The Effect Of Down-regulation MPPE1 Expression On The Tumor Malignant Behaviors Of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC)in China is one of the more malignant tumors,with a high incidence and mortality.The development of hepatocellular carcinoma is a multi-step,multi-factor,multi-gene involved in the complex pathological process,in which gene mutation is a very important mechanism.Gene mutation causes synthesis or structural dysfunction of gene product and leads to the imbalance of oncogene and tumor suppressor gene,so that the malignant proliferation of cells forms tumors;Gene mutation can cause signal path change.It affects proliferation,cycle,apoptosis,adhesion,migration and invasion of tumor cells and leads to the occurrence and development of tumor.Therefore,it is of great significance to the screening of possible regulatory genes in the prevention and treatment of liver tumor.It was early found that the mutations of metallophosphoesterase 1(MPPE1)gene in hepatocellular carcinoma tissues had relative higher frequency by high-throughput exon sequencing and PCR-MassArray sequencing.The mutations of MPPE1 gene is related to recurrence of hepatocellular carcinoma(HCC).There is no report in the study of MPPE1 gene in tumor at home and abroad.Therefore,it is necessary to study the mutations of MPPE1 gene for the biological behavior of liver cancer cell.ObjectivesTo investigate the proliferation,cycle,apoptosis,adhesion,migration and invasion of hepatocellular carcinoma cells,after down-regulated expression of MPPE1 gene by RNA interference technology.MethodsIn this study,we performed IHC to observe the expression and localization of MPPE1 gene in hepatocellular carcinoma tissues.Real-time PCR was used to detect the expression of the mRNA level of MPPE1 gene in hepatocellular carcinoma.The LV-RNAi technique was used to construct lentivirus and build the corresponding stably transfected down-regulated MPPE1 in the cell lines of HCC.The Real-time PCR and Western blot were used to verify the stability of the down-regulated transfection of MPPE1 in the lines of liver cancer cell.This study is to research the biology behavior of liver cancer cell after down-regulated MPPE1 genes through the cell proliferation,apoptosis,cycle,adhesion,migration,and invasion experiment.And is necessary to set up the negative control group.Results1.It shows that MPPE1 gene positive expression in cancerous tissue is mainly located in the cytoplasm by IHC.2.The LV-RNAi technique was used to construct ShRNA(LV3-H1-GFP+PURO)lentivirus carrier,and build the corresponding stably transfected down-regulated MPPE1 in the cell lines of HCC.After the transfected hepatoma HuH-7 and HepG2 cells and high pressure filter,the Real-time PCR and Western blot were used to verify the interfering effect,we successfully build a stable down-regulated MPPE1 gene ShRNA1 and ShRNA2 in the hepatoma HuH-7 and HepG2 cells,while the negative control is LV3-NC.3.The cell proliferation and cycle,apoptosis experiments shows that the hepatoma HuH-7 and HepG2 proliferation are restrained after down-regulated MPPE1 gene(CCK-8 detected 1-4day OD values,comparison between the experimental group and control group shows P<0.01);The percentages of hepatoma HuH-7 and HepG2 cells in G1 phases were substantially decreased and S phasessignificantly increased after ShRNA1 and ShRNA2 down-regulation of MPPE1 compared with the control(P<0.05);The cell apoptosis increases.After the application of Western blot-detection of Caspase 3 activation index PARP protein,we found that PARP carboxyl end structure of catalytic domain(89kD)significantly increased the amount of protein.It hints that cell apoptosis pathway is activated.4.The cell invasion,migration and adhesion experiments shows that the migration ability of hepatocellular carcinoma cells decreased(both of the groups showed P <0.01),the invasion ability of hepatocellular carcinoma cells decreased(both of the groups showed P <0.01),the adhesion of HCC was enhanced after down-regulated MPPE1 gene(both of the groups showed P <0.01).ConclusionsThis study found that down-regulated MPPE1 gene of hepatocellular carcinoma cell restrains proliferation and migration and invasion ability,promotes apoptosis,affects the biological behavior of liver cancer cells,we propose that MPPE1 gene may be involved in the occurrence and development ofhepatocellular carcinoma.