The Preliminary Research About ALDOA,A Novel Anti-tumor Drug Therapy Target And Active Angelica Sinensis Polysaccharide Screening

BackgroundAs one of the major diseases,malignant tumor is serious harmful to people's health,and also causes the highest mortality in Chinese population.The reason why tumor cells are fatal to humans and difficult to overcome,is that compared with normal cells,it has so unique characteristics that can provide a great survival and invasion advantage.As early as 1927,Warburg et al had pointed that tumor cells metabolized more sugar to convert to lactate than normal cells.Aldolase A(EC4.1.2.13,ALDOA)is an important enzyme in the process of energy metabolism and plays an important role in glycolysis and gluconeogenesis.It has been repoted that ALDOA is overexpressed in many cancer cells,such as squamous cell lung cancer and colorectal cancer.The expression of ALDOA in serum of some cancer patients is significantly higher,indicating that ALDOA may be a key molecule in tumor progression and malignant transformation.What is more,ALDOA not only plays an important role in glycolysis,but also involves in other functions of cells,such as signal transduction,vesicle transport,cell movement and so on.However,the relationship between ALDOA and tumor development is still unclear.Whether it can be a diagnostic marker of tumor or whether it can be a therapy target of candidate compounds remain to be confirmed.Our previous study found that ALDOA could promote the transfer of liver cancer,and angelica polysaccharide could affect the expression of ALDOA in liver cancer cells.There is no literature reported related work.ObjectivesIn this study,we aimed to explore the role of ALDOA in the regulation of HCC by using molecular biology techniques and related cell functional assays,and to further screen the anti-tumor polysaccharide with ALDOA as the therapeutic target and evaluate its anti-tumor activity.Methods(1)The protein expression of ALDOA in human hepatocarcinoma tissue was detected by immunohistochemistry(IHC),and then the expression of ALDOA in the tissues of human hepatocellular carcinoma and its adjacent normal tissues was compared.The expression of epithelial-mesenchymal transition(EMT)markers E-cadherin and vimentin in human HCC tissues were detected and their correlations were analyzed by Spearman correlation analysis.(2)The expression of ALDOA in different HCC cell lines was detected,and the expression of ALDOA in HepG2 cells was significantly higher after giving additional glucose than in HepG2 cells cultured under normal condition.Then ALDOA RNA interference and overexpression HepG2 cell model were established to detect the expression of ALDOA in mRNA and protein level by real-time PCR and Western blotting(WB).(3)The protein expressions of EMT markers were detected by immunofluorescence(IF)and WB.The invasion abilities of four recombinant cells HepG2-ALDOA-NC,HepG2-ALDOA,Hep G2-shRNA-NC and HepG2-shRNA-ALDOA were analysed by transwell cell invasion assay.Lung metastasis was observed by tail vein injection in nude mice,and lung tissue lesion was observed after nude mice were sacrificed.The tumor growth curve was drawn by subcutaneously implanted tumor in nude mice.The effects of ALDOA on the proliferation were observed and the cell growth curves of four recombinant cells were drawn.Cell apoptosis was measured by flow cytometry(FCM).(4)HepG2 cells were cultured in different concentration of Angelica sinensis polysaccharides respectively and then the expression of ALDOA protein in cells was detected by WB.The effects of Angelica sinensis polysaccharides on HepG2 cell and HepG2-ALDOA cell proliferation and apoptosis were observed in order to determine their specific effects on ALDOA.Results(1)The expression of ALDOA in hepatocellular carcinoma tissue was significantly increased and the expression of vimentin and E-cadherin were also changed in the tissues of human hepatocarcinoma tissue compared with the adjacent tissues.The correlation analysis showed that the expression of ALDOA and vimentin was positive,but the expression of ALDOA and E-cadherin was correlated negatively.(2)ALDOA overexpression and RNA interference cell model,HepG2-ALDOA-NC,HepG2-ALDOA,HepG2-shRNA-NC,HepG2-shRNA-ALDOA,were successfully constructed.(3)Vimentin and E-cadherin expression changed in the HepG2-ALDOA cells and the results consistent with the tissue chip IHC results.The in vivo lung metastasis ability and tumorigenic ability of ALDOA overexpressing cells were also enhanced.Compared with the control cells,the cell migration ability was enhanced in HepG2-ALDOA cells.The proliferation ability of ALDOA overexpressing cells was stronger than that of the RNA interference recombinant cells.Contrast to the RNA interference recombinant cells,the expression of Vimentin and E-cadherin in the control cells was also changed.Furthermore,the in vivo lung metastatic capacity and tumorigenic ability of the RNA interference recombinant cells were significantly reduced.Compared with its control group,the proliferation ability,anti-apoptotic ability,and cell migration ability were relatively weakened in HepG2-shRNA-ALDOA cells.(4)Angelica polysaccharide AAPS-2II could significantly inhibit the expression of ALDOA and the proliferation of HepG2 cells.Conclusions(1)ALDOA is closely related to the occurrence and metastasis of HCC.(2)The ALDOA overexpression and RNA interference recombinant cell model was constructed.ALDOA can significantly promote the proliferation and metastasis of HCC in vitro and in vivo,and inhibit apoptosis of HepG2 cells.(3)ALDOA promotes HepG2 cell metastasis by association with EMT pathway.(4)Angelica polysaccharide AAPS-2II may have anti-tumor effect through regulation of ALDOA expression.