Effects Of Malate Dehydrogenase 2 On Proliferation,Apoptosis,Migration And Invasion Of Mouse Hepatocarcinoma Cells And Its Interaction With Annexin A7

Background:Hepatocellular carcinoma is one of the common malignant tumors in China.Lymphatic metastasis is an important route of early metastasis,which is one of the main factors that cause high recurrence and poor prognosis of hepatocellular carcinoma.Hca-P cells(lymph node metastasis rate < 30%)and Hca-F cells(lymph node metastasis rate > 70%)are the liver cancer cell lines with different lymphatic metastasis potential from the same genetic background and the same parent cell,which were screened by the transplantable mouse ascites hepatoma cell line in the early stage of our research group.Malate dehydrogense 2 is an oxidoreductase which is present in cell mitochondria and participates in many important metabolic pathways including the tricarboxylic acid cycle,amino acid synthesis.Malate dehydrogenase 2 plays an important role in the tumor genesis,development and metastasis.Our previous study found that the protein expression level of malate dehydrogenase 2 in Hca-P cells was higher than that in Hca-F cells.The gene and protein expression levels of Annexin A7 in Hca-F cells were higher than that in Hca-P cells,and its expression level is directly proportional to the proliferation,metastasis and invasion ability of liver cancer cells.Therefore,the study of the interaction between malate dehydrogenase 2 and Annexin A7 is helpful to further explore their mechanism in tumors.Objective:1.To study the effects of malate dehydrogenase 2 on proliferation,apoptosis,migration and invasion ability of Hca-P cells.2.To study the interaction between malate dehydrogenase 2 and Annexin A7 in vitro and in vivo.Methods:1.To study the effects of malate dehydrogenase 2 on the proliferation,apoptosis,migration and invasion ability of Hca-P cells in mouse hepatocarcinoma.1.1 Expression vectors of malate dehydrogenase 2 up-regulated(pmu)and down-regulated(pmd)plasmids and expression vectors of Annexin A7 up-regulated(pa7u)and down-regulated(pa7d)plasmids and their corresponding negative controls(pun and pdn)were constructed and transfected into Hca-P cells stably.The m RNA and protein levels of malate dehydrogenase 2 in pmu/pmd cells were determined by q RT-PCR and Western Blot.1.2 Cell counting kit-8 was used to detect the effect of malate dehydrogenase 2 on the proliferation of Hca-P cells.1.3 The effect of malate dehydrogenase 2 on the apoptosis of Hca-P cells was measured by flow cytometry.1.4 Transwell assay was used to detect the effect of malate dehydrogenase 2 on the migration and invasion of Hca-P cells.2.To study the interaction between malate dehydrogenase 2 and Annexin A7 in vitro and in vivo.2.1 The interaction between malate dehydrogenase 2 and Annexin A7 was detected by co-immunoprecipitation.2.2 Establish Hca-P,pmu,pmd,pa7 u,pa7d,pun,pdn cell tumor animal models.2.3 The m RNA and protein expression levels of malate dehydrogenase 2 and Annexin A7 were detected by q RT-PCR,western blot,immunocytochemistry and immunohistochemistry,respectively.Results:1.QRT-PCR,western blot confirmed that the m RNA and protein expression level of MDH2 in pmu cells was significantly higher than that in Hca-P and pun cells,while the m RNA and protein expression level of malate dehydrogenase 2 in pmd cells was significantly lower than that in Hca-P and pdn cells(P< 0.05).There was no significant difference between Hca-P and pun/pdn cells(P>0.05).2.Cell counting kit-8 experiment showed that the number of cells was lower while upregulating malate dehydrogenase 2 after 48 h,and it was higher while downregulating malate dehydrogenase 2(P?0.05).3.The results of flow cytometry showed that the early apoptosis rate in pmu cells washigher than that in Hca-P and pun cells,and the early apoptosis rate in pmd cells was significantly lower than that in Hca-P and pdn cells(P <0.05).4.Transwell assay indicated that the invasion and migration rate was significantly decreased at 24 h while upregulating malate dehydrogenase 2,while the number of cells passing through the chamber in pmd cell group was significantly more than in Hca-P and pdn cells(P <0.05).5.Co-immunoprecipitate results showed that malate dehydrogenase 2 and Annexin A7 were interacting proteins.6.QRT-PCR,western blot,immunocytochemistry and immunohistochemistry confirmed that after upregulation of Annexin A7,the gene and protein expression levels of malate dehydrogenase 2 in pa7 u cells were lower than those in Hca-P and pun cells in vitro and in vivo.And after Annexin A7 was downregulated,the expression levels of malate dehydrogenase 2 in pa7 d cells were higher than those in Pdn cells in vitro and in vivo.7.The m RNA expression level of Annexin A7 in pmu cells was significantly higher than that in Hca-P and pun cells,and the m RNA expression level in pmd cells was significantly lower than that in Hca-P and pdn cells(P < 0.05).However,the protein expression level of Annexin A7 in pmu group was lower than that in Hca-P and pdn cells,and the protein expression level in pmd group was significantly higher than that in Hca-P and pdn cells(P < 0.05).The results in vivo and in vitro were consistent.Conclusions:1.Malate dehydrogenase 2 weakened the ability of proliferation,invasion,and migration,and enhanced the ability of apoptosis in Hca-P cells.2.Malate dehydrogenase 2 and Annexin A7 are interacting proteins in hepatocarcinoma cells,which may be involved in the regulation of tumor occurrence and development.