Effect Mechanism Of Malate Dehydrogenase 2 On Migration And Invasion Of Mice Hepatocarcinoma Cells

Background:Hepatocellular carcinoma is one of most prevalent cancers all over the world.It is subjected to lymphatic metastasis in the early stage,with high mortality and poor prognosis.Malate dehydrogenase 2 is a NAD/NADH-dependent oxidoreductase located in the mitochondria of eukaryotic cells.Compared with the expression level of malate dehydrogenase 2 in mice liver cancer ascites cells with high lymphatic metastasis potential,the low is higher,and it can inhibit the ability of proliferation,migration and invasion of Hca-P cells in vitro.E-cadherin,N-cadherin and CD44 are all closely related to tumor metastasis.E-cadherin is a maker of epithelium and N-cadherin is a maker of interstitium.Epithelial-mesenchymal transition drives the phenotypic transformations and increases the plasticity of epithelial cells,which can improve the adaptability of tumor cells to dynamic microenvironment and give them more malignant characteristics.It is generally recognized that the loss of E-cadherin and the expression of N-cadherin are the common features of epithelial-mesenchymal transition.CD44,an adhesive molecule,is a classic surface marker being recognized of cancer stem cells,which plays a vital part in regulating adaptability,plasticity and epithelial-mesenchymal transition of cancer cells.The overexpression of CD44 is supposed to be of great importance in stimulating the migration and invasion of cancer.We explored the relationship between malate dehydrogenase 2 and E-cadherin,N-cadherin and CD44 in the subject,which helped us to study the influencing mechanism of how malate dehydrogenase 2 affects the migration and invasion of mice liver cancer cells.Objective: This study has used Hca-P cells as the research object.To explore the effect mechanism of malate dehydrogenase 2 on migration and invasion of mice hepatocarcinoma cells preliminarily,we studied the relationship between malate dehydrogenase 2 and E-cadherin,N-cadherin and CD44.Methods:1.Hca-P cells lysate was used to perform immunoprecipitation experiment to detect whether malate dehydrogenase 2 could co-precipitate with E-cadherin,N-cadherin and CD44 in Hca-P cells.2.Constructed plasmid vectors with malate dehydrogenase 2 overexpression and knockdown,then imported the successfully constructed vectors with expression of target gene and its corresponding negative control plasmids into Hca-P cells through liposome transfection.Then the cells were collected after 48 hours to detect m RNA expression of malate dehydrogenase 2,and 72 hours to detect protein expression to verify whether the transient transfection succeeded in increasing or decreasing the expression level of malate dehydrogenase 2.3.After the up-regulated and down-regulated cell lines of malate dehydrogenase 2 were successfully constructed,each group of experiments were divided into three groups,namely Control group/ MU group/ MU-NC group,and Control group/ MD group/ MDNC group.Real-time fluorescent quantitative PCR,Western blotting and immunocytochemistry were used to detect the effects of change in expression level of malate dehydrogenase 2 on expression level of E-cadherin,N-cadherin,and CD44 in cells of each group.Then compared the MU group with control groups,and the MD group with control groups to observe whether there exist differences between the two groups.Results:1.The results of co-immunoprecipitation: Precipitated the Hca-P cell lysate with antimalate dehydrogenase 2 antibody,we discovered that the proteins of E-cadherin,Ncadherin and CD44 can be detected in the co-immunoprecipitation complex.And then the anti-E-cadherin antibody,anti-N-cadherin antibody and anti-CD44 antibody were precipitated with the cell lysate respectively,the malate dehydrogenase 2 can be detected by western blotting.2.The results of real-time fluorescent quantitative PCR and Western blotting showed that,the expression level of malate dehydrogenase 2 in m RNA and protein in the MU group were significantly higher than in the control groups after the malate dehydrogenase 2 upregulated plasmids were transfected(P<0.05).After the plasmids with malate dehydrogenase 2 down-regulated were transfected,the expression level of malate dehydrogenase 2 in the MD group was significantly lower than in the control groups(P<0.05).3.The results of real-time fluorescent quantitative PCR,western blotting,and immunocytochemistry experiments showed that there were several differences in the m RNA and protein expression level of E-cadherin and CD44 within the malate dehydrogenase 2 up-regulated group and malate dehydrogenase 2 down-regulated group(P<0.05).Compared with the Control group and MU-NC group,higher m RNA and protein expression level of E-cadherin,and lower expression of CD44 were discovered in the MU group.Compared with the Control group and MD-NC group,the m RNA expression level of E-cadherin was down-regulated in the MD group,and protein expression was decreased as well.However,the expression level of CD44 was increased significantly.Nevertheless,insignificant difference of N-cadherin expression was be found within the malate dehydrogenase 2 up-regulated group and malate dehydrogenase2 down-regulated group(P>0.05).Conclusion: The migration and invasion of mice liver cancer cells may be inhibited by malate dehydrogenase 2 via promoting the expression of E-cadherin and reducing the expression of CD44.