hepatocyte proliferation.Hepatic Aldoa knockdown decreased the protein expression of p-c-Jun（Thr93）and the m RNA expression of Cxcl1,Dusp1,Fgb and Ppp1r15a in liver tumor tissues.Conclusion:ALDOA knockout decreased the proliferation of hepatoma cells in vivo and in vitro,and overexpression of ALDOA promoted the proliferation of hepatoma cells in vivo and in vitro.Both ALDOA knockout or overexpression had no significant effect on the invasion and migration of hepatoma cells in vivo and in vitro.The biosynthesis of serine and glycine was inhibited in hepatoma cells after ALDOA knockout.ALDOA enhances hepatoma cells proliferation through facilitating PAK2 to phosphorylate Thr93 c-Jun,and affects the transcriptional regulation of ALDOA,CXCL8,DUSP1,FGB and PPP1R15A which are c-Jun target genes.Hepatic Aldoa knockdown inhibited DEN-induced hepatic hepatocarcinogenesis,ALDOA is a potential target for clinical treatment strategies in HCC.