Malate Dehydrogenase 2 Regulates Proliferation,Apoptosis And Metastasis Of Hepatocellular Carcinoma

Background:Primary liver cancer is a relatively malignant tumor of epithelial origin,and it is also the most common tumor in our country.Its treatment and prognosis recovery is still a major problem in scientific research.Lymphatic metastasis,as an early mode of metastasis,is an important factor affecting the prognosis of liver cancer.Therefore,studying the molecular mechanism of lymphatic metastasis of liver cancer is an important step in the search for lymphatic metastasis-associated proteins.Malate dehydrogenase 2 is a citrate recirculating enzyme that promotes the bidirectional conversion of malate to oxaloacetate.Although it is not a key enzyme in the tricarboxylic acid cycle,it also plays an irreplaceable role.Malate dehydrogenase 2also involves gluconeogenesis through the mitochondrial inner membrane through malic acid.In addition to this,the energy required for cell oxidation transfers oxidative phosphorylation of NADH from the cytoplasm by the action of malic acid and Clostridium Caspase to produce mitochondrial NADH.Provide energy for pumping chemotherapeutic drugs for P-glycoprotein.Decreased expression levels of malate dehydrogenase 2 also result in a significant decrease in DNA synthesis efficiency.In the early stage,we established two similar cell lines Hca-F cells and Hca-P cells from the same parent and the same genetic background.The difference is that we found that the lymphatic metastasis rate of Hca-F cells is more than 70%,and Hca-P cells have a lymphatic metastasis rate of less than 30%.At the protein level,Hca-P cells were 2.73times more potent than Hca-F cells.The biological characteristics and functions of tumor cells were verified by immunoblotting,Transwell chamber,flow cytometry and other techniques.Studying the relationship between malate dehydrogenase 2 and mouse ascites liver cancer is helpful to further discuss its mechanism of action in tumor proliferation and metastasis,and provide a new platform for targeted intervention of liver cancer.Objective:This study used Hca-P cells as the research object to investigate the effect of down-regulation of malate dehydrogenase 2 gene expression on cell proliferation,apoptosis,migration and invasion.Methods:Hca-P and Hca-F cells previously established and preserved in this group were used to detect the expression of MDH₂ at mRNA and protein levels by qRT-PCR and Western Blot.The pGPU6/GFP/Neo-shRNA-MDH₂ plasmid expression vector and its negative control pGPU6/GFP/Neo-NC-shRNA were constructed,and Hca-P cells were divided into blank control group?P?and transfected into pGPU6/GFP/Neo-MDH₂.The shRNA MDH₂-shRNA stable transfection group?Pmd?and the negative control group?Pmn?stable transfected with pGPU6/GFP/Neo-NC were transfected for 48h and the expression of green fluorescent protein was detected by fluorescence microscope.In the experiment,blank control group?P?,MDH₂ shRNA stable transfection group?Pmd?and the negative control group?Pmn?were used to control Hca-P cells.Screening for stable transfected cells,qRT-PCR and Western Blot were used to detect the above groups of cells.The expression of MDH₂ gene at mRNA and protein levels was detected by CCK-8 method,flow cytometry and Transwell chamber assay to detect the effects of low expression of MDH₂ on proliferation,apoptosis,migration and invasion of Hca-P cells.Results:1.Expression of MDH₂ at the gene level Hca-P cells were 2.574 times higher than Hca-F cells,and Hca-P cells were 2.248 times higher than Hca-F cells at the protein level.2.Green fluorescence detection of MDH₂ targeting shRNA successfully down-regulated the expression of MDH₂ gene in mouse hepatoma Hca-P cells.qRT-PCR and Western Blot showed that the expression of MDH₂ in Pmd transfection group was significantly lower than that in P group and Pmn group?P<0.05?.There was no significant difference in the expression between the P group and the Pmn group?P>0.05?.3.MDH₂ targeting sh-RNA successfully down-regulated the expression of MDH₂ gene in mouse liver cancer P cells.Transwell experients showed that the number of migrated cells in the P group,Pmd group and Pmn group was 208.5±13.44,860.5±53.03,278±2.83.Compared with the P group and the Pmn group,the cell migration ability of the Pmd group was significantly enhanced and the difference was statistically significant?P<0.05?.In the Transwell matrigel experimental group,the number of migrated cells in the P group,the Pmd group,and the Pmn group was 168±12.34,460±37.2,and 149.5±17.9.Compared with the P group and the Pmn group,the cell invasion ability of the Pmd group was significantly enhanced.The difference was statistically significant?P<0.05?.4.Cell counting kit8 proliferation assay was used to detect the absorbance of cells in P cell group,Pmd group and Pmn group at 0 hours,24 hours,48 hours,72 hours,96 hours,etc.The reaction time of proliferation reagent was 1 hour.The results showed that the values of mean absorbance±standard deviation of the cells in group P were 0.186±0.011,0.341±0.016,0.609±0.009,0.639±0.013,and 0.814±0.029,respectively,in five different time periods.The absorbance values of the Pmd cell group were 0.121±0.002,0.363±0.024,1.002±0.089,1.706±0.012,and 1.832±0.042,respectively.The absorbance values of the cells in the Pmn group were 0.129±0.034,0.349±0.026,0.648±0.038,0.736±0.086,and 0.893±0.034,respectively.The results showed that the proliferation rate of P,Pmd and Pmn cells was similar before the culture for 24 hours.The cell growth rate was similar,and the total number of cells was not significantly different?P>0.05?.The cells were cultured for nearly 48 hours.The total number of cells began to differ,and the 48-hour,72-hour,and 96-hour time points were statistically significant?P<0.05?.The proliferation ability of Pmd cells was higher than that of Pmn cell group and P cell group.There was no significant difference in proliferation ability between Pmn group and P group in 5 time periods?P>0.05?.5.Flow cytometry showed that the apoptosis rates of P group,Pmd group and Pmngroup were 5.98%±0.11%,3.07%±0.3%,6.03%±0.27%,respectively.The early apoptosis of cells in Pmd group was significantly lower than that in P group and Pmn group?P<0.05?.Conclusions:MDH₂ protein may play an important role in inhibiting the proliferation,invasion and migration of mouse hepatoma cells,and promote the early apoptosis ability.MDH₂ may be used as a link in the mechanism of lymphatic metastasis of liver cancer,providing new ideas for the study of targeted molecular drug therapy.